WO2022234175A1 - In vitro method for predicting and/or prognosing the ability of a biomaterial to induce tissue regeneration - Google Patents

In vitro method for predicting and/or prognosing the ability of a biomaterial to induce tissue regeneration Download PDF

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Publication number
WO2022234175A1
WO2022234175A1 PCT/ES2022/070279 ES2022070279W WO2022234175A1 WO 2022234175 A1 WO2022234175 A1 WO 2022234175A1 ES 2022070279 W ES2022070279 W ES 2022070279W WO 2022234175 A1 WO2022234175 A1 WO 2022234175A1
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Prior art keywords
biomaterial
markers
under study
cfah
clus
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PCT/ES2022/070279
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Spanish (es)
French (fr)
Inventor
Julio José SUAY ANTÓN
Francisco Javier ROMERO GAVILÁN
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Universitat Jaume I
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Publication of WO2022234175A1 publication Critical patent/WO2022234175A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0003—Not used, see subgroups
    • A61C8/0004—Consolidating natural teeth
    • A61C8/0006—Periodontal tissue or bone regeneration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans

Definitions

  • the present invention relates to a set of proteins that is particularly useful for predicting and/or forecasting the ability of a biomaterial to induce tissue regeneration.
  • the biomaterial to be studied is brought into contact with an isolated biological sample obtained from a subject and the peptide profile proposed here is analyzed and differentially adhered to the surface of said biomaterial, where said biomaterial is used as an implantable medical device , such as any type of prosthesis, such as stents, cannulas, pacemakers, dental implants, percutaneous medical devices, etc.
  • the present invention comprises an in vitro method to predict and/or forecast the ability to induce tissue regeneration by said biomaterial by determining and quantifying the peptide profile from an isolated biological sample adhered to it, as well as a kit to carry out this method.
  • an implant depends on its biological acceptability, that is, that it produces minimal damage and a minimal immune reaction in the implanted subject.
  • One of the essential requirements that said implant must meet is biocompatibility with the tissue of the subject receiving the implant, and, therefore, the absence of an immunological reaction to a foreign body.
  • the success of said implant depends directly on the regeneration capacity of the tissue that differentially adheres to the implant.
  • the tests that the biomaterial has to pass to be used as an implant are multiple and complicated, including in vitro tests against different cell lines (those of the tissue that is going to be in contact with the implant), to determine its cytotoxicity, cell proliferation, etc., to in vivo tests with those prototypes that have shown good in vitro properties.
  • preclinical studies and clinical trials in humans are necessary. All this process implies a long period of realization, in addition to the sacrifice of a significant number of animals, and an enormous economic cost. Additionally, clinical trials are not exempt from problems and risk for patients who give their consent to participate in them.
  • a regeneration process preferably bone regeneration or soft tissue regeneration
  • certain conditions are required, such as cell signaling, the presence of progenitor cells, as well as the processes of vascularization, healing, and, in the case of bone tissue, osteoinduction.
  • the interaction of the biomaterial-implant surface with biological systems determines the success or failure of the implant in the subject.
  • the surface properties of the implant (hydrophilicity, roughness or surface energy) affect its interaction with the body fluids of the individual.
  • this interaction can be positive and promote the integration of the biomaterial-implant (direct interaction between the surface of the biomaterial and the tissue without mediating fibrous tissue), or negative, giving rise to chronic and acute inflammation, high production of macrophages that constitute a response called foreign body reaction, which in most cases involves failure of the implantation.
  • Understanding the sequence of biological events that take place after implantation, such as blood coagulation processes, the development of infections, the immune response to the foreign body, and finally the rejection of the implant, is crucial to determine the compatibility of implants. a biomaterial, and, therefore, of prostheses perse. In this sense, the first protein layer adsorbed on the biomaterial and/or implant is responsible for triggering the cellular response to it.
  • Patent application WO2018/115553 describes an in vitro method to predict and/or predict the compatibility of biomaterials in a subject.
  • Said method describes a set of markers, preferably proteins, that are particularly useful for predicting and/or prognosticating the in vitro compatibility of a biomaterial.
  • the method is not designed to predict and/or determine the ability of a biomaterial to induce tissue regeneration; furthermore, certain markers discovered by the inventors and described herein are not considered.
  • US2009/258002 describes methods for the diagnosis and monitoring of cancer and tissue damaged by ischemia, as well as therapeutic and drug screening methods for said therapeutic methods.
  • the document describes a method for qualifying the status of a tissue in a subject, comprising the measurement of at least one marker in a sample of the subject, where the marker is selected from a list of 1325 genes whose expression varies depending on the tissue condition in Table 9, and the correlation of the measurement with the tissue condition.
  • Listed genes include CO3, C1QB, C1QC, TENT, CLUS, FLAK and APOE.
  • tissue to be diagnosed it is selected from cancer risk, tissue regeneration/repair, acute organ failure, organ transplant, presence or absence of diseases, stage of diseases, and efficacy of treatment of diseases.
  • tissue regeneration or any other type of biological sample in contact with biomaterials there are no specific references to tissue regeneration or any other type of biological sample in contact with biomaterials.
  • WO2015/110989 describes methods for predicting the probability of mucosal recovery in individuals with inflammatory bowel disease, including those with Crohn's or ulcerative colitis.
  • the method comprises the measurement of the concentrations or levels of liver growth factor (HGF), betacellulin (BTC), vascular cell adhesion molecule (VCAM-1) and anti-TNF compounds ⁇ ⁇ in a sample obtained from the subject.
  • HGF liver growth factor
  • BTC betacellulin
  • VCAM-1 vascular cell adhesion molecule
  • anti-TNF compounds ⁇ ⁇ in a sample obtained from the subject.
  • the present invention differs from WO2015/110989 in the marker panel that is used; furthermore, in the method of the present invention, the biological sample is adhered to the surface of a biomaterial.
  • WO2016/074068 describes chemical compounds that promote tissue regeneration in organs. These compounds can stimulate the production of certain molecular markers, including metalloproteinases (MMP1, MMP2, MMP9, MMP13), liver growth factor (HGF), lysyl oxidase (LOX), and E1 and A1 serpins.
  • MMP1, MMP2, MMP9, MMP13 metalloproteinases
  • HGF liver growth factor
  • LOX lysyl oxidase
  • E1 and A1 serpins E1 and A1 serpins.
  • the inventors of the present invention have identified a profile of markers, preferably protein markers, whose determination and/or quantification is capable of predicting and/or predicting the ability of a biomaterial to induce tissue regeneration.
  • a profile of markers preferably protein markers, whose determination and/or quantification is capable of predicting and/or predicting the ability of a biomaterial to induce tissue regeneration.
  • an isolated biological sample obtained from a subject is brought into contact with the biomaterial and said profile of markers adhered differentially to the surface of the biomaterial is analyzed, where the biomaterial is preferably an implantable biomaterial, and, more preferably , an implantable medical device, such as prostheses, stents, cannulas, pacemakers, dental implants, percutaneous medical devices, etc.
  • CRP C-reactive protein
  • SAMP serum amyloid P component
  • C1QC C subunit of the C1q subcomponent of complement
  • C1QB complement subcomponent C1q subunit B
  • C7 CO7
  • C5 CO5
  • IC1 inhibitor IC1
  • C3 complement component C3
  • C1S Complement factor H
  • C4BPA C4b binding protein > chain
  • C1R complement subcomponent C1 r
  • FCN2 Ficolin-2
  • VTNC Vitronectin
  • CLUS Clusterin
  • these proteins are markers of the ability of a biomaterial to induce tissue regeneration when they are adhered to it.
  • the in vitro identification and quantification of the presence of this set of markers represents a substantial improvement of the current state of the art, since until now no has identified no type of protein pattern that can be determined and quantified in vitro and that predicts what result will be obtained in vivo once the biomaterial is implanted.
  • the first aspect of the invention refers to an in vitro method ("method of the invention”) to predict and/or predict the ability to induce tissue regeneration by a biomaterial under study from a biological sample. isolated obtained from a subject and differentially adhered to the surface of said biomaterial, comprising determining and quantifying the presence of at least one of the markers adhered to the biomaterial under study, where said marker is selected from the list consisting of: CRP (UniProtKB P02741; entry version 234); SAMP (UniProtKB P02743; entry version 224); C1QC (UniProtKB P02747; entry version 210); C1QB (UniProtKB P02746; entry version 216); CO7 (UniProtKB P10643; entry version 209); C1S (UniProtKB P09871; entry version 245); VTNC (UniProtKB P04004; entry version 241); CO5 (UniProtKB P06684; entry version 185); IC1 (
  • the in vitro method for predicting and/or forecasting the ability to induce tissue regeneration by a biomaterial under study from an isolated biological sample obtained from a subject and differentially adhered to the surface of said biomaterial comprises the following stages: a. Incubate the biomaterial under study with the isolated biological sample; b.
  • markers attached to the biomaterial under study where said marker is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; NCTV; CO5; IC1; CO3; CFAH; C4BPA; C1R; COBA; FCN2; and CLUS; preferably in this stage the presence of markers adhered to the biomaterial under study is determined and quantified, where said markers are: component C5 of the complement (CO5), inhibitor of C1 (IC1), component C3 of the complement (CO3), Factor H of the complement (CFAH), complement subcomponent C1r (C1R), Ficolin-2 (FCN2) and Clusterin (CLUS); and more preferably a additional marker selected from the list consisting of: C-reactive protein (CRP); serum amyloid P component (SAMP); C subunit of the C1q subcomponent of complement (C1QC); complement subcomponent C1q subunit B (C1QB); complement component
  • CRP C-reactive protein
  • the method of the invention is characterized in that it comprises the simultaneous determination and quantification of one; two; three; four; five; six; seven; eight; nine; ten; eleven; twelve o'clock;thirteen;fourteen; fifteen or sixteen markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; NCTV; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; and CLUS.
  • the presence of the markers CRP; SAMP; C1QC; C1QB; CO7; C1S; NCTV; CO5; IC1; CO3; CFAH; C4BPA; C1R; CO8A; FCN2; and CLUS.
  • the method of the invention comprises the determination and quantification of at least one of the ratios between the IC1 markers; C4BPA; CFAH; NCTV; and CLUS, with respect to the C1QB markers; C1S; C1R; C1QC; CO5 and CO3.
  • at least one of the ratios selected from any of the following list is determined: IC1/C1QB; IC1/C1QC; IC1/C1S; IC1/C1R; CFAH/CO3; C4BPA/CO3; NCVT/CO5; CLUS/CO5. Values of these ratios equal to or greater than the values obtained in the reference biomaterial would indicate that the biomaterial has a good regeneration capacity when the sample is adhered to the biomaterial of interest or problem biomaterial.
  • the marker(s) that are identified and quantified are adsorbed to the biomaterial, once it has been in contact with the biological sample isolated from a subject, particularly of a subject to which the biomaterial has to be implanted.
  • Said markers are preferably markers present in the biological sample isolated from the subject.
  • the presence of these markers adsorbed to the surface of the analyzed biomaterials will determine the regeneration capacity of the biomaterial.
  • the term "adsorption” or “adherence” and variants refer to a process by which atoms, ions or molecules are trapped or retained on the surface of a material.
  • the term "regeneration” refers to any tissue repair process consisting of the neoformation of pre-existing tissue.
  • the term can be used for any type of tissue, including bone and soft tissue, connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports and binds other tissues such as bone, blood, and lymph.
  • the epithelial tissue serves as a cover; These include the skin and the lining of various ducts inside the body.
  • Muscular tissue consists of striated or voluntary muscles that move the skeleton and smooth muscle, such as that around the stomach.
  • Nervous tissue is made up of nerve cells or neurons and serves to carry "messages" to and from various parts of the body.
  • bone tissue or “bone tissue” refers to any type of tissue that gives strength and structure to bones, including compact tissue and cancellous or trabecular tissue.
  • prostheses/implants/bone substitutes that can improve their osseointegration, that is, that can contribute to generating new quality bone, more easily and in less time. It is also necessary to continue progressing towards a personalized implant design for each patient according to the characteristics of their clinical history and particular circumstances, such as lack of bone or the presence of osteoporotic bone, circumstances that are particularly frequent in postmenopausal women.
  • the improvement of the osseointegration capacity of bone prostheses depends on the physicochemical characteristics of the surfaces to be developed.
  • the different physicochemical properties of the newly developed surfaces will regulate the proteins adsorbed on the surface in contact with blood. Proteins adsorbed on the surfaces of the materials will regulate the main regenerative processes that will lead to the formation of new bone and ultimately to achieve optimal osseointegration of the implant.
  • soft tissue refers to muscle tissue, fibrous tissue, blood vessels, or any other supportive tissue of the body.
  • Percutaneous medical devices are being used more and more, for a wide variety of conditions, and in various medical fields. They can be defined as foreign bodies that cross the epithelial barrier and, therefore, connect the external environment with the internal structure of the body.
  • Examples of percutaneous devices include catheters and other long-term intubation systems or osseointegrated implants (for example, dental and orthopedic implants), where the device is permanently implanted in the body.
  • Soft tissue sealing is essential for stable osseointegration and long-term implant survival.
  • the goal of strategies to optimize soft tissue sealing around osseointegrated implants is to achieve implant surface properties that can promote soft tissue adhesion around percutaneous/permucosal implants, which would not only provide the implant with some stability , but it would also establish a permanent barrier against the invasion of bacteria and, therefore, infection.
  • fibroblasts produce collagen that serves to increase the mechanical resistance of the secreted extracellular matrix.
  • fibroblasts under stress conditions have the capacity to form pro-inflammatory environments (through expression of cytokines), produce reactive oxygen species (ROS) and decrease the levels of type I collagen and fibronectin, preventing tissue regeneration.
  • ROS reactive oxygen species
  • the response of fibroblasts to percutaneous implants may support tissue regeneration and wound healing (environments without chronic inflammation), or, conversely, the formation of non-regenerative microenvironments. , characterized by profiles of abnormal growth factors, inflammatory states and generation of ROS (environments with chronic inflammation).
  • NF-kp will be activated, as well as the expression of IL-ip type cytokines, and there will be a recruitment of neutrophils and macrophages.
  • the improvement of the soft tissue seal with the abutments depends on the physicochemical characteristics of the surfaces to be developed. The different physicochemical properties of the newly developed surfaces will regulate the proteins adsorbed on the surface in contact with blood. The proteins adsorbed on each of the surfaces of the material will regulate the main initial regenerative processes and, in particular, inflammation.
  • the in vitro method of the invention comprises the incubation of an isolated biological sample from a subject, preferably in which the biomaterial under study will be implanted, with a biomaterial whose capacity is to be determined. of regeneration.
  • biological sample refers to any biological material from a subject or multiple subjects.
  • the sample is preferably a tissue and/or biological fluid sample.
  • tissue sample may comprise, for example, kidney tissue, joint tissue, vascular tissue, and/or heart tissue, among others.
  • Biological fluid includes, but is not limited to, lymph, cerebrospinal fluid (SCF), amniotic fluid, pleural effusion, bone marrow, lymph node, lymphatic fluid, synovial fluid, a single cell suspension prepared from solid tissue or cell lines urine, saliva , sweat, tears, blood, serum and blood plasma.
  • the sample can be, and is preferably, peripheral blood, whole blood, plasma, or serum. In certain specific embodiments of the invention, the sample is serum.
  • the sample is human in origin, but can also be from any other animal.
  • the sample is from a mammal, including, without limitation, domestic animals, farm animals, and primates.
  • the terms "subject”, “individual” or “patient”, used interchangeably throughout this document, refer to any mammalian animal and includes, but is not restricted to domestic animals, farm animals, primates and humans, preferably humans. Said terms are not intended to be limiting in any aspect, and they may be of any age, sex and physical condition.
  • the subject is a subject suffering from a pathology that can be treated by implanting a biomaterial, as described herein.
  • a biomaterial as described herein.
  • the biological sample is isolated from the subject, said sample is incubated with the problem biomaterial to determine in a later stage of the method (stage b) the markers that adhere to said biomaterial.
  • the test biomaterial is incubated with the biological sample isolated from the subject for a range of 2 to 10 hours, preferably for a range of 3 to 6 hours, more preferably for a range of 3 to 4 hours.
  • the method of the invention comprises a step b where the presence of the markers described here, which have adhered to the problem biomaterial in contact, are identified, or determined and quantified. with the isolated biological sample.
  • the determination of the marker profile described in the present invention is carried out by identifying and determining the values of said markers that have remained attached to the problem biomaterial once it has been in contact with the biological sample of the subject to which said biomaterial is preferably going to be implanted.
  • the biological sample of the subject with the problem biomaterial After the incubation period of the biological sample of the subject with the problem biomaterial, it is preferably subjected to a series of washes to eliminate the markers that are not strongly adhered to the biomaterial.
  • said washes are preferably carried out with bidistilled water and more preferably, a final wash with a buffer solution preferably comprising sodium chloride at a concentration of between 50-500 mM.
  • the markers adhered to the problem biomaterial are collected or eluted from said biomaterial, preferably by treating the surface of the biomaterial with a buffer solution, preferably where the buffer solution is a triethylammonium bicarbonate solution, although it can be use any buffer solution known to those skilled in the art and useful for obtaining the proteins or markers adhered to said biomaterial.
  • a buffer solution preferably where the buffer solution is a triethylammonium bicarbonate solution, although it can be use any buffer solution known to those skilled in the art and useful for obtaining the proteins or markers adhered to said biomaterial.
  • the markers that form the protein profile of the invention are determined and quantified.
  • the determination and quantification of the presence of the markers of the invention is carried out, preferably by determining the amount of marker adhered to the material in contact with the biological sample, preferably in the eluates. obtained as described above, and also alternatively, by determining the protein expression levels of said markers, by any method known in the state of the art.
  • the authors of the present invention have shown that the detection of the quantity and/or concentration of these expression products in a semi-quantitative or quantitative manner allows to differentiate between a biomaterial with greater or lesser regenerative capacity of a certain type of tissue.
  • the presence, as well as the amount, of the detected marker establishes a differential profile between biomaterials with greater or lesser efficiency in terms of regeneration.
  • the measurement of the amount or concentration can be carried out directly or indirectly.
  • Direct measurement refers to the measurement of the amount and/or concentration of the proteins or markers of the invention.
  • Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said markers.
  • Indirect measurement includes measurement obtained from a secondary component or biological measurement system (eg, measurement of cellular responses, ligands, "tags,” or enzymatic reaction products).
  • Quantity refers to, but is not limited to, the absolute or relative quantity of the markers of the invention, but also refers to any other value or parameter related to them. or that may derive from them.
  • Said values or parameters comprise signal intensity values obtained from any of the physical or chemical properties of said markers, obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
  • the detection and quantification of the markers of the present invention can be carried out by quantifying the levels of proteins or peptides by conventional methods, known to those skilled in the art. , including, but not limited to, chemiluminescent methods, immunohistochemical staining or biochemical detection (eg, immunohistochemical assays), immunological techniques, flow cytometry, immunoprecipitation (or their equivalents for reagents other than antibodies), high performance liquid chromatography (HPLC), mass spectrometry (MS), plasma resonance absorbance measurement, etc.
  • the Quantification of the levels of the markers identified in the present invention associated with the ability to induce regeneration of a biomaterial is performed by immunological techniques, such as ELISA ("Enzyme-Linked Immunosorbent Assay), Western-blot, RIA (radioimmunoassay), Competitive EIA (competitive enzyme immunoassay), DAS-ELISA ("Double Antibody Sandwich ELISA”), mass spectroscopy, immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays that include specific antibodies or tests based on the colloidal precipitation in formats such as "dipsticks".
  • immunological techniques such as ELISA ("Enzyme-Linked Immunosorbent Assay), Western-blot, RIA (radioimmunoassay), Competitive EIA (competitive enzyme immunoassay), DAS-ELISA ("Double Antibody Sandwich ELISA”), mass spectroscopy, immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips
  • the proteins are detected by Western blot, ELISA, a protein array or matrix or by two-dimensional electrophoresis, more preferably the detection of the protein profile described in the present invention is carried out by ELISA.
  • the present invention relates not only to the markers described in the invention, but also to their fragments that could be generated, for example, by alternative processing or splicing phenomena, proteolytic rupture of said peptides or proteins, etc. Said fragments retain the capacity to be used as markers for the determination of the regenerative efficiency of a medical material as described in this document.
  • the markers of the invention, as well as their fragments can include post-translational modifications, such as phosphorylation, glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.
  • step c the determination and quantification value of the markers of the invention obtained in step b is compared with the reference values obtained for said markers in a reference biomaterial, that has also been contacted with a biological sample isolated from the subject, where
  • biological sample refers to a sample or set of isolated samples obtained from a subject or a group of subjects, either a fluid or a tissue as explained above.
  • problem biomaterial or "biomaterial of interest” or “biomaterial under study” refers to a biomaterial in which it is intended to determine the regeneration capacity of a biological sample.
  • the term "reference biomaterial” refers to a biomaterial in which the regeneration capacity of a biological sample has been previously determined.
  • the reference biomaterial is titanium
  • the reference biomaterial is medical grade titanium.
  • the reference biomaterial is smooth grade 4 titanium.
  • the reference quantity or value is obtained from a reference biomaterial.
  • the term "reference quantity” or “reference value”, used interchangeably throughout this document, is obtained from the values, preferably quantity values of the markers of the invention, analyzed in a reference biomaterial, of the that its compatibility with a subject is known.
  • a biocompatible material from which to obtain the reference quantity for the markers of the invention may be titanium, preferably medical grade.
  • the term “comparison”, as used in the description refers to, but is not limited to, the comparison of the amount of markers as described in the invention, adhered to the problem biomaterial that is cultured or incubated. together with the biological sample of a subject, with respect to the amount of the same markers that have adhered to a cultured reference biomaterial also together with a biological sample obtained from the same subject.
  • the reference biomaterial can be analyzed, for example, simultaneously or consecutively, together with the problem biomaterial.
  • the comparison described in step c of the method of the present invention can be performed manually or computer assisted.
  • step c the value obtained for at least one of the markers adhered to the problem biomaterial is compared, where said marker is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
  • the biological sample of the invention is preferably a tissue sample and/or a biological fluid.
  • the sample is a biological fluid.
  • the sample is a commercial biological fluid.
  • the sample is a biological fluid obtained from cell culture.
  • the sample is a biological fluid obtained from tissue.
  • the sample is a biological fluid obtained from bone tissue.
  • the sample is a biological fluid obtained from soft tissue.
  • the sample is blood serum, more preferably from the subject to which the biomaterial under study is going to be implanted.
  • the following parameters are considered as indicative of a good capacity for bone tissue regeneration: a level of SAMP markers; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; and C1QC up to 1.5 times higher than the reference value obtained for the reference biomaterial; a CRP marker value lower than the reference value obtained for the reference biomaterial; and ratios between the values obtained for the IC1 markers; C4BPA; CFAH; NCTV; and CLUS, and the C1QB markers; C1S; C1R; C1QC; and CO3 equal to or greater than the reference values obtained in the reference biomaterial.
  • the following parameters are considered as indicative of a good capacity for soft tissue regeneration: a value of the SAMP markers; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; and FCN2; lower than the reference value obtained for the reference biomaterial; and a value of IC1 markers; CFAH; C4BPA; NCTV; and CLUS equal to or greater than the reference value obtained for the reference biomaterial.
  • the amount and/or level of the marker(s) of the invention is said to be “increased” relative to the amount and/or level of said marker(s). marker(s) in the reference biomaterial”, when the quantity and/or level of expression of said marker(s) is elevated (or higher) in the problem biomaterial with respect to the quantity and/or level expression of said marker(s) in the reference biomaterial.
  • the amount and/or level of the marker(s) in a test biomaterial is considered to be increased relative to the amount and/or level of said marker(s) in the biomaterial.
  • the amount and/or level of a marker(s) is said to be "decreased” relative to the amount and/or level of said marker(s). es) in the reference biomaterial when the quantity and/or level of said markers) is reduced (or lower) with respect to the quantity and/or level of said markers) in the reference biomaterial. According to the present invention, it is considered that the quantity and/or the level of a marker(s) in a problem biomaterial under study is decreased with respect to the quantity and/or the level of said marker(s) in the reference biomaterial.
  • the terms "predict” or “forecast” refer to the assessment of the probability according to which a biomaterial is capable of inducing regeneration in a tissue of a subject that is going to be implanted with said biomaterial.
  • Such an assessment may not typically be correct for 100% of the biomaterials to be predicted for regenerative ability.
  • the term requires that a statistically significant portion of the biomaterials be identified as being capable of or available to induce regeneration. Whether a part is statistically significant can be determined without further ado by those skilled in the art using various well-known statistical evaluation tools, e.g., determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test. , etc.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%.
  • the p values are preferably 0.2, 0.1, 0.05.
  • the term “osseointegration” refers to the ability of the biomaterial or implant to provide a direct structural and functional bond with bone tissue through the formation of new bone or a new bone-like structure in the vicinity. of the biomaterial/tissue interface. Osteogenesis refers here to the process of formation and growth of new bone. It can also assume the respective formation of other tissues such as fibrous tissue or cartilage.
  • tissue regeneration soft refers to the ability of the biomaterial, implant or medical device to allow a healing between the percutaneous device and the adjacent tissue to close, for example, the possible entry of bacteria.
  • the compatibility analysis of a system made up of a biomaterial and the tissue with which it is in contact includes a large number of different in vitro and in vivo tests that are included in ISO10993.
  • biomaterial for the purposes of this invention, the terms "biomaterial”, “medical material”, “medical device”, “implant” or “prosthesis” are used interchangeably throughout the herein, and refer to any biomaterial and/or device that is surgically implantable.
  • Implants are generally used in combination with body tissues or organs temporarily, permanently, or semi-permanently to remedy a problem and may optionally be surgically removed or biodegrade within the body.
  • Examples of implants include bandages, sutures, staples, braces, fabric, mesh, nets, artificial bones, screws, bone plates, orthopedic rods, pins, hip implants, knee implants, artificial hearts, teeth, dental implants, catheters, and the like. .
  • marker refers to a molecule, or the expression product of a gene, preferably proteins, or fragments and variants of these that show substantial changes in a determined condition and that can be used both for the detection, diagnosis and/or prognosis of a state and/or condition of an object or individual. Said terms particularly refer to the detection and quantification of said markers, to predict and/or predict the ability of biomaterials to induce tissue regeneration, as described herein.
  • the changes in the marker are changes in the amount thereof, as well as, alternatively, changes in the expression levels thereof, with respect to a reference value.
  • expression refers particularly to the determination of the amount of each marker relative to a reference value. It will be appreciated that the amount and/or levels of a marker can be established by determining the levels of the corresponding polypeptide, or of polypeptides generated after its digestion in the event that proteomic means are used for its determination. Alternatively, the peptide tags may be variants resulting from post-translational modifications, including fragments thereof.
  • step b of the method of the invention further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Prothrombin (THRB; UniProtKB P00734); Antithrombin (ANT3; UniProtKB P01008; entry version 252); Kininogen 1 (KNG1; UniProtKB P01042; entry version 231);; Factor XII (FA12; UniProtKB P00748; entry version 238); Protein C dependent vitamin K (PROC; UniProtKB P04070); Protein S dependent vitamin K (PROS; UniProtKB P07225); Platelet factor 4 (PF4V; UniProtKB P10720); and Factor X (FA10; UniProtKB P00742; entry version 265); where:
  • a value of the THRB markers; PROC; PROS; and ANT3 at least 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
  • step b of the method of the invention comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; KNG1; PF4V; ANT3; PROC; PROS; FA10; and FA12.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list comprising THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
  • step b of the method of the invention may also comprise the determination and quantification of at least one of the markers selected from the list consisting of: Filaggrin-2 (FILA2; UniProtKB Q5D862; entry version 140); Desmoplakin (DESP; UniProtKB P15924; entry version 246); Fibronectin (FINC; UniProtKB P02751; entry version 266); Desmocolin 1 (DSC1; UniProtKB Q08554; entry version 180); tetranectin (TETN; UniProtKB P05452; entry version 198); and Placoglobin (PLAK; UniProtKB P14923; entry version 214); where a value of the markers DESP; ROW2; TETN; PLAK; DSC1; and FINC at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce bone regeneration of the biomaterial under study.
  • Filaggrin-2 FILA2; UniProtKB Q5D862; entry version
  • step b of the method of the invention comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one of the markers selected from any of the list consisting of FILA2; OFF; DSC1; TETN; FINC; and PLAK.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; and from at least one of the markers selected from any of the list consisting of THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and from at least one of the markers selected from any of the list consisting of FILA2; OFF; DSC1; TETN; FINC; and PLAK.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • step b of the method of the invention may also comprise the determination and quantification of at least one of the markers selected from the list consisting of: pigment epithelium-derived factor (PEDF; UniProtKB P36955; entry version 209); leudin-rich alpha-2-glycoprotein (A2GL; UniProtKB P02750; entry version 189); angiopoietin-1 (ANG-1; UniProtKB Q15389; entry version 185); and hypoxia-inducible factor 1- ⁇ (HIF- ⁇ ; UniProtKB Q16665; entry version 239), where a value of PEDF markers; A2GL; ANG-1; yHIF- ⁇ at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
  • PEDF pigment epithelium-derived factor
  • A2GL pigment epithelium-derived factor
  • ANG-1 angiopoietin-1
  • ANG-1 UniProtKB Q15389
  • step b comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • step b of the method of the invention further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Plasminogen (PLMN; UniProtKB P00747; entry version 255); fibrinogen a-chain (PIBA; UniProtKB P02671; entry version 251); plasminogen activator inhibitor-1 (PAI2; UniProtKB P05120; entry version 212); histidine-rich glycoprotein (HRG; UniProtKB Q6P1 K1; entry version 116); and kallikrein (KLKB1; UniProtKB P03952; entry version 218), where a value of the markers PLMN; GDPA; PAI2; HRG and KLKB1 at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
  • PLMN Plasminogen
  • PIBA UniProtKB P02671; entry version 251
  • PAI2 plasminogen activator
  • step b comprises the identification and quantification of the presence of at least one of the markers selected from any of the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • the markers selected from any of the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and at least one from markers selected from any of the list consisting of PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; and KLKB1.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • the biomaterial under study is intended for the regeneration of bone tissue and step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Apolipoprotein E (APOE; UniProtKB P02649); Lactotransferrin (TRFL; UniProtKB P02788); Staterin (STATH; UniProtKB P02808); and Peptidyl-prolyl cis-trans isomerase B (PPIB; UniProtKB P23284), where a value of APOE markers; TRFL; STATH; and PPIB at least 2 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
  • APOE Apolipoprotein E
  • TRFL Lactotransferrin
  • Staterin STATH; UniProtKB P02808
  • Peptidyl-prolyl cis-trans isomerase B Peptidyl-prolyl cis-trans isomerase B
  • step b of the method of the invention may comprise the determination and quantification of at least one of the markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • the markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: APOE; TRFL; STATH and PPIB.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b of the method of the invention comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; and of at least one of the markers selected from any of the list consisting of: APOE; TRFL; STATH and PPIB.
  • step b of the method of the invention is the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • the biomaterial under study is intended for soft tissue regeneration and step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Cathepsin B (CATB; UniProtKB P07858;entry version 234); and Callistatin (SERPINA4; UniProtKB P29622; entry version 178), where a CATB value lower than the reference value obtained for the reference biomaterial, and a SERPINA4 value lower than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
  • CAB Cathepsin B
  • SERPINA4 UniProtKB P29622
  • the biomaterial under study is intended for soft tissue regeneration and stage b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Insulin-like growth factors (IGF) and/or IGF-binding proteins such as insulin-like growth factor II (IGF2; UniProtKB-P01344) and insulin-like growth factor-binding protein 4 (IBP4; UniProtKB-P22692); serine protease inhibitors such as alpha-2-antiplasmin (A2AP; UniProtKB - P08697), alpha-1-antitrypsin (A1AT; UniProtKB - P01009) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3; UniProtKB - Q06033); CYTA; and procollagen C-endopeptidase enhancers such as procollagen C-endopeptidase enhancer 1 (PCOC1; UniProtKB - Q15113); where IGF2; UniProtKB-P
  • step b of the method of the invention may comprise the determination and quantification of at least one of the markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • the markers selected from
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOCI.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PC
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBP, including within IBPs, but not limited to, IBP4); A2AP; A1AT; CYTA; ITIH3
  • step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; and from at least one marker selected from any of the list consisting of: CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and
  • step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • IGF-binding proteins including but not limited to IGF2 and IBPs
  • a second aspect of the invention refers to the in vitro use ("use of the invention") of the determination and quantification of the presence of CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS markers, preferably CO5 markers , IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12.
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12.
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TE
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • markers CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10;
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • at least one marker that is selected from the
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
  • markers including but not limited to IGF2 and IBPs, including within IBPs, but
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2
  • the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • markers CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1;
  • the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; ApoE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITI
  • a third aspect of the invention refers to a kit ("kit of the invention”) to predict and/or predict the regeneration induction capacity of a biomaterial comprising the antibodies, the reagents, or any combination of the above, capable of to detect and quantify the presence of markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS, preferably markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPI
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
  • at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12;
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS;
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF- ⁇ .
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NC
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; and KLKB1.
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4B
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; IGF-binding
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; Y
  • the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP
  • the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF- ⁇ ; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A2AP; A
  • the determination and quantification of the markers of the invention by means of the kit of the invention is carried out after contacting the problem biomaterial with a biological sample isolated from a subject, as described here, preferably being the isolated biological sample a serum sample.
  • the kit of the invention can also include, without any type of limitation, buffers, protein extraction solutions, contamination prevention agents, protein degradation inhibitors, etc.
  • the kit can include all the necessary supports and containers for its start-up and optimization.
  • the kit further comprises instructions for carrying out the methods of the invention.
  • FIGURES Fig. 1 A. L929 fibroblasts cultured for 3 days on surface A (left) and on surface B (right). B. Cell area occupied by fibroblasts on a control surface when cultured on surface A and surface B for 3 days.
  • Fig. 2 ALP activity of MC3T3-E1 cells at 7 and 14 days.
  • Fig. 3. Measurements of cell proliferation at 1, 3 and 7 days. Results are shown as mean ⁇ SE. The asterisk (p ⁇ 0.05 (*)) indicates statistical differences.
  • Fig. 5 Adhesion of human fibroblasts after 1 day of culture on surfaces A (left) and B (right).
  • A reference sample of smooth grade 4 titanium
  • B sample with combined treatment that causes roughness
  • sample X is the grade 4 titanium reference with a surface treatment of shot blasting and acid attack, widely used in titanium prostheses for bone tissue.
  • type Y which is a titanium sample like X with a surface coating with greater osteogenic capacity. Both samples present differences in terms of their physicochemical properties and can be used as bone implants.
  • the materials are prepared in the form of a 12 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions.
  • Cell cultures Cell culture for the evaluation of A and B was performed with mouse fibroblasts (line L-929) in a culture medium composed of DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/ streptomycin (Gibco) and 10% FBS (Gibco).
  • the in vitro evaluation of X and Y was carried out using a cell culture with mouse osteoblasts (MC3T3-E1).
  • the cells were cultured in a medium composed of DMEM (Gibco, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco) and 10% FBS (Gibco).
  • the medium was replaced by osteogenic medium (DMEM, 1% penicillin/streptomycin, 10% FBS, 1% ascorbic acid and 0.21% ⁇ -glycerol phosphate.
  • fibroblast cytoskeleton To study the effect of the materials on the organization of the cytoskeleton of the L-929 cell line, the cells were seeded on the materials with a density of 1 x 10 4 cells per cm -2 . After 24 hours, the samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature. Cells were then permeabilized with 0.1% Triton X-100 for 5 min and incubated with Phaloidin (1:100; Abeam, Cambridge, UK) diluted in 0.1% w/v bovine serum albumin (BSA)-PBS for 1 h at room temperature. . Nuclei were stained with DAPI medium (Abeam). Fluorescence detection was performed with a Leica TCS SP8 Confocal Laser Scanning Microscope at 20x magnification. Images were analyzed with LAS X software (Leica) and Image J software (National Institutes of Health).
  • ALP activity in X and Y was carried out to evaluate the effect of the study samples on cell mineralization.
  • the MC3T3-E1 cells were seeded in the different samples in 24-well NUNC plates (Thermo Fisher Scientific). After 7 and 14 days of culture, lysis buffer (0.2% Triton X-100, 10 mM Tris-HC1, pH 7.2) was added. Next, 100 pL of p-NPP (1 mg mL -1 ) in buffer (50 mM glycine, 1 mM MgCl2, pH 10.5) were added to the samples. After 2 h of incubation, the absorbance at 405 nm was measured using a microplate reader. ALP activity was obtained using the standard curve of p-nitrophenol in 0.02 mM sodium hydroxide. It was normalized to the protein content obtained using a Pierce BCA assay kit (Thermo Fisher Scientific).
  • Protein analysis was performed using a tandem of equipment: nanoACQUITY UPLC mass spectrometer (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al. to [same reference]. Each sample was analyzed in quadruplicate. The analysis of the different proteins was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
  • Table 1 shows the proteins found differentially more adsorbed on surface B relative to surface A:
  • surface B is less susceptible to proteins linked to inflammation being adsorbed on its surface. Additionally, the main protein linked to oxidative stress is adsorbed significantly less on surface B. Similarly, proteins linked to coagulation processes are adsorbed in a controlled manner.
  • the evaluation of the influence of the two types of material (A and B) in the organization of the cytoskeleton is carried out by staining the cells with phalloidin (green) and staining the nuclei with DAPI (blue), after one day of culture. cell ( Figure 1A). It can be seen that the cytoskeleton of the fibroblasts cultured on material A has a globular shape without any type of adhesion to the substrate due to the absence of lamellipodia.
  • the fibroblasts cultured on material B do present multiple lamellipodia and an enlarged appearance, very different from the globular one found on surface A.
  • the presence of these lamellipodia in the cells cultured on B is essential for multiple biological processes, and specifically due to the ability of fibroblasts with this conformation to produce collagen and achieve wound healing (and in particular gingival sealing).
  • soft tissue regeneration depends on the ability of fibroblasts to generate collagen. Collagen generation is only possible if the fibroblast is located on a surface where a process of cellular stress is not triggered, there is a low inflammatory response, as well as regulated coagulation and fibrinolysis.
  • Surface B presents the markers that, according to the patent, reduce oxidative stress and inflammation, as well as giving rise to a contained coagulation response. It is this surface B that has a morphology that is not globular but widened, with greater focal adhesions and greater area. It will be these fibroblasts cultivated on surface B that will generate collagen and manage to regenerate the soft tissue and the consequent healing.
  • Proteins linked to the activation of the inflammation cascade appear (CO8A, CO5, CO3, C1R, C1S), inflammation regulators (C4BPA, VTNC, CLUS, CFAH, IC1), related to coagulation (A2MG, KNG1, THRB, FA11, PROC, PF4V), and linked to osteogenesis (APOE, VTNC).
  • the Y surface is less susceptible to proteins linked to inflammation being adsorbed on its surface. Additionally, there is a greater adsorption of proteins linked to osteogenesis and coagulation.
  • Figure 2 shows how the type Y sample gives rise to a greater secretion of ALP both at 7 and 14 days.
  • This higher ALP activity observed in samples Y may be related to a higher osteogenic capacity and a higher mineralization capacity compared to sample X (reference Ti).
  • the osseointegration capacity of implants intended for the regeneration of bone tissue depends on the physicochemical characteristics of the material.
  • the characteristics of this set of properties condition the interaction between the prosthesis and the surrounding tissues/fluids after its implantation.
  • each biomaterial depending on its properties, will give rise to the formation of a specific protein layer on its surface and these characteristic proteins will in turn condition the biological response of the material by influencing the activation and development of key regenerative processes ( immune reaction, inflammation, coagulation, osteogenesis, fibrinolysis, angiogenesis) that will lead to optimal implant osseointegration.
  • samples designed to favor the regeneration of bone tissue. Both samples consist of sol-gel borosilicate coatings applied to grade 4 titanium with a surface treatment of shot blasting and acid attack, widely used in titanium prostheses for bone tissue. The difference between samples C and D lies in the composition of these coatings, which lead to differences in their physicochemical properties and give rise to different biological responses.
  • the materials are prepared in the form of a 10 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions.
  • HOb cells were seeded in 48-well plates at a density of 7.5 x 10 3 cells cm -2 and proliferation was assessed following the manufacturer's instructions after 1, 3 and 7 days. Absorbance was measured at 490 nm in a Multiskan FC microplate reader (Thermo Fisher Scientific).
  • ALP activity was carried out to assess the effect of study samples on cell mineralization.
  • the osteoblasts were seeded in the different samples in 48-well plates. After 7 and 14 days of culture, lysis buffer (0.2% Triton X-100, 10 mM Tris-HCl, pH 7.2) was added. Next, 100 ⁇ l of p-NPP (1 mg mL 1 ) in buffer (50 mM glycine, 1 mM MgCl2, pH 10.5) were added to the samples. After 2 h of incubation, the absorbance at 405 nm was measured using a microplate reader. ALP activity was obtained using the standard curve of p-nitrophenol in 0.02 mM sodium hydroxide. It was normalized to the protein content obtained using a Pierce BCA assay kit (Thermo Fisher Scientific).
  • the proteomic analysis of the adsorbed protein layer was carried out using the protocol established by Romero-Gavilán et al. [F. Romero-Gavilan et al.; Biofouling. 33 (2017) 676-689] and described here. Materials were incubated for 3 h (37 °C, 5% CO 2 ) in a 24-well plate (Thermo Fisher Scientific) with 1 ml human serum AB plasma (Sigma-Aldrich). To remove unadsorbed proteins, the coatings were washed 5 times with ddH 2 O and then with buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.0). The layer of proteins adsorbed to the surfaces was obtained with a buffer (2M Thiourea, 7M Urea, 4% Chaps, 200 mM DTT).
  • Protein analysis was performed using a tandem of equipment: mass spectrometer with nanoACQUITY UPLC (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al [same reference]. Each sample was analyzed in quadruplicate. Differential protein analysis was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
  • test data were treated considering a normal distribution and an equivalent variance, using the ANOVA software with the Tukey post hoc test.
  • ANOVA ANOVA software
  • Tukey post hoc test a t-student analysis was performed. The data are expressed as the mean value ⁇ the standard error (SE).
  • SE standard error
  • proteomics data the t-student test was performed to evaluate the differences in adsorbed proteins using the Pyrogenesis software. The differences were considered statistically significant with a p ⁇ 0.05.
  • Table 3 shows how coating C gave rise to a greater adsorption of proteins associated with osteogenesis (TRLF, APOE, PEDF). This coating also showed a higher affinity both for pro-coagulatory proteins (KNG1, PF4V, FA12, FA10, HRG, THRB) and for proteins capable of regulating the activation of this process (PROC, PROS, ANT3).
  • A2GL, associated with a positive effect on angiogenesis, and PLMN, a key protein in the fibrinolysis process, were also detected as more adsorbed to coating C than to coating D.
  • DESP, TETN, PLAK, FINC, FIBA associated with functions in cell adhesion , also showed a higher affinity for the C-type surface with C/D abundance ratios ⁇ 1.5.
  • the ratios between the values of the markers C4BPA, IC1, CFAH and CLUS, and the values of the markers C1R and CO3 for surface C are higher than the values obtained for surface D, since that the ratios obtained are greater than 1. This fact would indicate that an immune response is present, but regulated.
  • the HOb cultured on the type C surface showed a significantly higher proliferation capacity at 1 and 7 days, compared to D ( Figure 3).
  • Figure 4 shows how osteoblasts cultured with type C coating give rise to higher ALP activity both at 7 and 14 days, compared to those cultured with samples D.
  • This higher ALP activity may be related to a higher ALP activity. greater capacity for osteogenic maturation and mineralization by this coating.
  • Ti titanium
  • A Ti grade 4 with nano-texturing type A
  • B Ti grade 4 with nano-texturing type B
  • the materials are prepared in the form of a 10 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions.
  • Cell cultures were performed with human fibroblasts (PSC-201-018 line) in a culture medium composed of DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco) and a 10% FBS (Gibco).
  • DMEM Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA
  • penicillin/streptomycin Gibco
  • FBS FBS
  • fibroblast cytoskeleton To study the ability of the surfaces to favor the adhesion of human fibroblasts, the cells were seeded on the materials with a density of 1 x 10 4 cells cm -2 . After 24 hours, samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. Fibroblasts were treated with 0.1% Triton X-100 (5 min) and incubated with Phaloidin (1:100; Abeam, Cambridge, UK) diluted in 0.1% w/v bovine serum albumin for 1 h at room temperature. The nuclei were marked with DAPI medium (Abeam). Cells were analyzed by fluorescence with a Leica TCS SP8 confocal microscope.
  • PFA paraformaldehyde
  • Protein analysis was performed using a tandem of equipment: nanoACQUITY UPLC mass spectrometer (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al. to [same reference]. Each sample was analyzed in quadruplicate. Differential protein analysis was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
  • the ratios between the abundance values of the key biomarkers were calculated to assess the regeneration capacity of soft tissue associated with both surfaces.
  • the identified biomarkers are shown in Table 5.
  • type A surface has a greater potential for soft tissue regeneration compared to type B, since this treatment showed a greater adhesion capacity of human fibroblasts, key in the regenerative process of the biomaterial.
  • This greater regenerative capacity of the type A surface correlates with the movements in the absorption patterns of the protein biomarkers claimed in the present invention.
  • a greater adsorption of IGF2, IBP4, A2AP, A1AT, CYTA, ITIH3, PCOC1 proteins was detected on the type A surface (A/B ratio>1), these molecules being associated with key functions in the tissue regenerative process. soft.
  • DESP, FILA2, TETN, PLAK, DSC1 and FINC, proteins with key functions in adhesion showed a higher affinity for the A surface with A/B abundance ratios >1.5.
  • the adsorption of DSC1 on type A treatment (with ratio 5.2) stands out.
  • Soft tissue regeneration depends on the ability of fibroblasts to generate collagen. For this process to be carried out in the presence of a biomaterial, it must show a low immune response, as well as avoid the activation of cellular stress. In addition, the activation of coagulation around the prosthesis must be controlled.
  • Surface A in addition to meeting these criteria, showed a higher affinity for key proteins in the process of adhesion, proliferation and collagen production (compared to B). The proteomics results showed a good correlation between the effect of the tested surfaces on protein adsorption and cellular response, since type A treatment was the material that gave rise to a markedly greater adhesion of human fibroblasts on the surface.

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Abstract

The present invention relates to a set of markers, preferably proteins, which are particularly useful for predicting and/or prognosing the ability of a biomaterial to induce tissue regeneration from an isolated biological sample obtained from a subject and differentially adhered to the surface of said biomaterial, such as, for example: joint prostheses, dental prostheses, valves, stents, percutaneous devices, etc. Additionally, the present invention comprises an in vitro method for predicting and/or prognosing the regenerative capacity of the biomaterial by determining the peptide profile described in the invention, in addition to a kit for carrying out said method.

Description

Método in vitro para predecir y/o pronosticar la capacidad de inducir regeneración de tejidos por parte de un biomaterial In vitro method to predict and/or forecast the ability to induce tissue regeneration by a biomaterial
SECTOR DE LA TÉCNICA TECHNICAL SECTOR
La presente invención se refiere a un conjunto de proteínas particularmente útil para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial. En la invención se pone en contacto el biomaterial a estudiar con una muestra biológica aislada obtenida de un sujeto y se analiza el perfil peptídico aquí propuesto y adherido de manera diferencial a la superficie de dicho biomaterial, donde dicho biomaterial es utilizado como un dispositivo médico implantable, como por ejemplo cualquier tipo de prótesis, tales como stents, cánulas, marcapasos, implantes dentales, dispositivos médicos percutáneos etc. Además, la presente invención comprende un método in vitro para predecir y/o pronosticar la capacidad de inducir regeneración de tejidos por parte de dicho biomaterial mediante la determinación y cuantificación del perfil peptídico procedente de una muestra biológica aislada adherido al mismo, así como un kit para llevar a cabo dicho método. The present invention relates to a set of proteins that is particularly useful for predicting and/or forecasting the ability of a biomaterial to induce tissue regeneration. In the invention, the biomaterial to be studied is brought into contact with an isolated biological sample obtained from a subject and the peptide profile proposed here is analyzed and differentially adhered to the surface of said biomaterial, where said biomaterial is used as an implantable medical device , such as any type of prosthesis, such as stents, cannulas, pacemakers, dental implants, percutaneous medical devices, etc. In addition, the present invention comprises an in vitro method to predict and/or forecast the ability to induce tissue regeneration by said biomaterial by determining and quantifying the peptide profile from an isolated biological sample adhered to it, as well as a kit to carry out this method.
ESTADO DE LA TÉCNICA STATE OF THE ART
El éxito de un implante depende de su aceptabilidad biológica, es decir, que produzca un mínimo daño y una mínima reacción inmune en el sujeto implantado. Uno de los requisitos imprescindibles que debe cumplir dicho implante es la biocompatibilidad con el tejido del sujeto que recibe el implante, y, por tanto, la ausencia de una reacción inmunológica a cuerpo extraño. Además, el éxito de dicho implante depende directamente de la capacidad de regeneración del tejido que se adhiera de manera diferencial sobre el implante. The success of an implant depends on its biological acceptability, that is, that it produces minimal damage and a minimal immune reaction in the implanted subject. One of the essential requirements that said implant must meet is biocompatibility with the tissue of the subject receiving the implant, and, therefore, the absence of an immunological reaction to a foreign body. In addition, the success of said implant depends directly on the regeneration capacity of the tissue that differentially adheres to the implant.
En este sentido, las pruebas que tiene que pasar el biomaterial para ser empleado como implante son múltiples y complicadas, incluyendo desde ensayos in vitro frente a diferentes líneas celulares (las propias del tejido que vaya a estar en contacto con el implante), para determinar su citotoxicidad, proliferación celular, etc., hasta ensayos in vivo con aquellos prototipos que han demostrado unas buenas propiedades in vitro. Además, son necesarios estudios preclínicos y ensayos clínicos en humanos. Todo este proceso implica un largo periodo de realización, además del sacrificio de un número significativo de animales, y un enorme coste económico. Adicionalmente, los ensayos clínicos no están exentos de problemática y riesgo para los pacientes que dan su consentimiento para participar en los mismos. En la actualidad, en la práctica clínica, no existe ningún tipo de ensayo personalizado que permita predecir y/o pronosticar el éxito o fracaso de un implante, sea del tipo que sea (por ejemplo, implante dental, prótesis de rodilla, de cadera o tornillería diversa en contacto con hueso, tales como estabilizadores de columna vertebral o placas de unión para huesos con rotura, stents, válvulas cardiacas, implantes dérmicos...). De la misma forma, tampoco existe actualmente ningún ensayo acelerado que sea capaz de predecir y/o pronosticar la capacidad de regeneración del tejido adherido de manera diferencial a la superficie de un biomaterial, que se esté desarrollando por parte de los fabricantes productores de implantes y/o biomateriales, teniendo que llevar a cabo todos los ensayos descritos anteriormente para llevar al mercado dicho material. Adicionalmente hay una presión cada vez mayor para reducir el número de animales de experimentación por cuestiones éticas, pero la opción de realizar la selección del biomaterial mediante experimentación in vitro es compleja, por la no existencia de una buena correlación entre ambos tipos de experimentación, in vitro vs in vivo. In this sense, the tests that the biomaterial has to pass to be used as an implant are multiple and complicated, including in vitro tests against different cell lines (those of the tissue that is going to be in contact with the implant), to determine its cytotoxicity, cell proliferation, etc., to in vivo tests with those prototypes that have shown good in vitro properties. In addition, preclinical studies and clinical trials in humans are necessary. All this process implies a long period of realization, in addition to the sacrifice of a significant number of animals, and an enormous economic cost. Additionally, clinical trials are not exempt from problems and risk for patients who give their consent to participate in them. Currently, in clinical practice, there is no type of personalized test that allows predicting and/or forecasting the success or failure of an implant, whatever its type (for example, dental implant, knee, hip or hip prosthesis). various screws in contact with bone, such as spinal column stabilizers or joint plates for broken bones, stents, heart valves, dermal implants...). In the same way, there is currently no accelerated test that is capable of predicting and/or predicting the regeneration capacity of the tissue differentially adhered to the surface of a biomaterial, which is being developed by manufacturers producing implants and / or biomaterials, having to carry out all the tests described above to bring said material to the market. Additionally, there is an increasing pressure to reduce the number of experimental animals for ethical reasons, but the option of selecting the biomaterial through in vitro experimentation is complex, due to the lack of a good correlation between both types of experimentation, in vitro vs. in vivo.
Hay que tener en cuenta que, para llevar a cabo un proceso de regeneración, preferiblemente regeneración ósea o regeneración de tejido blando, se requieren ciertas condiciones, como la señalización celular, la presencia de células progenitoras, así como los procesos de vascularización, cicatrización, y, en el caso de tejido óseo, la osteoinducción. La interacción de la superficie del biomaterial-implante con los sistemas biológicos determina el éxito o el fracaso del implante en el sujeto. Las propiedades superficiales del implante (hidrofilia, rugosidad o energía superficial) afectan a la interacción de este con los fluidos corporales del individuo. Así, esta interacción puede ser positiva y promover la integración del biomaterial-implante (interacción directa entre la superficie del biomaterial y el tejido sin mediar tejido fibroso), o bien negativa dando lugar a una inflamación crónica y aguda, alta producción de macrófagos que constituyen una respuesta denominada reacción a cuerpo extraño, que supone en la mayor parte de los casos el fracaso de la implantación. La comprensión de la secuencia de eventos biológicos que tienen lugar tras la implantación, tales como procesos de coagulación de la sangre, el desarrollo de infecciones, la respuesta inmune al cuerpo extraño, y finalmente el rechazo del implante, es crucial para determinar la compatibilidad de un biomaterial, y, por lo tanto, de las prótesis perse. En este sentido, la primera capa de proteínas adsorbida sobre el biomaterial y/o implante es la responsable de desencadenar la respuesta celular a este. It must be taken into account that, in order to carry out a regeneration process, preferably bone regeneration or soft tissue regeneration, certain conditions are required, such as cell signaling, the presence of progenitor cells, as well as the processes of vascularization, healing, and, in the case of bone tissue, osteoinduction. The interaction of the biomaterial-implant surface with biological systems determines the success or failure of the implant in the subject. The surface properties of the implant (hydrophilicity, roughness or surface energy) affect its interaction with the body fluids of the individual. Thus, this interaction can be positive and promote the integration of the biomaterial-implant (direct interaction between the surface of the biomaterial and the tissue without mediating fibrous tissue), or negative, giving rise to chronic and acute inflammation, high production of macrophages that constitute a response called foreign body reaction, which in most cases involves failure of the implantation. Understanding the sequence of biological events that take place after implantation, such as blood coagulation processes, the development of infections, the immune response to the foreign body, and finally the rejection of the implant, is crucial to determine the compatibility of implants. a biomaterial, and, therefore, of prostheses perse. In this sense, the first protein layer adsorbed on the biomaterial and/or implant is responsible for triggering the cellular response to it.
En la literatura se pueden encontrar diferentes ensayos in vitro en los que se pone en contacto el implante con una muestra obtenida de un individuo y se analiza el patrón de proteínas adsorbidas en la superficie de los implantes para determinar la compatibilidad de éstos en el sujeto, así como la capacidad de regeneración de determinados tejidos. Different in vitro tests can be found in the literature in which the implant is put in contact with a sample obtained from an individual and the pattern of proteins adsorbed on the surface of the implants to determine their compatibility in the subject, as well as the regeneration capacity of certain tissues.
En la solicitud de patente WO2018/115553 se describe un método in vitro para predecir y/o pronosticar la compatibilidad de biomateriales en un sujeto. En dicho método se describe un conjunto de marcadores, preferentemente proteínas, que son particularmente útiles para predecir y/o pronosticar la compatibilidad in vitro de un biomaterial. Sin embargo, el método no está diseñado para predecir y/o determinar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial; además, no se consideran ciertos marcadores descubiertos por los inventores y descritos en el presente documento. Patent application WO2018/115553 describes an in vitro method to predict and/or predict the compatibility of biomaterials in a subject. Said method describes a set of markers, preferably proteins, that are particularly useful for predicting and/or prognosticating the in vitro compatibility of a biomaterial. However, the method is not designed to predict and/or determine the ability of a biomaterial to induce tissue regeneration; furthermore, certain markers discovered by the inventors and described herein are not considered.
US2009/258002 describe métodos para el diagnóstico y monitorización de cáncer y tejido dañado por isquemia, así como métodos terapéuticos y de screening de medicamentos para dichos métodos terapéuticos. En el documento se describe un método de cualificación del estado de un tejido en un sujeto, comprendiendo la medida de al menos un marcador en una muestra del sujeto, donde el marcador se selecciona de entre un listado de 1325 genes cuya expresión varía en función del estado del tejido en la tabla 9, y la correlación de la medida con el estado del tejido. Los genes del listado comprenden CO3, C1QB, C1QC, TENT, CLUS, FLAK and APOE. US2009/258002 describes methods for the diagnosis and monitoring of cancer and tissue damaged by ischemia, as well as therapeutic and drug screening methods for said therapeutic methods. The document describes a method for qualifying the status of a tissue in a subject, comprising the measurement of at least one marker in a sample of the subject, where the marker is selected from a list of 1325 genes whose expression varies depending on the tissue condition in Table 9, and the correlation of the measurement with the tissue condition. Listed genes include CO3, C1QB, C1QC, TENT, CLUS, FLAK and APOE.
Con respecto al tejido a ser diagnosticado, se selecciona entre riesgo de cáncer, regeneración/ reparación del tejido, fallo agudo de órganos, trasplante de órganos, presencia o ausencia de enfermedades, estadio de las enfermedades y eficacia del tratamiento de las enfermedades. Sin embargo, no hay referencias específicas a la regeneración de tejidos o cualquier otro tipo de muestra biológica en contacto con biomateriales. With respect to the tissue to be diagnosed, it is selected from cancer risk, tissue regeneration/repair, acute organ failure, organ transplant, presence or absence of diseases, stage of diseases, and efficacy of treatment of diseases. However, there are no specific references to tissue regeneration or any other type of biological sample in contact with biomaterials.
WO2015/110989 describe métodos para la predicción de la probabilidad de recuperación de la mucosa en individuos con enfermedad intestinal inflamatoria, incluyendo aquellos con Crohn o colitis ulcerosa. El método comprende la medida de las concentraciones o niveles de factor de crecimiento hepático (HGF), betacelulina (BTC), molécula de adhesión de células vasculares (VCAM-1) y compuestos anti-TNF □ □en una muestra obtenida del sujeto. La presente invención se diferencia de WO2015/110989 en el panel de marcadores que se utiliza; además, en el método de la presente invención, la muestra biológica está adherida a la superficie de un biomaterial. WO2015/110989 describes methods for predicting the probability of mucosal recovery in individuals with inflammatory bowel disease, including those with Crohn's or ulcerative colitis. The method comprises the measurement of the concentrations or levels of liver growth factor (HGF), betacellulin (BTC), vascular cell adhesion molecule (VCAM-1) and anti-TNF compounds □ □ in a sample obtained from the subject. The present invention differs from WO2015/110989 in the marker panel that is used; furthermore, in the method of the present invention, the biological sample is adhered to the surface of a biomaterial.
WO2016/074068 describe compuestos químicos que promueven la regeneración de tejidos en órganos. Dichos compuestos pueden estimular la producción de determinados marcadores moleculares, entre ellos metaloproteinasas (MMP1, MMP2, MMP9, MMP13), factor de crecimiento hepáticos (HGF), lisil oxidasa (LOX), y serpinas E1 y A1. Sin embargo, en el documento no se describen ninguno de los marcadores de la presente invención y tampoco se describen métodos para determinar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial. WO2016/074068 describes chemical compounds that promote tissue regeneration in organs. These compounds can stimulate the production of certain molecular markers, including metalloproteinases (MMP1, MMP2, MMP9, MMP13), liver growth factor (HGF), lysyl oxidase (LOX), and E1 and A1 serpins. However, none of the markers of the present invention are described in the document, nor are methods for determining the ability of a biomaterial to induce tissue regeneration.
Teniendo en cuenta lo anterior, existe en el estado de la técnica la necesidad de desarrollar métodos in vitro fiables, rápidos y comparables con los resultados obtenidos a nivel in vivo, que permitan determinar y predecir la capacidad de inducción de regeneración de tejidos por parte de un biomaterial para su traslado a la clínica. Taking into account the foregoing, there is a need in the state of the art to develop reliable, fast and comparable in vitro methods with the results obtained at the in vivo level, which allow determining and predicting the ability to induce tissue regeneration by a biomaterial for transfer to the clinic.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Los inventores de la presente invención han identificado un perfil de marcadores, preferiblemente marcadores proteicos, cuya determinación y/o cuantificación es capaz de predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial. Para ello, se pone en contacto una muestra biológica aislada obtenida de un sujeto con el biomaterial y se analiza dicho perfil de marcadores adheridos de manera diferencial a la superficie del biomaterial, donde el biomaterial es de manera preferente un biomaterial implantable, y, más preferentemente, un dispositivo médico implantable, como por ejemplo prótesis, stents, cánulas, marcapasos, implantes dentales, dispositivos médicos percutáneos etc. The inventors of the present invention have identified a profile of markers, preferably protein markers, whose determination and/or quantification is capable of predicting and/or predicting the ability of a biomaterial to induce tissue regeneration. To do this, an isolated biological sample obtained from a subject is brought into contact with the biomaterial and said profile of markers adhered differentially to the surface of the biomaterial is analyzed, where the biomaterial is preferably an implantable biomaterial, and, more preferably , an implantable medical device, such as prostheses, stents, cannulas, pacemakers, dental implants, percutaneous medical devices, etc.
En particular, los inventores han identificado y cuantificado los marcadores seleccionados de la lista que consiste en: proteína C reactiva (CRP); componente amiloide P sérico (SAMP); subunidad C del subcomponente C1q del complemento (C1QC); subunidad B del subcomponente C1q del complemento (C1QB); componente C7 del complemento (CO7); componente C5 del complemento (CO5); inhibidor de C1 (IC1); componente C3 del complemento (CO3); subcomponente C1 s del complemento (C1S); Factor H del complemento (CFAH); cadena > de la proteína de unión a C4b (C4BPA); subcomponente C1 r del complemento (C1R); cadena > del componente C8 del complemento (CO8A); Ficolina-2 (FCN2); Vitronectina (VTNC); y Clusterina (CLUS); como marcadores proteicos asociados a la predicción y/o pronóstico de la capacidad de inducción de regeneración de tejidos por parte de un biomaterial. En otras palabras, estas proteínas son marcadores de la capacidad de inducción de regeneración de tejidos por parte de un biomaterial cuando se encuentran adheridos al mismo. La identificación y cuantificación in vitro de la presencia de este conjunto de marcadores, supone una mejora sustancial del actual estado de la técnica, ya que hasta ahora no se ha identificado ningún tipo de patrón de proteínas que puedan ser determinadas y cuantificadas in vitro y que predigan qué resultado va a ser obtenido in vivo una vez implantado el biomaterial. In particular, the inventors have identified and quantified markers selected from the list consisting of: C-reactive protein (CRP); serum amyloid P component (SAMP); C subunit of the C1q subcomponent of complement (C1QC); complement subcomponent C1q subunit B (C1QB); complement component C7 (CO7); complement component C5 (CO5); C1 inhibitor (IC1); complement component C3 (CO3); complement subcomponent C1 s (C1S); Complement factor H (CFAH); C4b binding protein > chain (C4BPA); complement subcomponent C1 r (C1R); chain > of the complement component C8 (CO8A); Ficolin-2 (FCN2); Vitronectin (VTNC); and Clusterin (CLUS); as protein markers associated with the prediction and/or prognosis of the tissue regeneration induction capacity of a biomaterial. In other words, these proteins are markers of the ability of a biomaterial to induce tissue regeneration when they are adhered to it. The in vitro identification and quantification of the presence of this set of markers represents a substantial improvement of the current state of the art, since until now no has identified no type of protein pattern that can be determined and quantified in vitro and that predicts what result will be obtained in vivo once the biomaterial is implanted.
Así, el primer aspecto de la invención se refiere a un método in vitro (“método de la invención”) para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial en estudio a partir de una muestra biológica aislada obtenida de un sujeto y adherida de manera diferencial a la superficie de dicho biomaterial, que comprende determinar y cuantificar la presencia de al menos uno de los marcadores adheridos al biomaterial en estudio, donde dicho marcador se selecciona de la lista que consiste en: CRP (UniProtKB P02741; entry version 234); SAMP (UniProtKB P02743; entry version 224); C1QC (UniProtKB P02747; entry version 210); C1QB (UniProtKB P02746; entry version 216); CO7 (UniProtKB P10643; entry version 209); C1S (UniProtKB P09871; entry version 245); VTNC (UniProtKB P04004; entry version 241); CO5 (UniProtKB P06684; entry version 185); IC1 (UniProtKB P05155; entry version 240); CO3 (UniProtKB P01024; entry versión 250); CFAH (UniProtKB P06909, entry versión 166); C4BPA (UniProtKB P04003; entry version 199); C1R (UniProtKB P00736; entry version 238); CO8A (UniProtKB P07357; entry version 213); FCN2 (UniProtKB Q15485; entry version 181); GLUS (UniProtKB P10909; entry version 234); para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte del biomaterial que ha estado en contacto con la muestra biológica aislada del sujeto. Thus, the first aspect of the invention refers to an in vitro method ("method of the invention") to predict and/or predict the ability to induce tissue regeneration by a biomaterial under study from a biological sample. isolated obtained from a subject and differentially adhered to the surface of said biomaterial, comprising determining and quantifying the presence of at least one of the markers adhered to the biomaterial under study, where said marker is selected from the list consisting of: CRP (UniProtKB P02741; entry version 234); SAMP (UniProtKB P02743; entry version 224); C1QC (UniProtKB P02747; entry version 210); C1QB (UniProtKB P02746; entry version 216); CO7 (UniProtKB P10643; entry version 209); C1S (UniProtKB P09871; entry version 245); VTNC (UniProtKB P04004; entry version 241); CO5 (UniProtKB P06684; entry version 185); IC1 (UniProtKB P05155; entry version 240); CO3 (UniProtKB P01024; entry version 250); CFAH (UniProtKB P06909, entry version 166); C4BPA (UniProtKB P04003; entry version 199); C1R (UniProtKB P00736; entry version 238); CO8A (UniProtKB P07357; entry version 213); FCN2 (UniProtKB Q15485; entry version 181); GLUS (UniProtKB P10909; entry version 234); to predict and/or predict the tissue regeneration induction capacity of the biomaterial that has been in contact with the biological sample isolated from the subject.
Específicamente, el método in vitro para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial en estudio a partir de una muestra biológica aislada obtenida de un sujeto y adherida de manera diferencial a la superficie de dicho biomaterial, comprende las siguientes etapas: a. Incubar el biomaterial en estudio con la muestra biológica aislada; b. Determinar y cuantificar la presencia de al menos uno de los marcadores adheridos al biomaterial en estudio, donde dicho marcador se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; C1S; VTNC; CO5; IC1; CO3; CFAH; C4BPA; C1R; COBA; FCN2; y CLUS; preferiblemente en esta etapa se determina y cuantifica la presencia de marcadores adheridos al biomaterial en estudio, donde dichos marcadores son: componente C5 del complemento (CO5), inhibidor de C1 (IC1), componente C3 del complemento (CO3), Factor H del complemento (CFAH), subcomponente C1 r del complemento (C1R), Ficolina-2 (FCN2) y Clusterina (CLUS); y más preferiblemente un marcador adicional seleccionado de la lista que consiste en: proteína C reactiva (CRP); componente amiloide P sérico (SAMP); subunidad C del subcomponente C1q del complemento (C1QC); subunidad B del subcomponente C1q del complemento (C1QB); componente C7 del complemento (CO7); subcomponente C1 s del complemento (C1S); cadena > de la proteína de unión a C4b (C4BPA); cadena > del componente C8 del complemento (CO8A); y Vitronectina (VTNC); y c. Comparar el valor obtenido en la etapa b. con un valor de referencia obtenido de un biomaterial de referencia; donde el biomaterial en estudio está destinado a regeneración de un tejido óseo o blando, y donde: Specifically, the in vitro method for predicting and/or forecasting the ability to induce tissue regeneration by a biomaterial under study from an isolated biological sample obtained from a subject and differentially adhered to the surface of said biomaterial, It comprises the following stages: a. Incubate the biomaterial under study with the isolated biological sample; b. Determine and quantify the presence of at least one of the markers attached to the biomaterial under study, where said marker is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; NCTV; CO5; IC1; CO3; CFAH; C4BPA; C1R; COBA; FCN2; and CLUS; preferably in this stage the presence of markers adhered to the biomaterial under study is determined and quantified, where said markers are: component C5 of the complement (CO5), inhibitor of C1 (IC1), component C3 of the complement (CO3), Factor H of the complement (CFAH), complement subcomponent C1r (C1R), Ficolin-2 (FCN2) and Clusterin (CLUS); and more preferably a additional marker selected from the list consisting of: C-reactive protein (CRP); serum amyloid P component (SAMP); C subunit of the C1q subcomponent of complement (C1QC); complement subcomponent C1q subunit B (C1QB); complement component C7 (CO7); complement subcomponent C1 s (C1S); C4b binding protein > chain (C4BPA); chain > of the complement component C8 (CO8A); and Vitronectin (VTNC); and c. Compare the value obtained in step b. with a reference value obtained from a reference biomaterial; where the biomaterial under study is intended for the regeneration of bone or soft tissue, and where:
(i) un valor de los marcadores SAMP; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; y C1QC de hasta 1 ,5 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio va destinado a regeneración de tejido óseo; (i) a value of the SAMP markers; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; and C1QC up to 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(ii) un valor del marcador CRP inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio va destinado a regeneración de tejido óseo; (ii) a CRP marker value lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
(iii) ratios entre los valores de los marcadores C4BPA; CFAH; IC1; VTNC; CLUS, y los valores de los marcadores C1QB; C1S; C1R; C1QC; y CO3 ¡guales o superiores a los valores de referencia obtenidos para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (iii) ratios between the values of the C4BPA markers; CFAH; IC1; NCTV; CLUS, and C1QB marker values; C1S; C1R; C1QC; and CO3 equal to or greater than the reference values obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(iv) un valor de los marcadores SAMP; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R;CO8A; y FCN2; inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; y/o (v) un valor de los marcadores IC1; CFAH; C4BPA; VTNC; y CLUS superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando, son indicativos de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. (iv) a value of the SAMP markers; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R;CO8A; and FCN2; lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; and/or (v) a value of the IC1 markers; CFAH; C4BPA; NCTV; and CLUS higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration, are indicative of a good ability to induce tissue regeneration by the biomaterial under study.
En ciertas realizaciones, el método de la invención se caracteriza porque comprende la determinación y cuantificación simultánea de uno; dos; tres; cuatro; cinco; seis; siete; ocho; nueve; diez; once; doce; trece; catorce; quince o dieciséis marcadores seleccionados de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; VTNC; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; y CLUS. In certain embodiments, the method of the invention is characterized in that it comprises the simultaneous determination and quantification of one; two; three; four; five; six; seven; eight; nine; ten; eleven; twelve o'clock;thirteen;fourteen; fifteen or sixteen markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; NCTV; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; and CLUS.
En ciertas realizaciones específicas de la invención, se identifica y cuantifica la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; C1S; VTNC; CO5; IC1; CO3; CFAH; C4BPA; C1R; CO8A; FCN2; y CLUS. In certain specific embodiments of the invention, the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; C1S; NCTV; CO5; IC1; CO3; CFAH; C4BPA; C1R; CO8A; FCN2; and CLUS.
En ciertas realizaciones del método de la invención, éste se caracteriza porque comprende la determinación y cuantificación de al menos uno de los ratios entre los marcadores IC1; C4BPA; CFAH; VTNC; y CLUS, respecto a los marcadores C1QB; C1S; C1R; C1QC; CO5 y CO3. En una realización más particular, se determinan al menos uno de los ratios seleccionados de entre cualquiera de la siguiente lista: IC1/C1QB; IC1/C1QC; IC1/C1S; IC1/C1R; CFAH/CO3; C4BPA/CO3; VTNC/CO5; CLUS/CO5. Valores de estos ratios igual o superiores a los valores obtenidos en el biomaterial de referencia indicarían que el biomaterial presenta una buena capacidad de regeneración cuando la muestra se encuentra adherida al biomaterial de interés o biomaterial problema. In certain embodiments of the method of the invention, it is characterized in that it comprises the determination and quantification of at least one of the ratios between the IC1 markers; C4BPA; CFAH; NCTV; and CLUS, with respect to the C1QB markers; C1S; C1R; C1QC; CO5 and CO3. In a more particular embodiment, at least one of the ratios selected from any of the following list is determined: IC1/C1QB; IC1/C1QC; IC1/C1S; IC1/C1R; CFAH/CO3; C4BPA/CO3; NCVT/CO5; CLUS/CO5. Values of these ratios equal to or greater than the values obtained in the reference biomaterial would indicate that the biomaterial has a good regeneration capacity when the sample is adhered to the biomaterial of interest or problem biomaterial.
En ciertas realizaciones del primer aspecto de la invención, este se caracteriza porque el(los) marcador(es) que se identifican y cuantifican se encuentran adsorbidos al biomaterial, una vez que este ha estado en contacto con la muestra biológica aislada de un sujeto, particularmente de un sujeto al que se le tiene que implantar el biomaterial. Dichos marcadores son preferiblemente marcadores presentes en la muestra biológica aislada del sujeto. Además, la presencia de dichos marcadores adsorbidos a la superficie de los biomateriales analizados determinará la capacidad de regeneración del biomaterial. In certain embodiments of the first aspect of the invention, it is characterized in that the marker(s) that are identified and quantified are adsorbed to the biomaterial, once it has been in contact with the biological sample isolated from a subject, particularly of a subject to which the biomaterial has to be implanted. Said markers are preferably markers present in the biological sample isolated from the subject. In addition, the presence of these markers adsorbed to the surface of the analyzed biomaterials will determine the regeneration capacity of the biomaterial.
En la presente invención, el término “adsorción” o “adherencia" y variantes se refiere a un proceso por el cual átomos, iones o moléculas son atrapadas o retenidas en la superficie de un material. In the present invention, the term "adsorption" or "adherence" and variants refer to a process by which atoms, ions or molecules are trapped or retained on the surface of a material.
En la presente invención, el término “regeneración” se refiere a cualquier proceso de reparación tisular consistente en la neoformación del tejido preexistente. El término se puede utilizar para cualquier tipo de tejido, incluyendo tejido óseo y blando, tejido conectivo, tejido epitelial, tejido muscular y tejido nervioso. El tejido conectivo sostiene y une otros tejidos como el óseo, el sanguíneo y el linfático. El tejido epitelial sirve de cobertura; entre éstos se encuentran la piel y el revestimiento de varios conductos en el interior del cuerpo. El tejido muscular consta de músculos estriados o voluntarios que mueven el esqueleto y de músculo liso, tal como el que rodea al estómago. El tejido nervioso está formado por células nerviosas o neuronas y sirve para llevar "mensajes" hacia y desde varias partes del cuerpo. In the present invention, the term "regeneration" refers to any tissue repair process consisting of the neoformation of pre-existing tissue. The term can be used for any type of tissue, including bone and soft tissue, connective tissue, epithelial tissue, muscle tissue, and nervous tissue. Connective tissue supports and binds other tissues such as bone, blood, and lymph. The epithelial tissue serves as a cover; These include the skin and the lining of various ducts inside the body. Muscular tissue consists of striated or voluntary muscles that move the skeleton and smooth muscle, such as that around the stomach. Nervous tissue is made up of nerve cells or neurons and serves to carry "messages" to and from various parts of the body.
El término “tejido óseo” o “tejido de hueso” se refiere a cualquier tipo de tejido que da fuerza y estructura a los huesos, incluyendo tejido compacto y tejido esponjoso o trabecular. The term "bone tissue" or "bone tissue" refers to any type of tissue that gives strength and structure to bones, including compact tissue and cancellous or trabecular tissue.
Una consecuencia directa de la pérdida de la calidad del tejido óseo (por ejemplo, en mujeres menopáusicas, pacientes con enfermedades sistémicas, pacientes irradiados) es el aumento en la tasa de fracaso implantes tanto ortopédicos (cadera o rodilla) como dentales, lo que se agrava además por problemas derivados de la mayor incidencia de otras enfermedades como la periodontitis en dicha población. Estos factores además se exacerban considerablemente con la edad. Así, tanto la menopausia como el aumento progresivo en la esperanza de vida hacen que haya un mayor número de pacientes que requieran la utilización de prótesis óseas incluyendo prótesis de cadera, rodilla, maxilofaciales, o la reposición de los dientes perdidos por fallos en su sistema de anclaje óseo. Los condicionantes de edad y los déficits hormonales en las mujeres agravan el problema y van a condicionar la respuesta frente a un implante. A direct consequence of the loss of bone tissue quality (for example, in menopausal women, patients with systemic diseases, irradiated patients) is the increase in the failure rate of both orthopedic (hip or knee) and dental implants, which is It is also aggravated by problems derived from the higher incidence of other diseases such as periodontitis in this population. These factors are further exacerbated considerably with age. Thus, both menopause and the progressive increase in life expectancy mean that there is a greater number of patients who require the use of bone prostheses, including hip, knee, maxillofacial prostheses, or the replacement of teeth lost due to failures in their system. bone anchoring. The determining factors of age and hormonal deficits in women aggravate the problem and will condition the response to an implant.
Se necesita, por tanto, seguir avanzando en la obtención de prótesis/implantes/sustitutos óseos que puedan mejorar su osteointegración, es decir, que puedan contribuir a generar nuevo hueso de calidad, con más facilidad y en menor tiempo. Se necesita también seguir progresando hacia un diseño de implantes personalizados para cada paciente según las características de su historial clínico y circunstancias particulares, como son la falta de hueso o la presencia de un hueso osteoporótico, circunstancias estas particularmente frecuentes en las mujeres postmenopáusicas. Therefore, it is necessary to continue advancing in obtaining prostheses/implants/bone substitutes that can improve their osseointegration, that is, that can contribute to generating new quality bone, more easily and in less time. It is also necessary to continue progressing towards a personalized implant design for each patient according to the characteristics of their clinical history and particular circumstances, such as lack of bone or the presence of osteoporotic bone, circumstances that are particularly frequent in postmenopausal women.
Así, resulta clave el estudio de los procesos biológicos que tienen lugar en la interfase entre las prótesis/implantes/sustitutos y el entorno celular, procesos que determinan la aceptación/rechazo de las prótesis, así como los tiempos de regeneración y osteointegración que son necesarios. Thus, the study of the biological processes that take place at the interface between the prostheses/implants/substitutes and the cellular environment is key, processes that determine the acceptance/rejection of the prostheses, as well as the regeneration and osseointegration times that are necessary. .
El estudio en profundidad de los procesos biológicos que suceden en la interfase entre prótesis e implantes necesitará de un diseño experimental interdisciplinary muy potente. The in-depth study of the biological processes that occur at the interface between prostheses and implants will require a very powerful interdisciplinary experimental design.
La mejora de la capacidad de osteointegración de prótesis óseas depende de las características fisicoquímicas de las superficies a desarrollar. Las diferentes propiedades fisicoquímicas de las nuevas superficies desarrolladas regularán las proteínas adsorbidas en la superficie en contacto con la sangre. Las proteínas adsorbidas en las superficies de los materiales regularán los principales procesos regenerativos que darán lugar a la formación de nuevo hueso y en definitiva a lograr una óptima osteointegración del implante. The improvement of the osseointegration capacity of bone prostheses depends on the physicochemical characteristics of the surfaces to be developed. The different physicochemical properties of the newly developed surfaces will regulate the proteins adsorbed on the surface in contact with blood. Proteins adsorbed on the surfaces of the materials will regulate the main regenerative processes that will lead to the formation of new bone and ultimately to achieve optimal osseointegration of the implant.
El término “tejido blando" se refiere al tejido muscular, tejido fibroso, los vasos sanguíneos o cualquier otro tejido de sostén del cuerpo. The term "soft tissue" refers to muscle tissue, fibrous tissue, blood vessels, or any other supportive tissue of the body.
Los dispositivos médicos percutáneos se utilizan cada vez más, para una amplia variedad de afecciones, y en diversos campos médicos. Se pueden definir como cuerpos extraños que atraviesan la barrera epitelial y, por tanto, conectan el entorno externo con la estructura intema del cuerpo. Ejemplos de dispositivos percutáneos incluyen los catéteres y otros sistemas de intubación a largo plazo o implantes osteointegrados (por ejemplo, implantes dentales y ortopédicos), donde el dispositivo se implanta de forma permanente en el cuerpo. Percutaneous medical devices are being used more and more, for a wide variety of conditions, and in various medical fields. They can be defined as foreign bodies that cross the epithelial barrier and, therefore, connect the external environment with the internal structure of the body. Examples of percutaneous devices include catheters and other long-term intubation systems or osseointegrated implants (for example, dental and orthopedic implants), where the device is permanently implanted in the body.
El sellado del tejido blando es imprescindible para una osteointegración estable y para la supervivencia a largo plazo de los implantes. El objetivo de las estrategias para optimizar el sellado del tejido blando alrededor de los implantes osteointegrados es conseguir propiedades de la superficie del implante que puedan promover la adhesión del tejido blando alrededor de los implantes percutáneos / permucosos, lo que no solo proporcionaría al implante cierta estabilidad, sino que también establecería una barrera permanente contra la invasión de las bacterias y, por tanto, la infección. Soft tissue sealing is essential for stable osseointegration and long-term implant survival. The goal of strategies to optimize soft tissue sealing around osseointegrated implants is to achieve implant surface properties that can promote soft tissue adhesion around percutaneous/permucosal implants, which would not only provide the implant with some stability , but it would also establish a permanent barrier against the invasion of bacteria and, therefore, infection.
Las nuevas investigaciones llevadas a cabo en cuanto a la cicatrización de heridas se centran en la regulación de la actividad de los fibroblastos. Así, en heridas sin inflamación crónica, los fibroblastos producen colágeno que sirve para incrementar la resistencia mecánica de la matriz extracelular secretada. Sin embargo, los fibroblastos bajo condiciones de estrés tienen capacidad de conformar ambientes pro-inflamatorios (mediante expresión de citokinas), producir especies reactivas con oxígeno (ROS) y decrecer los niveles de colágeno tipo I y fibronectina, impidiendo la regeneración de los tejidos. New research in wound healing focuses on the regulation of fibroblast activity. Thus, in wounds without chronic inflammation, fibroblasts produce collagen that serves to increase the mechanical resistance of the secreted extracellular matrix. However, fibroblasts under stress conditions have the capacity to form pro-inflammatory environments (through expression of cytokines), produce reactive oxygen species (ROS) and decrease the levels of type I collagen and fibronectin, preventing tissue regeneration.
Por tanto, la respuesta de los fibroblastos ante implantes percutáneos (tales como los implantes dentales) puede ayudar a la regeneración de tejidos y la cicatrización de la herida (ambientes sin inflamación crónica), o, por el contrario, la formación de microambientes no regenerativos, caracterizados por perfiles de factores de crecimiento anormales, estados inflamatorios y generación de ROS (ambientes con inflamación crónica). En este último estado se activará el NF-kp, así como la expresión de citokinas tipo IL-ip, y habrá un reclutamiento de neutrófilos y macrófagos. La mejora de sellado de los tejidos blandos con los pilares depende de las características fisicoquímicas de las superficies a desarrollar. Las diferentes propiedades fisicoquímicas de las nuevas superficies desarrolladas regularán las proteínas adsorbidas en la superficie en contacto con la sangre. Las proteínas adsorbidas en cada una de las superficies del material regularán los principales procesos regenerativos iniciales y, en particular, la inflamación. Therefore, the response of fibroblasts to percutaneous implants (such as dental implants) may support tissue regeneration and wound healing (environments without chronic inflammation), or, conversely, the formation of non-regenerative microenvironments. , characterized by profiles of abnormal growth factors, inflammatory states and generation of ROS (environments with chronic inflammation). In this last state, NF-kp will be activated, as well as the expression of IL-ip type cytokines, and there will be a recruitment of neutrophils and macrophages. The improvement of the soft tissue seal with the abutments depends on the physicochemical characteristics of the surfaces to be developed. The different physicochemical properties of the newly developed surfaces will regulate the proteins adsorbed on the surface in contact with blood. The proteins adsorbed on each of the surfaces of the material will regulate the main initial regenerative processes and, in particular, inflammation.
En una primera etapa (etapa a), el método in vitro de la invención comprende la incubación de una muestra biológica aislada procedente de un sujeto, preferiblemente en el cual se implantará el biomaterial en estudio, con un biomaterial del que se pretende determinar la capacidad de regeneración. In a first stage (stage a), the in vitro method of the invention comprises the incubation of an isolated biological sample from a subject, preferably in which the biomaterial under study will be implanted, with a biomaterial whose capacity is to be determined. of regeneration.
Cualquier muestra biológica puede ser usada para poner en práctica el método de la invención. El término “muestra biológica” está referido a cualquier material biológico procedente de un sujeto o varios sujetos. La muestra es preferiblemente una muestra de tejido y/o de un fluido biológico. Tal muestra de tejido puede comprender, por ejemplo, tejido renal, tejido articular, tejido vascular y/o tejido del corazón, entre otros. El fluido biológico incluye, sin limitarse, linfa, fluido cerebroespinal (FCS), fluido amniótico, efusión pleural, médula ósea, nódulo linfático, fluido linfático, fluido sinovial, una suspensión celular única preparada a partir de tejido sólido o líneas celulares orina, saliva, sudor, lágrimas, sangre, suero y plasma sanguíneo. La muestra puede ser, y es preferiblemente, sangre periférica, sangre, plasma o suero. En ciertas realizaciones específicas de la invención, la muestra es suero. Any biological sample can be used to put the method of the invention into practice. The term "biological sample" refers to any biological material from a subject or multiple subjects. The sample is preferably a tissue and/or biological fluid sample. Such a tissue sample may comprise, for example, kidney tissue, joint tissue, vascular tissue, and/or heart tissue, among others. Biological fluid includes, but is not limited to, lymph, cerebrospinal fluid (SCF), amniotic fluid, pleural effusion, bone marrow, lymph node, lymphatic fluid, synovial fluid, a single cell suspension prepared from solid tissue or cell lines urine, saliva , sweat, tears, blood, serum and blood plasma. The sample can be, and is preferably, peripheral blood, whole blood, plasma, or serum. In certain specific embodiments of the invention, the sample is serum.
Típicamente, la muestra es humana en origen, pero puede también proceder de cualquier otro animal. En ciertas realizaciones específicas, la muestra procede de un mamífero, incluyendo, sin limitación, animales domésticos, de granja, y primates. Typically, the sample is human in origin, but can also be from any other animal. In certain specific embodiments, the sample is from a mammal, including, without limitation, domestic animals, farm animals, and primates.
A efectos de la presente invención, los términos "sujeto", “individuo” o “paciente”, utilizados indistintamente a lo largo del presente documento, se refiere a cualquier animal mamífero e incluye, pero no se restringe a animales domésticos, de granja, primates y humanos, preferiblemente humanos. Dichos términos no pretenden ser limitativos en ningún aspecto, pudiendo ser éstos de cualquier edad, sexo y condición física. For the purposes of this invention, the terms "subject", "individual" or "patient", used interchangeably throughout this document, refer to any mammalian animal and includes, but is not restricted to domestic animals, farm animals, primates and humans, preferably humans. Said terms are not intended to be limiting in any aspect, and they may be of any age, sex and physical condition.
En una realización particular, el sujeto es un sujeto que padece una patología que es susceptible de ser tratada mediante la implantación de un biomaterial, tal y como se ha descrito en el presente documento. Una vez la muestra biológica es aislada del sujeto, dicha muestra se incuba con el biomaterial problema para determinar en una etapa posterior del método (etapa b) los marcadores que se adhieren a dicho biomaterial. En una realización preferida, el biomaterial problema se incuba con la muestra biológica aislada del sujeto durante un intervalo de 2 a 10 horas, preferiblemente durante un intervalo de entre 3 y 6 horas, más preferiblemente durante un intervalo de entre 3 y 4 horas. In a particular embodiment, the subject is a subject suffering from a pathology that can be treated by implanting a biomaterial, as described herein. Once the biological sample is isolated from the subject, said sample is incubated with the problem biomaterial to determine in a later stage of the method (stage b) the markers that adhere to said biomaterial. In a preferred embodiment, the test biomaterial is incubated with the biological sample isolated from the subject for a range of 2 to 10 hours, preferably for a range of 3 to 6 hours, more preferably for a range of 3 to 4 hours.
A continuación, una vez la muestra biológica se ha incubado con el biomaterial problema, el método de la invención comprende una etapa b donde se identifican, o determinan y cuantifican la presencia de los marcadores aquí descritos, que se han adherido al biomaterial problema en contacto con la muestra biológica aislada. Next, once the biological sample has been incubated with the problem biomaterial, the method of the invention comprises a step b where the presence of the markers described here, which have adhered to the problem biomaterial in contact, are identified, or determined and quantified. with the isolated biological sample.
Tal y como se ha mencionado anteriormente, la determinación del perfil de marcadores descrito en la presente invención se lleva a cabo identificando y determinando los valores de dichos marcadores que han quedado adheridos al biomaterial problema una vez éste ha estado en contacto con la muestra biológica del sujeto al que preferiblemente se le va a implantar dicho biomaterial. As mentioned above, the determination of the marker profile described in the present invention is carried out by identifying and determining the values of said markers that have remained attached to the problem biomaterial once it has been in contact with the biological sample of the subject to which said biomaterial is preferably going to be implanted.
Transcurrido el periodo de incubación de la muestra biológica del sujeto con el biomaterial problema, éste es sometido preferiblemente a una serie de lavados para eliminar los marcadores que no están fuertemente adheridos al biomaterial. En una realización preferida dichos lavados se llevan a cabo preferiblemente con agua bidestilada y más preferiblemente, un último lavado con una solución tampón que comprende preferiblemente cloruro sódico a una concentración de entre 50-500 mM. After the incubation period of the biological sample of the subject with the problem biomaterial, it is preferably subjected to a series of washes to eliminate the markers that are not strongly adhered to the biomaterial. In a preferred embodiment, said washes are preferably carried out with bidistilled water and more preferably, a final wash with a buffer solution preferably comprising sodium chloride at a concentration of between 50-500 mM.
En otra realización más particular, los marcadores adheridos al biomaterial problema se recolectan o eluyen de dicho biomaterial, preferiblemente mediante un tratamiento de la superficie del biomaterial con una solución tampón, preferiblemente donde la solución tampón es una solución de bicarbonato de trietilamonio, aunque se puede utilizar cualquier solución tampón conocida por el experto en la materia y útil para obtener las proteínas o marcadores adheridas a dicho biomaterial. In another more particular embodiment, the markers adhered to the problem biomaterial are collected or eluted from said biomaterial, preferably by treating the surface of the biomaterial with a buffer solution, preferably where the buffer solution is a triethylammonium bicarbonate solution, although it can be use any buffer solution known to those skilled in the art and useful for obtaining the proteins or markers adhered to said biomaterial.
A continuación, en otra realización particular, en dichos eluidos que comprenden los marcadores adheridos al biomaterial, se determinan y cuantifican los marcadores que forman el perfil proteico de la invención. Next, in another particular embodiment, in said eluates that comprise the markers adhered to the biomaterial, the markers that form the protein profile of the invention are determined and quantified.
La determinación y cuantificación de la presencia de los marcadores de la invención se lleva a cabo, preferiblemente mediante la determinación de la cantidad de marcador adherido al material en contacto con la muestra biológica, preferiblemente en los eluidos obtenidos según se ha descrito anteriormente, y también de manera alternativa, mediante la determinación de los niveles de expresión proteicos de dichos marcadores, mediante cualquier método conocido en el estado de la técnica. Los autores de la presente invención han demostrado que la detección de la cantidad y/o la concentración de estos productos de expresión de manera semicuantitativa o cuantitativa permiten diferenciar entre un biomaterial con mayor o menor capacidad regenerativa de un determinado tipo de tejido. La presencia, así como la cantidad, del marcador detectada establece un perfil diferencial entre biomateriales con mayor o menor eficiencia en términos de regeneración. The determination and quantification of the presence of the markers of the invention is carried out, preferably by determining the amount of marker adhered to the material in contact with the biological sample, preferably in the eluates. obtained as described above, and also alternatively, by determining the protein expression levels of said markers, by any method known in the state of the art. The authors of the present invention have shown that the detection of the quantity and/or concentration of these expression products in a semi-quantitative or quantitative manner allows to differentiate between a biomaterial with greater or lesser regenerative capacity of a certain type of tissue. The presence, as well as the amount, of the detected marker establishes a differential profile between biomaterials with greater or lesser efficiency in terms of regeneration.
La medida de la cantidad o la concentración, preferiblemente de manera semicuantitativa o cuantitativa, puede ser llevada a cabo de manera directa o indirecta. La medida directa se refiere a la medida de la cantidad y/o la concentración de las proteínas o marcadores de la invención. Dicha señal a la que también podemos referimos como señal de intensidad -puede obtenerse, por ejemplo, midiendo un valor de intensidad de una propiedad química o física de dichos marcadores. La medida indirecta incluye la medida obtenida de un componente secundario o un sistema de medida biológica (por ejemplo, la medida de respuestas celulares, ligandos, "etiquetas" o productos de reacción enzimática). The measurement of the amount or concentration, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly. Direct measurement refers to the measurement of the amount and/or concentration of the proteins or markers of the invention. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said markers. Indirect measurement includes measurement obtained from a secondary component or biological measurement system (eg, measurement of cellular responses, ligands, "tags," or enzymatic reaction products).
El término "cantidad" tal y como se utiliza en la descripción, se refiere, pero no se limita, a la cantidad absoluta o relativa de los marcadores de la invención, sino que también se refiere a cualquier otro valor o parámetro relacionado con los mismos o que pueda derivarse de éstos. Dichos valores o parámetros comprenden valores de intensidad de la señal obtenidos a partir de cualquiera de las propiedades físicas o químicas de dichos marcadores, obtenidos mediante medida directa. Adicionalmente, dichos valores o parámetros incluyen todos aquellos obtenidos mediante medida indirecta, por ejemplo, cualquiera de los sistemas de medida descritos en otra parte del presente documento. The term "quantity" as used in the description, refers to, but is not limited to, the absolute or relative quantity of the markers of the invention, but also refers to any other value or parameter related to them. or that may derive from them. Said values or parameters comprise signal intensity values obtained from any of the physical or chemical properties of said markers, obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
La detección y cuantificación de los marcadores de la presente invención, asociados a la determinación de la capacidad regenerativa de un biomaterial, puede llevarse a cabo mediante la cuantificación de los niveles de las proteínas o péptidos por métodos convencionales, conocidos por los técnicos en la materia, incluyendo, aunque sin limitarse a, métodos quimio luminiscentes, tinción inmunohistoquímica o detección bioquímica (erg., ensayos inmunohistoquímicos), técnicas inmunológicas, citometría de flujo, inmunoprecipitación (o sus equivalentes para reactivos que no sean anticuerpos), cromatografía líquida de alta eficacia (HPLC), espectrometría de masas (MS), medida de la absorbancia de resonancia de plasma, etc. En una realización particular, la cuantificación de los niveles de los marcadores identificados en la presente invendón asociados con la capaddad de inducdón de regeneración de un biomaterial se realiza mediante técnicas inmunológicas, tales como ELISA ("Enzyme-Linked Immunosorbent Assay), Western-blot, RIA (radioinmunoensayo), EIA competitivo (inmunoensayo de enzima competitivo), DAS-ELISA ("Double Antibody Sandwich ELISA"), espectroscopia de masas, técnicas inmunocitoquímicas e inmunohistoquímicas, técnicas basadas en el uso de biochips de proteínas o microarrays que induyan anticuerpos específicos o ensayos basados en la predpitación coloidal en formatos tales como los "dipsticks". The detection and quantification of the markers of the present invention, associated with the determination of the regenerative capacity of a biomaterial, can be carried out by quantifying the levels of proteins or peptides by conventional methods, known to those skilled in the art. , including, but not limited to, chemiluminescent methods, immunohistochemical staining or biochemical detection (eg, immunohistochemical assays), immunological techniques, flow cytometry, immunoprecipitation (or their equivalents for reagents other than antibodies), high performance liquid chromatography (HPLC), mass spectrometry (MS), plasma resonance absorbance measurement, etc. In a particular embodiment, the Quantification of the levels of the markers identified in the present invention associated with the ability to induce regeneration of a biomaterial is performed by immunological techniques, such as ELISA ("Enzyme-Linked Immunosorbent Assay), Western-blot, RIA (radioimmunoassay), Competitive EIA (competitive enzyme immunoassay), DAS-ELISA ("Double Antibody Sandwich ELISA"), mass spectroscopy, immunocytochemical and immunohistochemical techniques, techniques based on the use of protein biochips or microarrays that include specific antibodies or tests based on the colloidal precipitation in formats such as "dipsticks".
Preferiblemente, las proteínas se detectan por Westem-blot, ELISA, un array o matriz proteico o por electroforesis bidimensional, más preferiblemente la detecdón del perfil proteico descrito en la presente invendón se lleva a cabo mediante ELISA. Asimismo, el técnico en la materia reconocerá que la presente invención se reladona no sólo con los marcadores descritos en la invención, sino también con fragmentos de los mismos que pudieran generarse, por ejemplo, por fenómenos de procesamiento alternativo o splicing, ruptura proteolítica de dichos péptidos o proteínas, etc. Dichos fragmentos conservan la capaddad de ser utilizados como marcadores para la determinación de la efidenda regenerativa de un material médico tal y como se describe en el presente documento. Los marcadores de la invendón, así como sus fragmentos, pueden induir modificaciones post-traduccionales, tales como fosforilación, glicosilación, acetilación, isoprenilación, miristoilación, procesamiento proteolítico, etc. Preferably, the proteins are detected by Western blot, ELISA, a protein array or matrix or by two-dimensional electrophoresis, more preferably the detection of the protein profile described in the present invention is carried out by ELISA. Likewise, the person skilled in the art will recognize that the present invention relates not only to the markers described in the invention, but also to their fragments that could be generated, for example, by alternative processing or splicing phenomena, proteolytic rupture of said peptides or proteins, etc. Said fragments retain the capacity to be used as markers for the determination of the regenerative efficiency of a medical material as described in this document. The markers of the invention, as well as their fragments, can include post-translational modifications, such as phosphorylation, glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.
En el siguiente paso del método de la invención, (etapa c), la determinación y el valor de cuantificación de los marcadores de la invención obtenidos en la etapa b se compara con los valores de referencia obtenidos para dichos marcadores en un biomaterial de referenda, que también se ha puesto en contacto con una muestra biológica aislada del sujeto, en donde In the next step of the method of the invention, (step c), the determination and quantification value of the markers of the invention obtained in step b is compared with the reference values obtained for said markers in a reference biomaterial, that has also been contacted with a biological sample isolated from the subject, where
(i) un valor de los marcadores SAMP; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; y C1QC de hasta 1 ,5 veces superior al valor de referenda obtenido para el biomaterial de referenda cuando el biomaterial en estudio va destinado a regeneración de tejido óseo;(i) a value of the SAMP markers; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; and C1QC up to 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(ii) un valor del marcador CRP inferior al valor de referenda obtenido para el biomaterial de referencia cuando el biomaterial en estudio va destinado a regeneración de tejido óseo; (ii) a CRP marker value lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
(iii) ratios entre los valores de los marcadores C4BPA; CFAH; IC1; VTNC; CLUS, y los valores de los marcadores C1QB; C1S; C1R; C1QC; y CO3 iguales o superiores a los valores de referencia obtenidos para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (iv) un valor de los marcadores SAMP; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; y FCN2 inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; y/o(iii) ratios between the values of the C4BPA markers; CFAH; IC1; NCTV; CLUS, and C1QB marker values; C1S; C1R; C1QC; and CO3 equal to or greater than the reference values obtained for the reference biomaterial when the biomaterial under study is intended for the regeneration of bone tissue; (iv) a value of the SAMP markers; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; and FCN2 lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; me
(v) un valor de los marcadores IC1; CFAH; C4BPA; VTNC; y CLUS superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando, son indicativos de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. (v) an IC1 marker value; CFAH; C4BPA; NCTV; and CLUS higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration, are indicative of a good ability to induce tissue regeneration by the biomaterial under study.
A efectos de la presente invención el término “muestra biológica" se refiere a una muestra o conjunto de muestras aisladas obtenidas de un sujeto o un grupo de sujetos, bien sea un fluido o un tejido como se ha explicado anteriormente. For the purposes of the present invention, the term "biological sample" refers to a sample or set of isolated samples obtained from a subject or a group of subjects, either a fluid or a tissue as explained above.
El término “biomaterial problema" o “biomaterial de interés" o “biomaterial en estudio” se refiere a un biomaterial en el cual se pretende determinar la capacidad de regeneración de una muestra biológica. The term "problem biomaterial" or "biomaterial of interest" or "biomaterial under study" refers to a biomaterial in which it is intended to determine the regeneration capacity of a biological sample.
A efectos de la presente invención el término “biomaterial de referencia" se refiere a un biomaterial en el cual se ha determinado previamente la capacidad de regeneración de una muestra biológica. For the purposes of the present invention, the term "reference biomaterial" refers to a biomaterial in which the regeneration capacity of a biological sample has been previously determined.
En ciertas realizaciones de la invención, el biomaterial de referencia es titanio. In certain embodiments of the invention, the reference biomaterial is titanium.
En ciertas realizaciones de la invención, el biomaterial de referencia es titanio de grado médico. In certain embodiments of the invention, the reference biomaterial is medical grade titanium.
En ciertas realizaciones específicas de la invención, el biomaterial de referencia es titanio liso grado 4. In certain specific embodiments of the invention, the reference biomaterial is smooth grade 4 titanium.
En la presente invención, la cantidad o valor de referencia se obtiene a partir de un biomaterial de referencia. El término “cantidad de referencia” o “valor de referencia”, utilizados indistintamente a lo lardo del presente documento, se obtiene a partir de los valores, preferiblemente valores de cantidad de los marcadores de la invención, analizados en un biomaterial de referencia, del que se conoce su compatibilidad con un sujeto. Como se ha mencionado en el presente documento, a efectos de la presente invención, y a modo de ejemplo, un material biocompatible del que obtener la cantidad de referencia para los marcadores de la invención, puede ser titanio, preferentemente de grado médico. El término "comparación", tal y como se utiliza en la descripción, se refiere, pero no se limita, a la comparación de la cantidad de los marcadores tal y como se describe en la invención, adheridos al biomaterial problema que se cultiva o incuba junto con la muestra biológica de un sujeto, respecto a la cantidad de los mismos marcadores que se han adherido a un biomaterial de referencia cultivado también junto con una muestra biológica obtenida del mismo sujeto. In the present invention, the reference quantity or value is obtained from a reference biomaterial. The term "reference quantity" or "reference value", used interchangeably throughout this document, is obtained from the values, preferably quantity values of the markers of the invention, analyzed in a reference biomaterial, of the that its compatibility with a subject is known. As mentioned herein, for the purposes of the present invention, and by way of example, a biocompatible material from which to obtain the reference quantity for the markers of the invention, may be titanium, preferably medical grade. The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of markers as described in the invention, adhered to the problem biomaterial that is cultured or incubated. together with the biological sample of a subject, with respect to the amount of the same markers that have adhered to a cultured reference biomaterial also together with a biological sample obtained from the same subject.
El biomaterial de referencia puede ser analizado, por ejemplo, simultánea o consecutivamente, junto con el biomaterial problema. La comparación descrita en la etapa c del método de la presente invención, puede ser realizada manualmente o asistida por ordenador. The reference biomaterial can be analyzed, for example, simultaneously or consecutively, together with the problem biomaterial. The comparison described in step c of the method of the present invention can be performed manually or computer assisted.
En ciertas realizaciones del método de la invención, éste se caracteriza porque en la etapa c se compara el valor obtenido para al menos uno de los marcadores adheridos al biomaterial problema donde dicho marcador se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; y CLUS. In certain embodiments of the method of the invention, it is characterized in that in step c the value obtained for at least one of the markers adhered to the problem biomaterial is compared, where said marker is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
Como ya se ha indicado, la muestra biológica de la invención es preferiblemente una muestra de tejido y/o un fluido biológico. En ciertas realizaciones de la invención, la muestra es un fluido biológico. En ciertas realizaciones de la invención la muestra es un fluido biológico comercial. En ciertas realizaciones de la invención, la muestra es un fluido biológico obtenido a partir de un cultivo celular. En ciertas realizaciones de la invención, la muestra es un fluido biológico obtenido a partir de un tejido. En ciertas realizaciones específicas de la invención, la muestra es un fluido biológico obtenido de un tejido óseo. En ciertas realizaciones específicas de la invención, la muestra es un fluido biológico obtenido de un tejido blando. As already indicated, the biological sample of the invention is preferably a tissue sample and/or a biological fluid. In certain embodiments of the invention, the sample is a biological fluid. In certain embodiments of the invention the sample is a commercial biological fluid. In certain embodiments of the invention, the sample is a biological fluid obtained from cell culture. In certain embodiments of the invention, the sample is a biological fluid obtained from tissue. In certain specific embodiments of the invention, the sample is a biological fluid obtained from bone tissue. In certain specific embodiments of the invention, the sample is a biological fluid obtained from soft tissue.
En ciertas realizaciones específicas de la invención, la muestra es suero sanguíneo, más preferiblemente procedente del sujeto al que se le va a implantar el biomaterial en estudio. In certain specific embodiments of the invention, the sample is blood serum, more preferably from the subject to which the biomaterial under study is going to be implanted.
En aquellas realizaciones de la invención en las que el biomaterial está destinado a regeneración de tejido óseo, se consideran los siguientes parámetros como indicativos de una buena capacidad de regeneración del tejido óseo: un nivel de los marcadores SAMP; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; y C1QC de hasta 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia; un valor del marcador CRP inferior al valor de referencia obtenido para el biomaterial de referencia; y ratios entre los valores obtenidos para los marcadores IC1; C4BPA; CFAH; VTNC; y CLUS, y los marcadores C1QB; C1S; C1R; C1QC; y CO3 igual o superior a los valores de referencia obtenidos en el biomaterial de referencia. In those embodiments of the invention in which the biomaterial is intended for bone tissue regeneration, the following parameters are considered as indicative of a good capacity for bone tissue regeneration: a level of SAMP markers; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; and C1QC up to 1.5 times higher than the reference value obtained for the reference biomaterial; a CRP marker value lower than the reference value obtained for the reference biomaterial; and ratios between the values obtained for the IC1 markers; C4BPA; CFAH; NCTV; and CLUS, and the C1QB markers; C1S; C1R; C1QC; and CO3 equal to or greater than the reference values obtained in the reference biomaterial.
En aquellas realizaciones de la invención en las que el biomaterial está destinado a regeneración de tejido blando, se consideran los siguientes parámetros como indicativos de una buena capacidad de regeneración del tejido blando: un valor de los marcadores SAMP; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; y FCN2; inferior al valor de referencia obtenido para el biomaterial de referencia; y un valor de los marcadores IC1 ; CFAH; C4BPA; VTNC; y CLUS igual o superior al valor de referencia obtenido para el biomaterial de referencia. In those embodiments of the invention in which the biomaterial is intended for soft tissue regeneration, the following parameters are considered as indicative of a good capacity for soft tissue regeneration: a value of the SAMP markers; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; and FCN2; lower than the reference value obtained for the reference biomaterial; and a value of IC1 markers; CFAH; C4BPA; NCTV; and CLUS equal to or greater than the reference value obtained for the reference biomaterial.
En el contexto de la presente invención, se dice que la cantidad y/o el nivel del marcador o marcadores de la invención "está(n) aumentado(s)" con respecto a la cantidad y/o al nivel de dicho(s) marcador(es) en el biomaterial de referencia”, cuando la cantidad y/o el nivel de expresión de dicho(s) marcador(es) está elevado (o es superior) en el biomaterial problema con respecto la cantidad y/o al nivel de expresión de dicho marcador(s) en el biomaterial de referencia. De acuerdo con la presente invención, se considera que la cantidad y/o el nivel del marcador(es) en un biomaterial problema está aumentado con respecto a la cantidad y/o al nivel de dicho(s) marcador(es) en el biomaterial de referencia cuando la cantidad y/o el nivel de dicho(s) marcador(es) en el biomaterial problema está aumentado en al menos 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, (n) veces respecto a la cantidad y/o al nivel de expresión de dicho(s) marcador(es) en el biomaterial de referencia. In the context of the present invention, the amount and/or level of the marker(s) of the invention is said to be "increased" relative to the amount and/or level of said marker(s). marker(s) in the reference biomaterial”, when the quantity and/or level of expression of said marker(s) is elevated (or higher) in the problem biomaterial with respect to the quantity and/or level expression of said marker(s) in the reference biomaterial. In accordance with the present invention, the amount and/or level of the marker(s) in a test biomaterial is considered to be increased relative to the amount and/or level of said marker(s) in the biomaterial. of reference when the amount and/or the level of said marker(s) in the biomaterial in question is increased by at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, (n) times with respect to the amount and/or the level of expression of said marker(s) in the reference biomaterial.
Asimismo, en el contexto de la presente invención, se dice que la cantidad y/o el nivel de un(os) marcador(es) "está disminuido" con respecto a la cantidad y/o al nivel de dicho(s) marcador(es) en el biomaterial de referencia cuando la cantidad y/o el nivel de dicho marcadores) está reducido (o es inferior) con respecto a la cantidad y/o al nivel de dicho marcadores) en el biomaterial de referencia. De acuerdo con la presente invención, se considera que la cantidad y/o el nivel de un marcadores) en un biomaterial problema bajo estudio está disminuido con respecto a la cantidad y/o al nivel de dicho marcador(es) en el biomaterial de referencia cuando la cantidad y/o el nivel de dicho marcador(es) en el biomaterial problema está disminuido en al menos 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, (n) veces, respecto a la cantidad y/o al nivel de expresión de dicho marcador(es) en el biomaterial de referencia. En la presente invención los términos “predecir” o "pronosticar" se refieren a la valoración de la probabilidad según la cual un biomaterial es capaz de inducir regeneración en un tejido de un sujeto que va a ser implantado con dicho biomaterial. Also, in the context of the present invention, the amount and/or level of a marker(s) is said to be "decreased" relative to the amount and/or level of said marker(s). es) in the reference biomaterial when the quantity and/or level of said markers) is reduced (or lower) with respect to the quantity and/or level of said markers) in the reference biomaterial. According to the present invention, it is considered that the quantity and/or the level of a marker(s) in a problem biomaterial under study is decreased with respect to the quantity and/or the level of said marker(s) in the reference biomaterial. when the amount and/or the level of said marker(s) in the biomaterial under test is decreased by at least 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.2, 1.3, 1.4, 1.5 , 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, (n) times, regarding the amount and/or the level of expression of said marker(s) in the reference biomaterial. In the present invention, the terms "predict" or "forecast" refer to the assessment of the probability according to which a biomaterial is capable of inducing regeneration in a tissue of a subject that is going to be implanted with said biomaterial.
Como entenderán los expertos en la materia, tal valoración, aunque se prefiere que sea, normalmente puede no ser correcta para el 100% de los biomateriales que se van pronosticar para su capacidad de regeneración. El término, sin embargo, requiere que una parte estadísticamente significativa de los biomateriales se pueda identificar como que es capaz de inducir regeneración o que tiene disponibilidad para serlo. Si una parte es estadísticamente significativa se puede determinar sin más por el experto en la materia usando varias herramientas de evaluación estadística bien conocidas, por ejemplo, determinación de intervalos de confianza, determinación de valores p, prueba t de Student, prueba de Mann-Whitney, etc. Los intervalos de confianza preferidos son al menos el 50%, al menos el 60%, al menos el 70%, al menos el 80%, al menos el 90%, al menos el 95%. Los valores de p son, preferiblemente, 0.2, 0.1, 0.05. As will be understood by those skilled in the art, such an assessment, although preferred, may not typically be correct for 100% of the biomaterials to be predicted for regenerative ability. The term, however, requires that a statistically significant portion of the biomaterials be identified as being capable of or available to induce regeneration. Whether a part is statistically significant can be determined without further ado by those skilled in the art using various well-known statistical evaluation tools, e.g., determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test. , etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p values are preferably 0.2, 0.1, 0.05.
A efectos de la presente invención, el término “osteointegración" se refiere a la capacidad del biomaterial o implante para proporcionar una unión estructural y funcional directa con el tejido óseo mediante la formación de nuevo hueso o de una nueva estructura similar al hueso en la proximidad de la interfaz biomaterial/tejido. La osteogénesis se refiere aquí al proceso de formación y crecimiento del hueso nuevo. También puede asumir la formación respectiva de otros tejidos tales como tejido fibroso o cartílago. A efectos de la presente invención, el término regeneración de tejido blando se refiere a la capacidad del biomaterial, implante o dispositivo médico para permitir que exista una cicatrización entre dispositivo percutáneo y el tejido adyacente que cierre, por ejemplo, la posible entrada de bacterias. For the purposes of the present invention, the term "osseointegration" refers to the ability of the biomaterial or implant to provide a direct structural and functional bond with bone tissue through the formation of new bone or a new bone-like structure in the vicinity. of the biomaterial/tissue interface. Osteogenesis refers here to the process of formation and growth of new bone. It can also assume the respective formation of other tissues such as fibrous tissue or cartilage. For the purposes of the present invention, the term tissue regeneration soft refers to the ability of the biomaterial, implant or medical device to allow a healing between the percutaneous device and the adjacent tissue to close, for example, the possible entry of bacteria.
A efectos de la presente invención, el término “compatible" “compatibilidad", “biocompatibilidad” o “biocompatible”, utilizados indistintamente a lo largo del presente documento, se refiere a la capacidad de un material para estar en contacto con un sistema vivo sin generar un efecto adverso, tóxico o dañino, y adicionalmente desarrollar una actividad específica positiva y beneficiosa en el organismo. El análisis de compatibilidad de un sistema formado por un biomaterial y el tejido con el que se encuentra en contacto, incluye un elevado número de diferentes ensayos in vitro e in vivo que se recogen en ISO10993. For the purposes of this invention, the term "compatible" "compatibility", "biocompatibility" or "biocompatible", used interchangeably throughout this document, refers to the ability of a material to be in contact with a living system without generate an adverse, toxic or harmful effect, and additionally develop a specific positive and beneficial activity in the organism. The compatibility analysis of a system made up of a biomaterial and the tissue with which it is in contact includes a large number of different in vitro and in vivo tests that are included in ISO10993.
A efectos de la presente invención los términos “biomaterial", “material médico”, “dispositivo médico”, “implante" o “prótesis", se utilizan indistintamente a lo largo del presente documento, y hacen referencia a cualquier biomaterial y/o dispositivo que es implantable quirúrgicamente. Generalmente, los implantes se usan en combinación con tejidos u órganos corporales temporalmente, permanentemente o semipermanentemente, para remediar un problema y opcionalmente pueden ser retirados quirúrgicamente o desaparecer por biodegradación dentro del cuerpo. Ejemplos de implantes incluyen vendajes, suturas, grapas, abrazaderas, tela, mallas, redes, huesos artificiales, tomillos, placas óseas, varillas ortopédicas, clavos, implantes de cadera, implantes de rodilla, corazones artificiales, dientes, implantes dentales, catéteres y similares. For the purposes of this invention, the terms "biomaterial", "medical material", "medical device", "implant" or "prosthesis" are used interchangeably throughout the herein, and refer to any biomaterial and/or device that is surgically implantable. Implants are generally used in combination with body tissues or organs temporarily, permanently, or semi-permanently to remedy a problem and may optionally be surgically removed or biodegrade within the body. Examples of implants include bandages, sutures, staples, braces, fabric, mesh, nets, artificial bones, screws, bone plates, orthopedic rods, pins, hip implants, knee implants, artificial hearts, teeth, dental implants, catheters, and the like. .
Los términos “marcador”, “marcadores”, se usan indistintamente a lo largo del presente documento, y hacen referencia a una molécula, o al producto de expresión de un gen, preferentemente proteínas, o a fragmentos y variantes de éstas que muestran cambios sustanciales en una condición determinada y que se pueden usar tanto para la detección, como para el diagnóstico y/o pronóstico de un estado y/o condición de un objeto o individuo. Dichos términos hacen referencia particularmente a la detección y cuantificación de dichos marcadores, para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de biomateriales, tal y como se describe en el presente documento. The terms "marker", "markers", are used interchangeably throughout this document, and refer to a molecule, or the expression product of a gene, preferably proteins, or fragments and variants of these that show substantial changes in a determined condition and that can be used both for the detection, diagnosis and/or prognosis of a state and/or condition of an object or individual. Said terms particularly refer to the detection and quantification of said markers, to predict and/or predict the ability of biomaterials to induce tissue regeneration, as described herein.
En ciertas realizaciones, los cambios en el marcador son cambios en la cantidad de los mismos, así como también, alternativamente, cambios en los niveles de expresión de éstos, respecto a un valor de referencia. El término "expresión", como se usa aquí, se refiere particularmente a la determinación de la cantidad de cada marcador respecto a un valor de referencia. Se apreciará que la cantidad y/o niveles de un marcador se pueden establecer mediante la determinación de los niveles del polipéptido correspondiente, o de polipéptidos generados tras su digestión en el caso de que se utilicen medios proteómicos para su determinación. De forma alternativa, los marcadores peptídicos pueden ser variantes resultantes de modificaciones postraduccionales, incluyendo fragmentos de los mismos. In certain embodiments, the changes in the marker are changes in the amount thereof, as well as, alternatively, changes in the expression levels thereof, with respect to a reference value. The term "expression" as used herein refers particularly to the determination of the amount of each marker relative to a reference value. It will be appreciated that the amount and/or levels of a marker can be established by determining the levels of the corresponding polypeptide, or of polypeptides generated after its digestion in the event that proteomic means are used for its determination. Alternatively, the peptide tags may be variants resulting from post-translational modifications, including fragments thereof.
En ciertas realizaciones, la etapa b del método de la invención comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Protrombina (THRB; UniProtKB P00734); Antitrombina (ANT3; UniProtKB P01008; entry version 252); Kininógeno 1 (KNG1; UniProtKB P01042; entry version 231);; Factor XII (FA12; UniProtKB P00748; entry version 238); Vitamina K dependiente proteína C (PROC; UniProtKB P04070); Vitamina K dependiente proteína S (PROS; UniProtKB P07225); Factor plaquetario 4 (PF4V; UniProtKB P10720); y Factor X (FA10; UniProtKB P00742; entry version 265); donde: In certain embodiments, step b of the method of the invention further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Prothrombin (THRB; UniProtKB P00734); Antithrombin (ANT3; UniProtKB P01008; entry version 252); Kininogen 1 (KNG1; UniProtKB P01042; entry version 231);; Factor XII (FA12; UniProtKB P00748; entry version 238); Protein C dependent vitamin K (PROC; UniProtKB P04070); Protein S dependent vitamin K (PROS; UniProtKB P07225); Platelet factor 4 (PF4V; UniProtKB P10720); and Factor X (FA10; UniProtKB P00742; entry version 265); where:
(vi) un valor de los marcadores THRB; PROC; PROS; y ANT3 al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (vi) a value of the THRB markers; PROC; PROS; and ANT3 at least 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
(vii) un valor de los marcadores KNG1; PF4V; FA12; y FA10 al menos 2 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (vii) a value of the KNG1 markers; PF4V; FA12; and FA10 at least 2 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(viii) un valor de los marcadores THRB; PF4V; KNG1; FA12; y FA10 de hasta 1,5 veces inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; y/o (viii) a value of the THRB markers; PF4V; KNG1; FA12; and FA10 up to 1.5 times lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; me
(ix) un valor del marcador ANT3, PROC; y PROS de hasta 1,5 veces inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; son indicativos de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. (ix) an ANT3 marker value, PROC; and PROS up to 1.5 times lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; are indicative of a good ability to induce tissue regeneration by the biomaterial under study.
Así, en ciertas realizaciones, la etapa b del método de la invención comprende la identificación y cuantificación de la presencia de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que comprende: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; KNG1; PF4V; ANT3; PROC; PROS; FA10; y FA12. Thus, in certain embodiments, step b of the method of the invention comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; KNG1; PF4V; ANT3; PROC; PROS; FA10; and FA12.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que comprende THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; y FA12. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list comprising THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; y FA12. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
De manera similar, en ciertas realizaciones, la etapa b del método de la invención puede comprender también la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Filaggrin-2 (FILA2; UniProtKB Q5D862; entry version 140); Desmoplaquina (DESP; UniProtKB P15924; entry version 246); Fibronectina (FINC; UniProtKB P02751; entry version 266); Desmocolina 1 (DSC1; UniProtKB Q08554; entry version 180); tetranectina (TETN; UniProtKB P05452; entry version 198); y Placoglobina (PLAK; UniProtKB P14923; entry version 214); donde un valor de los marcadores DESP; FILA2; TETN; PLAK; DSC1; y FINC al menos 1,5 veces superior al valor de referenda obtenido para el biomaterial de referenda es indicativo de una buena capaddad de inducdón de regeneración ósea del biomaterial en estudio. Similarly, in certain embodiments, step b of the method of the invention may also comprise the determination and quantification of at least one of the markers selected from the list consisting of: Filaggrin-2 (FILA2; UniProtKB Q5D862; entry version 140); Desmoplakin (DESP; UniProtKB P15924; entry version 246); Fibronectin (FINC; UniProtKB P02751; entry version 266); Desmocolin 1 (DSC1; UniProtKB Q08554; entry version 180); tetranectin (TETN; UniProtKB P05452; entry version 198); and Placoglobin (PLAK; UniProtKB P14923; entry version 214); where a value of the markers DESP; ROW2; TETN; PLAK; DSC1; and FINC at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce bone regeneration of the biomaterial under study.
Por tanto, en ciertas realizadones del método de la invendón, la etapa b del método de la invendón comprende la identificación y cuantificación de la presencia de al menos uno de los marcadores selecdonados de entre cualquiera de la lista que comprende: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. Therefore, in certain embodiments of the method of the invention, step b of the method of the invention comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En dertas realizadones de la invendón, la etapa b del método de la invendón comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores selecdonados de entre cualquiera de la lista que consiste en FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one of the markers selected from any of the list consisting of FILA2; OFF; DSC1; TETN; FINC; and PLAK.
En dertas realizadones de la invendón, la etapa b del método de la invendón comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; y de al menos uno de los marcadores selecdonados de entre cualquiera de la lista que consiste en THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; and from at least one of the markers selected from any of the list consisting of THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En dertas realizadones de la invendón, la etapa b del método de la invendón comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; y de al menos uno de los marcadores selecdonados de entre cualquiera de la lista que consiste en FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and from at least one of the markers selected from any of the list consisting of FILA2; OFF; DSC1; TETN; FINC; and PLAK.
En dertas realizadones de la invendón, la etapa b del método de la invendón comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. De manera adicional, en ciertas realizaciones, la etapa b del método de la invención puede comprender también la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: factor derivado del epitelio pigmentario (PEDF; UniProtKB P36955; entry version 209); alfa-2-glicoproteína rica en leudna (A2GL; UniProtKB P02750; entry version 189); angiopoietina-1 (ANG-1; UniProtKB Q15389; entry version 185); y factor inducible por hipoxia 1-α (HIF-α; UniProtKB Q16665; entry version 239), donde un valor de los marcadores PEDF; A2GL; ANG-1; yHIF-α al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK. Of additionally, in certain embodiments, step b of the method of the invention may also comprise the determination and quantification of at least one of the markers selected from the list consisting of: pigment epithelium-derived factor (PEDF; UniProtKB P36955; entry version 209); leudin-rich alpha-2-glycoprotein (A2GL; UniProtKB P02750; entry version 189); angiopoietin-1 (ANG-1; UniProtKB Q15389; entry version 185); and hypoxia-inducible factor 1-α (HIF-α; UniProtKB Q16665; entry version 239), where a value of PEDF markers; A2GL; ANG-1; yHIF-α at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
Por tanto, en ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que comprende: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. Therefore, in certain embodiments of the method of the invention, step b comprises the identification and quantification of the presence of at least one of the markers selected from any of the list comprising: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; y HIF-α. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones, la etapa b del método de la invención comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Plasminógeno (PLMN; UniProtKB P00747; entry version 255); cadena a del fibrinógeno (PIBA; UniProtKB P02671; entry version 251); inhibidor del activador del plasminógeno- 1 (PAI2; UniProtKB P05120; entry version 212); glicoproteína rica en histidina (HRG; UniProtKB Q6P1 K1 ; entry version 116); y calicreína (KLKB1; UniProtKB P03952; entry version 218), donde un valor de los marcadores PLMN; PIBA; PAI2; HRG y KLKB1 al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. In certain embodiments, step b of the method of the invention further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Plasminogen (PLMN; UniProtKB P00747; entry version 255); fibrinogen a-chain (PIBA; UniProtKB P02671; entry version 251); plasminogen activator inhibitor-1 (PAI2; UniProtKB P05120; entry version 212); histidine-rich glycoprotein (HRG; UniProtKB Q6P1 K1; entry version 116); and kallikrein (KLKB1; UniProtKB P03952; entry version 218), where a value of the markers PLMN; GDPA; PAI2; HRG and KLKB1 at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
Por tanto, en ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. Therefore, in certain embodiments of the method of the invention, step b comprises the identification and quantification of the presence of at least one of the markers selected from any of the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and at least one from markers selected from any of the list consisting of PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1 ; HIF-α; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PLMN; FIBA; PAI2; HRG; y KLKB1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones del método de la invención, el biomaterial en estudio está destinado a regeneración de tejido óseo y la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Apolipoproteína E (APOE; UniProtKB P02649); Lactotransferrina (TRFL; UniProtKB P02788); Estaterina (STATH; UniProtKB P02808); y Peptidyl-prolyl cis-trans isomerasa B (PPIB; UniProtKB P23284), donde un valor de los marcadores APOE; TRFL; STATH; y PPIB al menos 2 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. In certain embodiments of the method of the invention, the biomaterial under study is intended for the regeneration of bone tissue and step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Apolipoprotein E (APOE; UniProtKB P02649); Lactotransferrin (TRFL; UniProtKB P02788); Staterin (STATH; UniProtKB P02808); and Peptidyl-prolyl cis-trans isomerase B (PPIB; UniProtKB P23284), where a value of APOE markers; TRFL; STATH; and PPIB at least 2 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
Por tanto, la etapa b del método de la invención puede comprender la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. Therefore, step b of the method of the invention may comprise the determination and quantification of at least one of the markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: APOE; TRFL; STATH y PPIB. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: APOE; TRFL; STATH and PPIB.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En otras realizaciones del método de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In other embodiments of the method of the invention, step b of the method of the invention comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: APOE; TRFL; STATH y PPIB. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; and of at least one of the markers selected from any of the list consisting of: APOE; TRFL; STATH and PPIB.
En ciertas realizaciones de la invención, la etapa b del método de la invención la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments of the invention, step b of the method of the invention is the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones del método de la invención, el biomaterial en estudio está destinado a regeneración de tejido blando y la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Catepsina B (CATB; UniProtKB P07858; entry version 234); y Calistatina (SERPINA4; UniProtKB P29622; entry version 178), donde un valor de CATB inferior al valor de referencia obtenido para el biomaterial de referencia, y un valor de SERPINA4 inferior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. In certain embodiments of the method of the invention, the biomaterial under study is intended for soft tissue regeneration and step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Cathepsin B (CATB; UniProtKB P07858;entry version 234); and Callistatin (SERPINA4; UniProtKB P29622; entry version 178), where a CATB value lower than the reference value obtained for the reference biomaterial, and a SERPINA4 value lower than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
En ciertas realizaciones del método de la invención, el biomaterial en estudio está destinado a regeneración de tejido blando y la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Insulin-like growth factors (IGF) y/o proteínas de unión a IGF como por ejemplo insulin-like growth factor II (IGF2; UniProtKB - P01344) e insulin-like growth factor-binding protein 4 (IBP4; UniProtKB - P22692); inhibidores de serina proteasas como alpha-2-antiplasmin (A2AP; UniProtKB - P08697), alpha-1-antitrypsin (A1AT; UniProtKB - P01009) e inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3; UniProtKB - Q06033); CYTA; y potenciadores de procolágeno C-endopeptidasa como procollagen C-endopeptidase enhancer 1 (PCOC1; UniProtKB - Q15113); donde cuando estos marcadores muestran un valor superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. In certain embodiments of the method of the invention, the biomaterial under study is intended for soft tissue regeneration and stage b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Insulin-like growth factors (IGF) and/or IGF-binding proteins such as insulin-like growth factor II (IGF2; UniProtKB-P01344) and insulin-like growth factor-binding protein 4 (IBP4; UniProtKB-P22692); serine protease inhibitors such as alpha-2-antiplasmin (A2AP; UniProtKB - P08697), alpha-1-antitrypsin (A1AT; UniProtKB - P01009) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3; UniProtKB - Q06033); CYTA; and procollagen C-endopeptidase enhancers such as procollagen C-endopeptidase enhancer 1 (PCOC1; UniProtKB - Q15113); where when these markers show a value higher than the reference value obtained for the reference biomaterial, it is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
Por tanto, la etapa b del método de la invención puede comprender la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. Therefore, step b of the method of the invention may comprise the determination and quantification of at least one of the markers selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitarnos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOCI. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOCI.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitarnos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the method of the invention, step b comprises identifying and quantifying the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; and from at least one marker selected from any of the list consisting of: THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; and of at least one of the markers selected from any of the list consisting of: ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; and of at least one of the markers selected from any of the list consisting of: PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBP, including within IBPs, but not limited to, IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En otras realizaciones del método de la invención, la etapa b comprende la identificación y cuantificación de la presencia de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1 ; HIF-α; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, I BP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In other embodiments of the method of the invention, step b comprises the identification and quantification of the presence of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; and from at least one marker selected from any of the list consisting of: PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; y de al menos uno de los marcadores seleccionados de entre cualquiera de la lista que consiste en: CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitarnos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; and from at least one marker selected from any of the list consisting of: CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones de la invención, la etapa b del método de la invención comprende la identificación y cuantificación de: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments of the invention, step b of the method of the invention comprises the identification and quantification of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
Un segundo aspecto de la invención se refiere al uso in vitro (“uso de la invención”) de la determinación y cuantificación de la presencia de los marcadores CO5, IC1 , CO3, CFAH, C1R, FCN2 y CLUS, preferiblemente de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS y al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; VTNC; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1; en una muestra biológica aislada obtenida de un sujeto y adherida de manera diferencial a la superficie de un biomaterial médico, para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de dicho biomaterial. A second aspect of the invention refers to the in vitro use ("use of the invention") of the determination and quantification of the presence of CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS markers, preferably CO5 markers , IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1; in an isolated biological sample obtained from a subject and differentially adhered to the surface of a medical biomaterial, to predict and/or predict the ability of said biomaterial to induce tissue regeneration.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; y CLUS. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1.In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. En ciertas realizaciones específicas, el uso de la invención comprende la determinación y cuantificación de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB. In certain specific embodiments, the use of the invention comprises the determination and quantification of markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones, el uso de la invención comprende la determinación y cuantificación de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; ApoE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments, the use of the invention comprises the determination and quantification of at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; ApoE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
Un tercer aspecto de la invención se refiere a un kit (“kit de la invención”) para predecir y/o pronosticar la capacidad de inducción de regeneración de un biomaterial que comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores CO5, IC1 , CO3, CFAH, C1R, FCN2 y CLUS, preferiblemente de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS y al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; VTNC; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. A third aspect of the invention refers to a kit ("kit of the invention") to predict and/or predict the regeneration induction capacity of a biomaterial comprising the antibodies, the reagents, or any combination of the above, capable of to detect and quantify the presence of markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS, preferably markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; y CLUS. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; and CLUS.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; y FA12. En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; y FA12. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; and FA12.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; y PLAK. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; and PLAK.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; y HIF-α. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; and HIF-α.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1.In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; y KLKB1. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; and KLKB1.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH y PPIB.In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH and PPIB.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitarnos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PC0C1. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; Y PC0C1.
En ciertas realizaciones, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitamos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain embodiments, the kit of the invention comprises antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of at least one marker selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; CO8A; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones específicas, el kit de la invención comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presencia de los marcadores: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; VTNC; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF (incluyendo pero sin limitarnos IGF2 y IBP, incluyendo dentro de las IBP, pero sin limitamos, IBP4); A2AP; A1AT; CYTA; ITIH3; y PCOC1. In certain specific embodiments, the kit of the invention comprises the antibodies, the reagents, or any combination of the above, capable of detecting and quantifying the presence of the markers: CRP; SAMP; C1QC; C1QB; CO7; CO5; IC1; CO3; C1S; CFAH; C4BPA; C1R; COBA; FCN2; NCTV; CLUS; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF-binding proteins (including but not limited to IGF2 and IBPs, including within IBPs, but not limited to IBP4); A2AP; A1AT; CYTA; ITIH3; and PCOC1.
En ciertas realizaciones específicas, la determinación y cuantificación de los marcadores de la invención mediante el kit de la invención se lleva a cabo tras contactar el biomaterial problema con una muestra biológica aislada de un sujeto, tal y como se ha descrito aquí, preferiblemente siendo la muestra biológica aislada una muestra de suero. In certain specific embodiments, the determination and quantification of the markers of the invention by means of the kit of the invention is carried out after contacting the problem biomaterial with a biological sample isolated from a subject, as described here, preferably being the isolated biological sample a serum sample.
En otras realizaciones específicas, el kit de la invención además puede incluir, sin ningún tipo de limitación, tampones, soluciones de extracción de proteínas, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado, el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo los métodos de la invención. In other specific embodiments, the kit of the invention can also include, without any type of limitation, buffers, protein extraction solutions, contamination prevention agents, protein degradation inhibitors, etc. On the other hand, the kit can include all the necessary supports and containers for its start-up and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention.
A Io largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps.
BREVE DESCRIPCIÓN DE LAS FIGURAS Fig. 1. A. Fibroblastos L929 cultivados durante 3 días sobre la superficie A (izquierda) y sobre la superficie B (derecha). B. Área celular ocupada por los fibroblastos en una superficie control al ser cultivados sobre la superficie A y la superficie B durante 3 días. BRIEF DESCRIPTION OF THE FIGURES Fig. 1. A. L929 fibroblasts cultured for 3 days on surface A (left) and on surface B (right). B. Cell area occupied by fibroblasts on a control surface when cultured on surface A and surface B for 3 days.
Fig. 2. Actividad ALP de células MC3T3-E1 a 7 y 14 días. Fig. 2. ALP activity of MC3T3-E1 cells at 7 and 14 days.
Fig. 3. Medidas de proliferación celular a 1 , 3 y 7 días. Los resultados se muestran como media ± SE. El asterisco (p ≤ 0,05 (*)) indica diferencias estadísticas. Fig. 3. Measurements of cell proliferation at 1, 3 and 7 days. Results are shown as mean ± SE. The asterisk (p ≤ 0.05 (*)) indicates statistical differences.
Fig. 4. Actividad ALP en HOb a 7 y 14 días. Los resultados se muestran como medias ± SE. Los asteriscos (p ≤ 0.01 (**)) indican diferencias estadísticas. Fig. 4. ALP activity in HOb at 7 and 14 days. Results are shown as means ± SE. Asterisks (p ≤ 0.01 (**)) indicate statistical differences.
Fig. 5. Adhesión de fibroblastos humanos tras 1 día de cultivo en las superficies A (izquierda) y B (derecha). Fig. 5. Adhesion of human fibroblasts after 1 day of culture on surfaces A (left) and B (right).
EJEMPLOS EXAMPLES
A continuación, se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la efectividad del método de la invención. Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness of the method of the invention.
EJEMPLO 1 EXAMPLE 1
Materiales y métodos Materials and methods
Materiales. Se han seleccionado, por un lado, dos muestras: A (muestra de referencia de titanio liso grado 4) y B (muestra con tratamiento combinado que provoca rugosidad), como materiales con propiedades fisicoquímicas muy diferentes y que pueden servir de base para la producción de distintos tipos de implantes percutáneos. Materials. On the one hand, two samples have been selected: A (reference sample of smooth grade 4 titanium) and B (sample with combined treatment that causes roughness), as materials with very different physicochemical properties and that can serve as the basis for the production of different types of percutaneous implants.
Por el otro, se han seleccionado otras dos muestras: la muestra X es la referencia de titanio grado 4 con un tratamiento superficial de granallado y ataque ácido, ampliamente utilizado en prótesis de titanio destinadas a tejido óseo. Y un segundo tipo de muestra, tipo Y, que es una muestra de titanio como la X con un recubrimiento superficial con mayor capacidad osteogénica. Ambas muestras presentan diferencias en cuanto a sus propiedades fisicoquímicas y pueden emplearse como implantes óseos. On the other hand, two other samples have been selected: sample X is the grade 4 titanium reference with a surface treatment of shot blasting and acid attack, widely used in titanium prostheses for bone tissue. And a second type of sample, type Y, which is a titanium sample like X with a surface coating with greater osteogenic capacity. Both samples present differences in terms of their physicochemical properties and can be used as bone implants.
Los materiales se preparan con forma de disco de 12 mm de diámetro para poder realizar los cultivos in vitro al ser dispuestos en el fondo de los pocilios de una placa de cultivo con dimensiones similares. Cultivos celulares. El cultivo celular para la evaluación de A y B se realizó con fibroblastos de ratón (línea L-929) en un medio de cultivo compuesto por DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) suplementado con 1% penicilina/streptomicina (Gibco) y un 10% FBS (Gibco). The materials are prepared in the form of a 12 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions. Cell cultures. Cell culture for the evaluation of A and B was performed with mouse fibroblasts (line L-929) in a culture medium composed of DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/ streptomycin (Gibco) and 10% FBS (Gibco).
Por otro lado, la evaluación in vitro de X e Y se realizó mediante un cultivo celular con osteoblastos de ratón (MC3T3-E1). Durante las primeras 24h, las células fueron cultivadas en un medio compuesto por DMEM (Gibco, Thermo Fisher Scientific, NY, USA) suplementado con 1% penicilina/streptomicina (Gibco) y un 10% FBS (Gibco). Tras este tiempo, el medio fue reemplazado por medio osteogénico (DMEM, 1% penicilina/streptomicina, 10% FBS, 1% ácido ascórbico y 0.21% β-glicerol fosfato. On the other hand, the in vitro evaluation of X and Y was carried out using a cell culture with mouse osteoblasts (MC3T3-E1). During the first 24h, the cells were cultured in a medium composed of DMEM (Gibco, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco) and 10% FBS (Gibco). After this time, the medium was replaced by osteogenic medium (DMEM, 1% penicillin/streptomycin, 10% FBS, 1% ascorbic acid and 0.21% β-glycerol phosphate.
Organización del citoesqueleto de los fibroblastos. Para estudiar el efecto de los materiales en la organización del citoesqueleto de la línea celular L-929, las células fueron sembradas en los materiales con una densidad de 1 x 104 células por cm-2. Después de 24 horas, las muestras se lavaron con PBS y fueron fijadas con un 4% paraformaldehido (PFA) durante 20 minutos a temperatura ambiente. Las células fueron entonces permeabilizadas con 0.1% Triton X-100 durante 5 min e incubadas con Phaloidina (1:100; Abeam, Cambridge, UK) diluida en 0.1% w/v bovine serum albumin (BSA)-PBS durante 1 h a temperatura ambiente. Los núcleos fueron tintados con el medio DAPI (Abeam). La detección mediante fluorescencia fue realizada con un Leica TCS SP8 Confocal Laser Scanning Microscope con 20x aumentos. Las imágenes fueron analizadas con un software LAS X (Leica) y con el Image J software (National Institutes of Health). Organization of the fibroblast cytoskeleton. To study the effect of the materials on the organization of the cytoskeleton of the L-929 cell line, the cells were seeded on the materials with a density of 1 x 10 4 cells per cm -2 . After 24 hours, the samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes at room temperature. Cells were then permeabilized with 0.1% Triton X-100 for 5 min and incubated with Phaloidin (1:100; Abeam, Cambridge, UK) diluted in 0.1% w/v bovine serum albumin (BSA)-PBS for 1 h at room temperature. . Nuclei were stained with DAPI medium (Abeam). Fluorescence detection was performed with a Leica TCS SP8 Confocal Laser Scanning Microscope at 20x magnification. Images were analyzed with LAS X software (Leica) and Image J software (National Institutes of Health).
Ensayo actividad ALP en osteoblastos ALP activity assay in osteoblasts
La actividad de ALP en X e Y se llevó a cabo para evaluar el efecto de las muestras de estudio en la mineralización celular. Las células MC3T3-E1 fueron sembradas en las diferentes muestras en placas NUNC de 24 pocilios (Thermo Fisher Scientific). Tras 7 y 14 días de cultivo, se añadió tampón de lisis (Triton X-100 al 0,2%, Tris-HC1 10 mM, pH 7,2). A continuación, se añadieron a las muestras 100 pL de p-NPP (1 mg mL-1) en el buffer (glicina 50 mM, MgCI2 1 mM, pH 10,5). Después de 2 h de incubación, se midió la absorbantia a 405 nm usando un lector de microplacas. La actividad ALP se obtuvo usando la curva estándar de p-nitrofenol en hidróxido de sodio 0,02 mM. Se normalizó al contenido de proteína obtenido empleando un kit de ensayo Pierce BCA (Thermo Fisher Scientific). ALP activity in X and Y was carried out to evaluate the effect of the study samples on cell mineralization. The MC3T3-E1 cells were seeded in the different samples in 24-well NUNC plates (Thermo Fisher Scientific). After 7 and 14 days of culture, lysis buffer (0.2% Triton X-100, 10 mM Tris-HC1, pH 7.2) was added. Next, 100 pL of p-NPP (1 mg mL -1 ) in buffer (50 mM glycine, 1 mM MgCl2, pH 10.5) were added to the samples. After 2 h of incubation, the absorbance at 405 nm was measured using a microplate reader. ALP activity was obtained using the standard curve of p-nitrophenol in 0.02 mM sodium hydroxide. It was normalized to the protein content obtained using a Pierce BCA assay kit (Thermo Fisher Scientific).
Análisis proteómico de la capa de proteínas adsorbidas. Para el análisis proteómico de la capa de proteínas adsorbidas se siguió el protocolo establecido por Romero- Gavilán et al. [F. Romero-Gavilan, A.M. Sánchez-Pérez, N. Araújo-Gomes, M. Azkargorta, I. lloro, F. Elortza, M. Gurruchaga, I. Goñi, J. Suay, Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo, Biofouling. 33 (2017) 676-689. doi:10.1080/08927014.2017.1356289] y descrito en esta patente. Los materiales fueron incubados durante 3 horas (37 °C, 5 % CO2) en una placa de 24 pocilios (Thermo Fisher Scientific) con 1 mL de suero humano AB plasma (Sigma-Aldrich). Para eliminar las proteínas no adsorbidas, los materiales fueron lavados 5 veces con ddH2O y entonces con un buffer (100 mM NaCI, 50 mM Tris- HCI, pH 7.0). La capa de proteínas adsorbidas a la superficie fue obtenida con un buffer (Thiourea 2M, Urea 7M, 4% Chaps, DTT 200 mM). Proteomic analysis of the adsorbed protein layer. For proteomic analysis of the layer of adsorbed proteins, the protocol established by Romero-Gavilán et al. [F. Romero-Gavilan, AM Sánchez-Pérez, N. Araújo-Gomes, M. Azkargorta, I. Lloro, F. Elortza, M. Gurruchaga, I. Goñi, J. Suay, Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo, Biofouling. 33 (2017) 676-689. doi:10.1080/08927014.2017.1356289] and described in this patent. Materials were incubated for 3 hours (37 °C, 5% CO 2 ) in a 24-well plate (Thermo Fisher Scientific) with 1 mL of human serum AB plasma (Sigma-Aldrich). To remove unadsorbed proteins, the materials were washed 5 times with ddH 2 O and then with buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.0). The protein layer adsorbed to the surface was obtained with a buffer (2M Thiourea, 7M Urea, 4% Chaps, 200 mM DTT).
Se analizaron 4 réplicas independientes de cada superficie y cada replica se obtuvo un u conjunto de 4 capas de proteínas adsorbidas en sus respectivos discos. La concentración total de proteína en el suero fue calculada con un kit de ensayo Pierce BCA (Thermo Fisher). Four independent replicas of each surface were analyzed and each replica obtained a set of 4 protein layers adsorbed on their respective discs. Total serum protein concentration was calculated with a Pierce BCA assay kit (Thermo Fisher).
El análisis de las proteínas fue realizado utilizando un tándem de equipos: espectrómetro de masas con nanoACQUITY UPLC (Waters, Milford, MA, USA) acoplado con un Orbitrap XL (Thermo Electron, Bremen, Germany) tal como fue descrito por Romero-Gavilán et al [misma referencia]. Cada muestra fue analizada por cuadriplicado. El análisis de las proteínas diferencias fue realizado mediante el software PEAKS (Bioinformatics Solutions Inc., Waterloo, Canada). Protein analysis was performed using a tandem of equipment: nanoACQUITY UPLC mass spectrometer (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al. to [same reference]. Each sample was analyzed in quadruplicate. The analysis of the different proteins was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
Análisis estadístico. En la caracterización in vitro se trataron los datos de los ensayos considerando una distribución normal y una varianza equivalente, mediante el software ANOVA con el test Tukey post hoc. Para confirmar los resultados después del test ANOVA se realizó un análisis t-student. Los datos son expresados con el valor medio ± el error standard (SE). Statistic analysis. In the in vitro characterization, the test data were treated considering a normal distribution and an equivalent variance, using the ANOVA software with the Tukey post hoc test. To confirm the results after the ANOVA test, a t-student analysis was performed. The data are expressed as the mean value ± the standard error (SE).
Para el análisis de los datos de proteómica, el test t-student fue realizado para evauar las diferencias en proteínas adsorbidas utilizando el software Progenesis. Las diferencias fueron consideras estadísticamente significativas con un p ≤ 0.05. For proteomics data analysis, the t-student test was performed to assess differences in adsorbed proteins using Progenesis software. The differences were considered statistically significant with a p ≤ 0.05.
Resultados. Results.
Tejido blando. Soft tissue.
Se presenta en la tabla 1 las proteínas encontradas de forma diferencialmente más adsorbidas en la superficie B respecto a la superficie A:
Figure imgf000040_0001
Table 1 shows the proteins found differentially more adsorbed on surface B relative to surface A:
Figure imgf000040_0001
Tabla 1. Nombre y descripción de las proteínas diferencialmente adsorbidas con significancia estadística, ratio entre las cantidades encontradas entre las muestras (B frente a A) y ratio de interés en la invención. Aparecen las proteínas ligadas a la activación de la cascada de inflamación (SAMP, C1S, C1QC, C1R, CO7, C1QB, FCN2, CO3, CO5, CRP), reguladoras de la inflamación (IC1, CLUS, CFAH), relativas a coagulación (THRB, KNG1, ANT3), ligadas al estrés oxidativo (CATB) y regeneración (CYTA). Table 1. Name and description of the differentially adsorbed proteins with statistical significance, ratio between the amounts found in the samples (B versus A) and ratio of interest in the invention. Proteins linked to the activation of the inflammation cascade appear (SAMP, C1S, C1QC, C1R, CO7, C1QB, FCN2, CO3, CO5, CRP), inflammation regulators (IC1, CLUS, CFAH), related to coagulation ( THRB, KNG1, ANT3), linked to oxidative stress (CATB) and regeneration (CYTA).
Tal y como se puede observar en la tabla la superficie B es menos susceptible a que sean adsorbidas en su superficie proteínas ligadas a la inflamación. Adicionalmente la principal proteína ligada al estrés oxidativo se adsorbe de forma muy significativa menos en la superficie B. De igual forma las proteínas ligadas a los procesos de coagulación se adsorben de forma controlada. La evaluación de la influencia de los dos tipos de material (A y B) en la organización del citoesqueleto se realiza mediante tinción de las células con phaloidina (verde) y tinción de los núcleos con DAPI (azul), después de un día de cultivo celular (Figura 1A). Se puede observar que el citoesqueleto de los fibroblastos cultivados sobre el material A presenta una forma globular sin ningún tipo de adhesión al substrato por la no presencia de lamelipodios. Por el contrario, los fibroblastos cultivados sobre el material B sí que presentan múltiples lamelipodios y un aspecto ensanchado, muy diferente al globular encontrado sobre la superficie A. La presencia de estos lamelipodios en las células cultivadas sobre B es fundamental para múltiples procesos biológicos, y en concreto por la capacidad de los fibroblastos con esta conformación de producir colágeno y conseguir la cicatrización de heridas (y en particular en sellado gingival). As can be seen in the table, surface B is less susceptible to proteins linked to inflammation being adsorbed on its surface. Additionally, the main protein linked to oxidative stress is adsorbed significantly less on surface B. Similarly, proteins linked to coagulation processes are adsorbed in a controlled manner. The evaluation of the influence of the two types of material (A and B) in the organization of the cytoskeleton is carried out by staining the cells with phalloidin (green) and staining the nuclei with DAPI (blue), after one day of culture. cell (Figure 1A). It can be seen that the cytoskeleton of the fibroblasts cultured on material A has a globular shape without any type of adhesion to the substrate due to the absence of lamellipodia. On the contrary, the fibroblasts cultured on material B do present multiple lamellipodia and an enlarged appearance, very different from the globular one found on surface A. The presence of these lamellipodia in the cells cultured on B is essential for multiple biological processes, and specifically due to the ability of fibroblasts with this conformation to produce collagen and achieve wound healing (and in particular gingival sealing).
En la figura 1B se ha calculado el área de las células de fibroblastos tanto para la superficie A como para la superficie B (células contenidas en un área control). Se puede observar de forma clara como el área ocupada por los fibroblastos cultivados sobre la superficie B es significativamente superior a la encontrada en los fibroblastos cultivados sobre la superficie A. In Figure 1B, the area of the fibroblast cells has been calculated for both surface A and surface B (cells contained in a control area). It can be clearly seen how the area occupied by the fibroblasts cultured on surface B is significantly higher than that found in the fibroblasts cultured on surface A.
Tal y como se ha descrito en la literatura la regeneración del tejido blando depende de la capacidad de generación de colágeno por parte de los fibroblastos. La generación de colágeno sólo es posible si el fibroblasto se encuentra en una superficie donde no se desencadene un proceso de estrés celular, haya una baja respuesta inflamatoria, asi como una coagulación y fibrinolisis regulada. As has been described in the literature, soft tissue regeneration depends on the ability of fibroblasts to generate collagen. Collagen generation is only possible if the fibroblast is located on a surface where a process of cellular stress is not triggered, there is a low inflammatory response, as well as regulated coagulation and fibrinolysis.
La superficie B presenta los marcadores que de acuerdo con la patente reducen el estrés oxidativo y la inflamación, así como da lugar a una respuesta en coagulación contenida. Es esta superficie B la que tiene una morfología no globular sino ensanchada, con mayores adhesiones focales y mayor área. Serán estos fibroblastos cultivados sobre la superficie B los que generen colágeno y consigan regenerar el tejido blando y la consiguiente cicatrización. Surface B presents the markers that, according to the patent, reduce oxidative stress and inflammation, as well as giving rise to a contained coagulation response. It is this surface B that has a morphology that is not globular but widened, with greater focal adhesions and greater area. It will be these fibroblasts cultivated on surface B that will generate collagen and manage to regenerate the soft tissue and the consequent healing.
Tejido óseo. Woven bone.
Se presenta en la siguiente tabla las proteínas encontradas de forma diferencialmente más adsorbidas en la superficie Y respecto a la superficie X:
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
The following table shows the proteins found to be differentially more adsorbed on surface Y with respect to surface X:
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Tabla 2. Nombre y descripción de las proteínas diferencialmente adsorbida con significancia estadística, ratio entre las cantidades encontradas entre las muestras (X frente a Y) y ratio establecida en la presente invención. Aparecen las proteínas ligadas a la activación de la cascada de inflamación (CO8A, CO5, CO3, C1R, C1S), reguladoras de la inflamación (C4BPA, VTNC, CLUS, CFAH, IC1), relativas a coagulación (A2MG, KNG1, THRB, FA11, PROC, PF4V), y ligadas a osteogénesis (APOE, VTNC). Table 2. Name and description of the differentially adsorbed proteins with statistical significance, ratio between the amounts found in the samples (X versus Y) and ratio established in the present invention. Proteins linked to the activation of the inflammation cascade appear (CO8A, CO5, CO3, C1R, C1S), inflammation regulators (C4BPA, VTNC, CLUS, CFAH, IC1), related to coagulation (A2MG, KNG1, THRB, FA11, PROC, PF4V), and linked to osteogenesis (APOE, VTNC).
Tal y como se puede observar en la tabla, la superficie Y es menos susceptible a que sean adsorbidas en su superficie proteínas ligadas a la inflamación. Adicionalmente, hay una mayor adsorción de proteínas ligadas a osteogénesis y a coagulación. As can be seen in the table, the Y surface is less susceptible to proteins linked to inflammation being adsorbed on its surface. Additionally, there is a greater adsorption of proteins linked to osteogenesis and coagulation.
La evaluación de la influencia de los dos tipos de material (X e Y) en la actividad de ALP, marcador ampliamente utilizado para medir potencial osteogénico en estudios in vitro, se muestra en la Figura 2. The evaluation of the influence of the two types of material (X and Y) on ALP activity, a marker widely used to measure osteogenic potential in in vitro studies, is shown in Figure 2.
En la figura 2 se puede observar como la muestra tipo Y da lugar a una mayor secreción de ALP tanto a 7 como a 14 dias. Esta mayor actividad de ALP observada en las muestras Y puede relacionarse con una capacidad osteogénica superior y una mayor capacidad de mineralización en comparación con la muestra X (referencia Ti). Figure 2 shows how the type Y sample gives rise to a greater secretion of ALP both at 7 and 14 days. This higher ALP activity observed in samples Y may be related to a higher osteogenic capacity and a higher mineralization capacity compared to sample X (reference Ti).
La mayor adsorción de marcadores que según la patente incrementan las propiedades osteogénicas en las muestras Y justifican esta mayor actividad de ALP. The greater adsorption of markers that, according to the patent, increase the properties osteogenic in the Y samples justify this higher ALP activity.
EJEMPLO 2: TEJIDO ÓSEO EXAMPLE 2: BONE TISSUE
La capacidad de osteointegración de implantes destinados a regeneración de tejido óseo depende de las características fisicoquímicas del material. Las características de este conjunto de propiedades condicionan la interacción entre la prótesis y los tejidos/ fluidos circundantes tras su implantación. Así, cada biomaterial, en función de sus propiedades, dará lugar a la formación de una capa de proteínas específica sobre su superficie y estas proteínas características condicionarán a su vez la respuesta biológica del material al influir en la activación y desarrollo de procesos regenerativos claves (reacción inmune, inflamación, coagulación, osteogenesis, fibrinólisis, angiogénesis) que darán lugar a una óptima osteointegración del implante. The osseointegration capacity of implants intended for the regeneration of bone tissue depends on the physicochemical characteristics of the material. The characteristics of this set of properties condition the interaction between the prosthesis and the surrounding tissues/fluids after its implantation. Thus, each biomaterial, depending on its properties, will give rise to the formation of a specific protein layer on its surface and these characteristic proteins will in turn condition the biological response of the material by influencing the activation and development of key regenerative processes ( immune reaction, inflammation, coagulation, osteogenesis, fibrinolysis, angiogenesis) that will lead to optimal implant osseointegration.
Materiales Materials
En este estudio se evaluaron dos tipos de muestras diseñadas para favorecer la regeneración de tejido óseo. Ambas muestras consisten en recubrimientos de borosilicato sol-gel aplicados sobre titanio grado 4 con un tratamiento superficial de granallado y ataque ácido, ampliamente utilizado en prótesis de titanio destinadas a tejido óseo. La diferencia entre las muestras C y D reside en la composición de dichos recubrimientos, que conllevan diferencias en cuanto a sus propiedades fisicoquímicas y dan lugar a distintas respuestas biológicas. In this study, two types of samples designed to favor the regeneration of bone tissue were evaluated. Both samples consist of sol-gel borosilicate coatings applied to grade 4 titanium with a surface treatment of shot blasting and acid attack, widely used in titanium prostheses for bone tissue. The difference between samples C and D lies in the composition of these coatings, which lead to differences in their physicochemical properties and give rise to different biological responses.
Los materiales se preparan con forma de disco de 10 mm de diámetro para poder realizar los cultivos in vitro al ser dispuestos en el fondo de los pocilios de una placa de cultivo con dimensiones similares. The materials are prepared in the form of a 10 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions.
Cultivos celulares Cell cultures
El cultivo celular se realizó con osteoblastos humanos (HOb). Durante las primeras 24h, las células fueron cultivadas en un medio compuesto por DMEM (Gibco, Thermo Fisher Scientific) suplementado con 1% penicilina/streptomicina (Gibco) y un 10% FBS (Gibco). Tras este tiempo, el medio fue reemplazado por medio osteogénico (DMEM, 1% penicilina/streptomicina, 10% FBS, 1% ácido ascórbico y 0.21% β-glicerol fosfato. Proliferación Cell culture was performed with human osteoblasts (HOb). During the first 24h, cells were cultured in a medium composed of DMEM (Gibco, Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Gibco) and 10% FBS (Gibco). After this time, the medium was replaced by osteogenic medium (DMEM, 1% penicillin/streptomycin, 10% FBS, 1% ascorbic acid and 0.21% β-glycerol phosphate. Proliferation
La proliferación celular se analizó mediante MTS (Promega). Las células HOb se sembraron en placas de 48 pocilios a una densidad de 7.5 x 103 células cm-2 y se evaluó la proliferación siguiendo las instrucciones del fabricante después de 1 , 3 y 7 días. La absorbancia se midió a 490 nm en un lector de microplacas Multiskan FC (Thermo Fisher Scientific). Cell proliferation was analyzed by MTS (Promega). HOb cells were seeded in 48-well plates at a density of 7.5 x 10 3 cells cm -2 and proliferation was assessed following the manufacturer's instructions after 1, 3 and 7 days. Absorbance was measured at 490 nm in a Multiskan FC microplate reader (Thermo Fisher Scientific).
Ensayo actividad ALP ALP activity assay
La actividad de ALP se llevó a cabo para evaluar el efecto de las muestras de estudio en la mineralización celular. Los osteoblastos fueron sembrados en las diferentes muestras en placas de 48 pocilios. Tras 7 y 14 días de cultivo, se añadió tampón de lisis (Triton X-100 al 0,2%, Tris-HCI 10 mM, pH 7,2). A continuación, se añadieron a las muestras 100 pl de p-NPP (1 mg mL1) en el buffer (glicina 50 mM, MgCI2 1 mM, pH 10,5). Después de 2 h de incubación, se midió la absorbancia a 405 nm usando un lector de microplacas. La actividad ALP se obtuvo usando la curva estándar de p-nitrofenol en hidróxido de sodio 0,02 mM. Se normalizó al contenido de proteína obtenido empleando un kit de ensayo Pierce BCA (Thermo Fisher Scientific). ALP activity was carried out to assess the effect of study samples on cell mineralization. The osteoblasts were seeded in the different samples in 48-well plates. After 7 and 14 days of culture, lysis buffer (0.2% Triton X-100, 10 mM Tris-HCl, pH 7.2) was added. Next, 100 µl of p-NPP (1 mg mL 1 ) in buffer (50 mM glycine, 1 mM MgCl2, pH 10.5) were added to the samples. After 2 h of incubation, the absorbance at 405 nm was measured using a microplate reader. ALP activity was obtained using the standard curve of p-nitrophenol in 0.02 mM sodium hydroxide. It was normalized to the protein content obtained using a Pierce BCA assay kit (Thermo Fisher Scientific).
Análisis proteómico de la capa de proteínas adsorbidas Proteomic analysis of the adsorbed protein layer
El análisis proteómico de la capa de proteínas adsorbidas se llevó a cabo mediante el protocolo establecido por Romero-Gavilán et al. [F. Romero-Gavilan et al.; Biofouling. 33 (2017) 676-689] y descrito aquí. Los materiales fueron incubados durante 3 h (37 °C, 5 % CO2) en una placa de 24 pocilios (Thermo Fisher Scientific) con 1 ml de suero humano AB plasma (Sigma-Aldrich). Para eliminar las proteínas no adsorbidas, los recubrimientos fueron lavados 5 veces con ddH2O y entonces con un buffer (100 mM NaCI, 50 mM Tris-HCI, pH 7.0). La capa de proteínas adsorbidas a las superficies fue obtenida con un buffer (Thiourea 2M, Urea 7M, 4% Chaps, DTT 200 mM). The proteomic analysis of the adsorbed protein layer was carried out using the protocol established by Romero-Gavilán et al. [F. Romero-Gavilan et al.; Biofouling. 33 (2017) 676-689] and described here. Materials were incubated for 3 h (37 °C, 5% CO 2 ) in a 24-well plate (Thermo Fisher Scientific) with 1 ml human serum AB plasma (Sigma-Aldrich). To remove unadsorbed proteins, the coatings were washed 5 times with ddH 2 O and then with buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.0). The layer of proteins adsorbed to the surfaces was obtained with a buffer (2M Thiourea, 7M Urea, 4% Chaps, 200 mM DTT).
Se analizaron 4 réplicas independientes de cada superficie y de cada replica se obtuvo un conjunto de 4 capas de proteínas adsorbidas en sus respectivos discos. La concentración total de proteína en el suero fue calculada con un kit de ensayo Pierce BCA (Thermo Fisher). Four independent replicas of each surface were analyzed and from each replica a set of 4 protein layers adsorbed on their respective discs was obtained. Total serum protein concentration was calculated with a Pierce BCA assay kit (Thermo Fisher).
El análisis de las proteínas fue realizado utilizando un tándem de equipos: espectrómetro de masas con nanoACQUITY UPLC (Waters, Milford, MA, USA) acoplado con un Orbitrap XL (Thermo Electron, Bremen, Germany) tal como fue descrito por Romero-Gavilán et al [misma referencia]. Cada muestra fue analizada por cuadriplicado. El análisis de las proteínas diferenciales fue realizado mediante el software PEAKS (Bioinformatics Solutions Inc., Waterloo, Canada). Protein analysis was performed using a tandem of equipment: mass spectrometer with nanoACQUITY UPLC (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al [same reference]. Each sample was analyzed in quadruplicate. Differential protein analysis was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
Análisis estadístico Statistic analysis
En la caracterización in vitro se trataron los datos de los ensayos considerando una distribución normal y una varianza equivalente, mediante el software ANOVA con el test Tukey post hoc. Para confirmar los resultados después del test ANOVA se realizó un análisis t-student. Los datos son expresados con el valor medio ± el error standard (SE). Para el análisis de los datos de proteómica, el test t-student fue realizado para evaluar las diferencias en proteínas adsorbidas utilizando el software Pirogénesis. Las diferencias fueron consideras estadísticamente significativas con un p ≤ 0.05. In the in vitro characterization, the test data were treated considering a normal distribution and an equivalent variance, using the ANOVA software with the Tukey post hoc test. To confirm the results after the ANOVA test, a t-student analysis was performed. The data are expressed as the mean value ± the standard error (SE). For the analysis of proteomics data, the t-student test was performed to evaluate the differences in adsorbed proteins using the Pyrogenesis software. The differences were considered statistically significant with a p ≤ 0.05.
Resultados Results
Análisis proteómico Proteomic analysis
Se presentan en la siguiente tabla las proteínas encontradas de forma diferencialmente más adsorbidas en la superficie D respecto a la superficie C:
Figure imgf000046_0001
Figure imgf000047_0001
The proteins found differentially more adsorbed on surface D with respect to surface C are presented in the following table:
Figure imgf000046_0001
Figure imgf000047_0001
Tabla 3. Uniprot ID (HUMAN), nombre y ratio de abundancia del grupo de biomarcadores identificados como diferencialmente adsorbidos en los recubrimientos C y D. Table 3. Uniprot ID (HUMAN), name and abundance ratio of the group of biomarkers identified as differentially adsorbed on coatings C and D.
En la Tabla 3 se muestra cómo el recubrimiento C dio lugar a una mayor adsorción de proteínas asociadas a osteogenesis (TRLF, APOE, PEDF). Este recubrimiento también mostró una mayor afinidad tanto hacía proteínas pro-coagulatorias (KNG1, PF4V, FA12, FA10, HRG, THRB) como por proteínas capaces de regular la activación de este proceso (PROC, PROS, ANT3). A2GL, asociada a un efecto positivo en angiogénesis, y PLMN, proteína clave en el proceso de fibrinólisis, fueron también detectadas como más adsorbidas al recubrimiento C que al D. DESP, TETN, PLAK, FINC, FIBA, asociadas a funciones en adhesión celular, mostraron también una mayor afinidad por la superficie tipo C con ratios de abundancia C/D ≥ 1.5. Table 3 shows how coating C gave rise to a greater adsorption of proteins associated with osteogenesis (TRLF, APOE, PEDF). This coating also showed a higher affinity both for pro-coagulatory proteins (KNG1, PF4V, FA12, FA10, HRG, THRB) and for proteins capable of regulating the activation of this process (PROC, PROS, ANT3). A2GL, associated with a positive effect on angiogenesis, and PLMN, a key protein in the fibrinolysis process, were also detected as more adsorbed to coating C than to coating D. DESP, TETN, PLAK, FINC, FIBA, associated with functions in cell adhesion , also showed a higher affinity for the C-type surface with C/D abundance ratios ≥ 1.5.
Respecto a la respuesta inmune/inflamatoria por parte de los recubrimientos, cabe destacar la presencia de C1R, CO5, FCN2, CO3 en ambas superficies; siendo la ratio C/D ≤ 1.5. Además, se calcularon los ratios entre las proteínas capaces de inhibir la respuesta inmune y proteínas pro-activadoras de esta señal como CO3 y C1R (Tabla 4). Regarding the immune/inflammatory response by the coatings, it is worth noting the presence of C1R, CO5, FCN2, CO3 on both surfaces; being the ratio C/D ≤ 1.5. In addition, the ratios between proteins capable of inhibiting the immune response and pro-activating proteins of this signal, such as CO3 and C1R, were calculated (Table 4).
Figure imgf000048_0001
Figure imgf000048_0001
Tabla 4. Ratios entre C/D del cociente de la abundancia de biomarcadores inhibidores de la respuesta inmune (C4BPA, IC1, CFAH, CLUS) y las proteínas pro-inflamatorias (CO3 y C1R) entre ambos tipos de recubrimientos. Table 4. Ratios between C/D of the quotient of the abundance of immune response inhibitory biomarkers (C4BPA, IC1, CFAH, CLUS) and pro-inflammatory proteins (CO3 and C1R) between both types of coatings.
Tal como se muestra en la Tabla 4, los ratios entre los valores de los marcadores C4BPA, IC1, CFAH y CLUS, y los valores de los marcadores C1R y CO3 para la superficie C son superiores a los valores obtenidos para la superficie D, ya que los cocientes obtenidos son superiores a 1. Este hecho indicaría que una respuesta inmune, presente, pero regulada. As shown in Table 4, the ratios between the values of the markers C4BPA, IC1, CFAH and CLUS, and the values of the markers C1R and CO3 for surface C are higher than the values obtained for surface D, since that the ratios obtained are greater than 1. This fact would indicate that an immune response is present, but regulated.
Ensayos in vitro in vitro tests
Respecto a los resultados de proliferación, los HOb cultivados sobre la superficie tipo C mostraron una significativa mayor capacidad de proliferación a 1 y 7 días, en comparación con D (Figura 3). Regarding the proliferation results, the HOb cultured on the type C surface showed a significantly higher proliferation capacity at 1 and 7 days, compared to D (Figure 3).
El efecto de las superficies C y D en la actividad de ALP, marcador ampliamente utilizado para medir potencial osteogénico en estudios in vitro, se muestra en la Figura 4. The effect of surfaces C and D on ALP activity, a marker widely used to measure osteogenic potential in in vitro studies, is shown in Figure 4.
En la Figura 4 puede observarse cómo los osteoblastos cultivados con el recubrimiento tipo C dan lugar a una mayor actividad de ALP tanto a 7 como a 14 días, en comparación con aquellos cultivados con las muestras D. Esta mayor actividad de ALP puede relacionarse con una mayor capacidad de maduración osteogénica y mineralización por parte de este recubrimiento. Figure 4 shows how osteoblasts cultured with type C coating give rise to higher ALP activity both at 7 and 14 days, compared to those cultured with samples D. This higher ALP activity may be related to a higher ALP activity. greater capacity for osteogenic maturation and mineralization by this coating.
Conclusión conclusion
De este modo, la adsorción diferencial de biomarcadores entre C y D se correlacionan con las diferencias observadas en sus propiedades osteogénicas. El recubrimiento C dio lugar a una mayor adsorción de proteínas asociadas a osteogenesis, angiogénesis, fibrinólisis y coagulación que el material tipo D. Además, la adsorción de proteínas pro- y anti- inflamatorias sobre esta superficie mostraron un ratio óptimo de regulación. A su vez, al comparar las respuestas de células HOb ante ambos recubrimientos, fue el tipo C el material que dio lugar a una mayor capacidad de proliferación y a una mayor actividad de ALP. Thus, the differential adsorption of biomarkers between C and D correlates with the differences observed in their osteogenic properties. Coating C resulted in a higher adsorption of proteins associated with osteogenesis, angiogenesis, fibrinolysis and coagulation than material type D. In addition, the adsorption of pro- and anti-inflammatory proteins on this surface showed an optimal ratio of regulation. In turn, when comparing the responses of HOb cells to both coatings, type C was the material that gave rise to a greater proliferation capacity and greater ALP activity.
EJEMPLO 3: TEJIDO BLANDO EXAMPLE 3: SOFT FABRIC
Materiales y métodos Materials and methods
Materiales. Se evaluaron dos superficies de titanio (Ti) destinadas a ser empleadas para el desarrollo de implantes percutáneos: A (Ti grado 4 con nano texturizado tipo A) y B (Ti grado 4 con nano texturizado tipo B). La aplicación de estos tratamientos conlleva diferencias morfológicas y físico-químicas que van a conllevar distintas respuestas biológicas. Materials. Two titanium (Ti) surfaces intended to be used for the development of percutaneous implants were evaluated: A (Ti grade 4 with nano-texturing type A) and B (Ti grade 4 with nano-texturing type B). The application of these treatments entails morphological and physical-chemical differences that will lead to different biological responses.
Los materiales se preparan con forma de disco de 10 mm de diámetro para poder realizar los cultivos in vitro al ser dispuestos en el fondo de los pocilios de una placa de cultivo con dimensiones similares. The materials are prepared in the form of a 10 mm diameter disk to be able to carry out in vitro cultures by being placed at the bottom of the wells of a culture plate with similar dimensions.
Cultivos celulares. El cultivo celular se realizó con fibroblastos humanos (línea PSC- 201-018) en un medio de cultivo compuesto por DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) suplementado con 1% penicilina/streptomicina (Gibco) y un 10% FBS (Gibco). Cell cultures. Cell culture was performed with human fibroblasts (PSC-201-018 line) in a culture medium composed of DMEM (Gibco, Life Technologies, Thermo Fisher Scientific, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco) and a 10% FBS (Gibco).
Organización del citoesqueleto de los fibroblastos. Para estudiar la capacidad de las superficies de favorecer la adhesión de los fibroblastos humanos, las células fueron sembradas en los materiales con una densidad de 1 x 104 células cm-2. Después de 24 horas, las muestras se lavaron con PBS y fueron fijadas con un 4% paraformaldehido (PFA) durante 20 min. Los fibroblastos fueron tratados con 0.1% Triton X-100 (5 min) e incubados con Phaloidina (1:100; Abeam, Cambridge, UK) diluida en 0.1% w/v bovine serum albumin durante 1 h a temperatura ambiente. Los núcleos fueron marcados con el medio DAPI (Abeam). Las células fueron analizadas mediante fluorescencia con un microscopio confocal Leica TCS SP8. Las imágenes tomadas fueron estudiadas con el software LAS X (Leica) y con Image J software (National Institutes of Health). Análisis proteómico de la capa de proteínas adsorbidas. Para el análisis proteómico de la capa de proteínas adsorbidas se siguió el protocolo establecido por Romero- Gavilán et al. [F. Romero-Gavilan, A.M. Sánchez-Pérez, N. Araújo-Gomes, M. Azkargorta, I. lloro, F. Elortza, M. Gurruchaga, I. Goñi, J. Suay, Biofouling. 33 (2017) 676-689] y descrito aquí. Los materiales fueron incubados durante 3 h (37 °C, 5 % CO2) en una placa de 24 pocilios (Thermo Fisher Scientific) con 1 ml de suero humano AB plasma (Merk). Para eliminar las proteínas no adsorbidas, los materiales fueron lavados 5 veces con ddH2O y a continuación se realizó un lavado adicional con 100 mM NaCI, 50 mM Tris-HCI, pH 7.0. La capa de proteínas adsorbidas a la superficie fue obtenida con un buffer (Thiourea 2M, Urea 7M, 4% Chaps, DTT 200 mM). Organization of the fibroblast cytoskeleton. To study the ability of the surfaces to favor the adhesion of human fibroblasts, the cells were seeded on the materials with a density of 1 x 10 4 cells cm -2 . After 24 hours, samples were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. Fibroblasts were treated with 0.1% Triton X-100 (5 min) and incubated with Phaloidin (1:100; Abeam, Cambridge, UK) diluted in 0.1% w/v bovine serum albumin for 1 h at room temperature. The nuclei were marked with DAPI medium (Abeam). Cells were analyzed by fluorescence with a Leica TCS SP8 confocal microscope. The images taken were studied with LAS X software (Leica) and with Image J software (National Institutes of Health). Proteomic analysis of the adsorbed protein layer. For the proteomic analysis of the adsorbed protein layer, the protocol established by Romero-Gavilán et al. [F. Romero-Gavilan, AM Sánchez-Pérez, N. Araújo-Gomes, M. Azkargorta, I. Lloro, F. Elortza, M. Gurruchaga, I. Goñi, J. Suay, Biofouling. 33 (2017) 676-689] and described here. Materials were incubated for 3 h (37 °C, 5% CO 2 ) in a 24-well plate (Thermo Fisher Scientific) with 1 ml human serum AB plasma (Merk). To remove unadsorbed proteins, the materials were washed 5 times with ddH 2 O followed by an additional wash with 100 mM NaCl, 50 mM Tris-HCl, pH 7.0. The protein layer adsorbed to the surface was obtained with a buffer (2M Thiourea, 7M Urea, 4% Chaps, 200 mM DTT).
Se analizaron 4 réplicas independientes de cada superficie y de cada replica se obtuvo un conjunto de 4 capas de proteínas adsorbidas en sus respectivos discos. La concentración total de proteína en el suero fue calculada con un kit de ensayo Pierce BCA (Thermo Fisher). Four independent replicas of each surface were analyzed and from each replica a set of 4 protein layers adsorbed on their respective discs was obtained. Total serum protein concentration was calculated with a Pierce BCA assay kit (Thermo Fisher).
El análisis de las proteínas fue realizado utilizando un tándem de equipos: espectrómetro de masas con nanoACQUITY UPLC (Waters, Milford, MA, USA) acoplado con un Orbitrap XL (Thermo Electron, Bremen, Germany) tal como fue descrito por Romero-Gavilán et al [misma referencia]. Cada muestra fue analizada por cuadriplicado. El análisis de las proteínas diferenciales fue realizado mediante el software PEAKS (Bioinformatics Solutions Inc., Waterloo, Canada). Protein analysis was performed using a tandem of equipment: nanoACQUITY UPLC mass spectrometer (Waters, Milford, MA, USA) coupled with an Orbitrap XL (Thermo Electron, Bremen, Germany) as described by Romero-Gavilán et al. to [same reference]. Each sample was analyzed in quadruplicate. Differential protein analysis was performed using PEAKS software (Bioinformatics Solutions Inc., Waterloo, Canada).
Para el análisis de los datos de proteómica, el test t-student fue realizado para evaluar las diferencias en proteínas adsorbidas utilizando el software Progenesis. Las diferencias fueron consideras estadísticamente significativas con un p ≤ 0.05. For proteomics data analysis, the t-student test was performed to assess differences in adsorbed proteins using Progenesis software. The differences were considered statistically significant with a p ≤ 0.05.
Resultados Results
Análisis proteómico Proteomic analysis
Tras realizar el análisis proteómico de las capas de proteínas adsorbidas a las superficies A y B, se calcularon las ratios entre valores de abundancia de los biomarcadores claves para valorar la capacidad de regeneración de tejido blando asociados a ambas superficies. Los biomarcadores identificados son mostrados en la Tabla 5.
Figure imgf000050_0001
After performing the proteomic analysis of the protein layers adsorbed to surfaces A and B, the ratios between the abundance values of the key biomarkers were calculated to assess the regeneration capacity of soft tissue associated with both surfaces. The identified biomarkers are shown in Table 5.
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000051_0001
Tabla 5. Uniprot ID (HUMAN), nombre y ratio de abundancia del grupo de biomarcadores identificados como diferencialmente adsorbidos en ambas superficies (p- valor<0.05). Table 5. Uniprot ID (HUMAN), name and abundance ratio of the group of biomarkers identified as differentially adsorbed on both surfaces (p-value <0.05).
Organización del citoesqueleto organization of the cytoskeleton
El efecto de ambos nano-texturizados (A y B) en la organización del citoesqueleto de los fibroblastos humanos se evaluó mediante tinción de las células con phaloidina (verde) y tinción de los núcleos con DAPI (azul) tras un día de cultivo celular (Figura 5). The effect of both nano-textures (A and B) on the organization of the cytoskeleton of human fibroblasts was evaluated by staining cells with phalloidin (green) and staining nuclei with DAPI (blue) after one day of cell culture ( Figure 5).
En las imágenes tomadas mediante el microscopio confocal puede observarse cómo el citoesqueleto de los fibroblastos cultivados sobre la superficie A presentan una forma marcadamente más extendida que aquellos cultivados con el tratamiento B. Además, las células en contacto con la superficie tipo A mostraron más puntos de adhesión al substrato que las cultivadas sobre la superficie B. Esta mayor afinidad y adhesión celular in vitro sobre la superficie tipo A puede asociarse a una mayor capacidad de cicatrización de tejido blando. In the images taken by means of the confocal microscope, it can be observed how the cytoskeleton of the fibroblasts cultured on surface A present a markedly more extended shape than those cultured with treatment B. In addition, the cells in contact with the type A surface showed more points of adhesion to the substrate than those cultured on the B surface. This greater affinity and in vitro cell adhesion on the type A surface may be associated with a greater capacity for soft tissue healing.
Discusión y conclusiones Discussion and Conclusions
Los estudios in vitro concluyeron que la superficie tipo A tiene un mayor potencial de regeneración de tejido blando en comparación con el tipo B, dado que este tratamiento mostró una mayor capacidad de adhesión de fibroblastos humanos, clave en el proceso regenerativo del biomaterial. In vitro studies concluded that type A surface has a greater potential for soft tissue regeneration compared to type B, since this treatment showed a greater adhesion capacity of human fibroblasts, key in the regenerative process of the biomaterial.
Esta mayor capacidad regenerativa de la superficie tipo A se correlaciona con los movimientos en los patrones de absorción de los biomarcadores proteicos reivindicados en la presente invención. This greater regenerative capacity of the type A surface correlates with the movements in the absorption patterns of the protein biomarkers claimed in the present invention.
Así, el análisis proteómico de las capas de proteínas adsorbidas sobre los tratamientos A y B permitieron identificar la presencia de CO5, CO3, C1R, COBA, y FCN2 en ratios de abundancia A/B inferiores a 1, indicando que el tratamiento tipo A mostró una menor afinidad por proteínas asociadas a inflamación. En este sentido, CATB y KAIN fueron detectadas en menor proporción en A que en B (Ratio A/B<1). Thus, the proteomic analysis of the protein layers adsorbed on treatments A and B allowed to identify the presence of CO5, CO3, C1R, COBA, and FCN2 in abundance ratios A/B less than 1, indicating that treatment type A showed decreased affinity for proteins associated with inflammation. In this sense, CATB and KAIN were detected in a lower proportion in A than in B (Ratio A/B<1).
Las proteínas IC1, C4BPA, CFAH y CLUS, asociadas a funciones de regulación de la respuesta inmune/inflamatoria, fueron detectadas más adsorbidas sobre A que sobre B (Ratio A/B>1). La menor adsorción de proteínas asociadas a funciones pro-inflamatorias en la superficie A, unida a esta mayor afinidad por proteínas capaces de inhibir esta respuesta, indicarían que el tratamiento A tiene un potencial antiinflamatorio mayor que B. Además, se detectó una mayor adsorción de las proteínas IGF2, IBP4, A2AP, A1AT, CYTA, ITIH3, PCOC1 en la superficie tipo A (Ratio A/B>1), estando estas moléculas asociadas con funciones clave en el proceso regenerativo de tejido blando. The IC1, C4BPA, CFAH and CLUS proteins, associated with immune/inflammatory response regulation functions, were detected more adsorbed on A than on B (Ratio A/B>1). The lower adsorption of proteins associated with pro-inflammatory functions on surface A, together with this greater affinity for proteins capable of inhibiting this response, would indicate that treatment A has a greater anti-inflammatory potential than B. In addition, a greater adsorption of IGF2, IBP4, A2AP, A1AT, CYTA, ITIH3, PCOC1 proteins was detected on the type A surface (A/B ratio>1), these molecules being associated with key functions in the tissue regenerative process. soft.
DESP, FILA2, TETN, PLAK, DSC1 y FINC, proteínas con funciones clave en adhesión, mostraron una mayor afinidad por la superficie A con ratios de abundancia A/B >1.5. Destaca entre este grupo la adsorción de DSC1 sobre el tratamiento tipo A (con ratio 5.2). DESP, FILA2, TETN, PLAK, DSC1 and FINC, proteins with key functions in adhesion, showed a higher affinity for the A surface with A/B abundance ratios >1.5. Among this group, the adsorption of DSC1 on type A treatment (with ratio 5.2) stands out.
Respecto al grupo de biomarcadores asociados a funciones de coagulación, ANT3, PROC, PROS, THRB, PF4V, KNG1, FA12, y FA10 fueron identificadas tanto en las capas de proteínas adsorbidas a la superficie A como a la B. Este grupo mostró Ratios A/B <1.5. Regarding the group of biomarkers associated with coagulation functions, ANT3, PROC, PROS, THRB, PF4V, KNG1, FA12, and FA10 were identified in both the protein layers adsorbed to surface A and B. This group showed Ratios A /B<1.5.
La regeneración del tejido blando depende de la capacidad de generación de colágeno por parte de los fibroblastos. Para que este proceso se pueda llevar a cabo en presencia de un biomaterial, éste debe mostrar una baja respuesta inmune, así como evitar la activación del estrés celular. Además, la activación de la coagulación en tomo a la prótesis ha de estar controlada. La superficie A además de cumplir estos criterios, mostró una mayor afinidad por proteínas claves en el proceso de adhesión, proliferación y producción de colágeno (en comparación con B). Los resultados de proteómica mostraron una buena correlación entre el efecto de las superficies testadas en la adsorción de proteínas y la respuesta celular, ya que fue el tratamiento tipo A el material que dio lugar a una marcada mayor adhesión de fibroblastos humanos sobre la superficie. Soft tissue regeneration depends on the ability of fibroblasts to generate collagen. For this process to be carried out in the presence of a biomaterial, it must show a low immune response, as well as avoid the activation of cellular stress. In addition, the activation of coagulation around the prosthesis must be controlled. Surface A, in addition to meeting these criteria, showed a higher affinity for key proteins in the process of adhesion, proliferation and collagen production (compared to B). The proteomics results showed a good correlation between the effect of the tested surfaces on protein adsorption and cellular response, since type A treatment was the material that gave rise to a markedly greater adhesion of human fibroblasts on the surface.

Claims

REIVINDICACIONES
1. Método in vitro para predecir y/o pronosticar la capacidad de inducción de regeneración de tejidos por parte de un biomaterial en estudio a partir de una muestra biológica aislada obtenida de un sujeto y adherida de manera diferencial a la superficie de dicho biomaterial, que comprende las siguientes etapas: a. Incubar el biomaterial en estudio con la muestra biológica aislada; b. Determinar y cuantificar la presencia de marcadores adheridos al biomaterial en estudio, donde dichos marcadores son: componente C5 del complemento (CO5), inhibidor de C1 (IC1), componente C3 del complemento (CO3), Factor H del complemento (CFAH), subcomponente C1 r del complemento (C1R), Ficolina-2 (FCN2) y Clusterina (CLUS); y preferiblemente un marcador adicional seleccionado de la lista que consiste en: proteína C reactiva (CRP); componente amiloide P sérico (SAMP); subunidad C del subcomponente C1q del complemento (C1QC); subunidad B del subcomponente C1q del complemento (C1QB); componente C7 del complemento (CO7); subcomponente C1 s del complemento (C1S); cadena > de la proteína de unión a C4b (C4BPA); cadena ■ del componente C8 del complemento (CO8A); y Vitronectina (VTNC); y c. Comparar el valor obtenido en la etapa b. con un valor de referencia obtenido de un biomaterial de referencia; donde el biomaterial en estudio está destinado a la regeneración de un tejido óseo o blando, y donde: 1. In vitro method to predict and/or predict the ability to induce tissue regeneration by a biomaterial under study from an isolated biological sample obtained from a subject and differentially adhered to the surface of said biomaterial, which It comprises the following stages: a. Incubate the biomaterial under study with the isolated biological sample; b. Determine and quantify the presence of markers adhered to the biomaterial under study, where said markers are: component C5 of the complement (CO5), inhibitor of C1 (IC1), component C3 of the complement (CO3), Factor H of the complement (CFAH), subcomponent C1 r of complement (C1R), Phycolin-2 (FCN2) and Clusterin (CLUS); and preferably an additional marker selected from the list consisting of: C-reactive protein (CRP); serum amyloid P component (SAMP); C subunit of the C1q subcomponent of complement (C1QC); complement subcomponent C1q subunit B (C1QB); complement component C7 (CO7); complement subcomponent C1 s (C1S); C4b binding protein > chain (C4BPA); ■ chain of complement component C8 (CO8A); and Vitronectin (VTNC); and c. Compare the value obtained in step b. with a reference value obtained from a reference biomaterial; where the biomaterial under study is intended for the regeneration of bone or soft tissue, and where:
(i) un valor de los marcadores SAMP; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; y C1QC de hasta 1 ,5 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio va destinado a regeneración de tejido óseo; (i) a value of the SAMP markers; CO7; C1QB; C1S; C1R; CO5; FCN2; CO3; and C1QC up to 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(ii) un valor del marcador CRP inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio va destinado a regeneración de tejido óseo; (ii) a CRP marker value lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
(¡ii) ratios entre los valores de los marcadores C4BPA; CFAH; IC1; VTNC; CLUS, y los valores de los marcadores C1QB; C1S; C1R; C1QC; y CO3 iguales o superiores a los valores de referencia obtenidos para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (iii) ratios between the values of the C4BPA markers; CFAH; IC1; NCTV; CLUS, and C1QB marker values; C1S; C1R; C1QC; and CO3 equal to or greater than the reference values obtained for the reference biomaterial when the biomaterial under study is intended for the regeneration of bone tissue;
(iv) un valor de los marcadores SAMP; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; y FCN2; inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; y/o (iv) a value of the SAMP markers; CO5; CO3; C1S; CRP; CO7; C1QB; C1QC; C1R; CO8A; and FCN2; lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; me
(v) un valor de los marcadores IC1; CFAH; C4BPA; VTNC; y CLUS superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando, son indicativos de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. (v) an IC1 marker value; CFAH; C4BPA; NCTV; and CLUS higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration, are indicative of a good ability to induce tissue regeneration by the biomaterial under study.
2. El método de la reivindicación 1, donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Protrombina (THRB); Antitrombina (ANT3); Kininógeno 1 (KNG1); Factor XII (FA12); Vitamina K dependiente proteína C (PROC); Vitamina K dependiente proteína S (PROS); Factor plaquetario 4 (PF4V); y Factor X (FA10); y donde: 2. The method of claim 1, wherein step b further comprises determining and quantifying at least one of the markers selected from the list consisting of: Prothrombin (THRB); Antithrombin (ANT3); Kininogen 1 (KNG1); Factor XII (FA12); Vitamin K dependent protein C (PROC); Vitamin K dependent protein S (PROS); Platelet factor 4 (PF4V); and Factor X (FA10); and where:
(vi) un valor de los marcadores THRB; PROC; PROS; y ANT3 al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (vi) a value of the THRB markers; PROC; PROS; and ANT3 at least 1.5 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for bone tissue regeneration;
(vii) un valor de los marcadores KNG1; PF4V; FA12; y FA10 al menos 2 veces superior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido óseo; (vii) a value of the KNG1 markers; PF4V; FA12; and FA10 at least 2 times higher than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for regeneration of bone tissue;
(viii) un valor de los marcadores THRB; PF4V; KNG1; FA12; y FA10 de hasta 1,5 veces inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; y/o(viii) a value of the THRB markers; PF4V; KNG1; FA12; and FA10 up to 1.5 times lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; me
(ix) un valor del marcador ANT3, PROC; y PROS de hasta 1 ,5 veces inferior al valor de referencia obtenido para el biomaterial de referencia cuando el biomaterial en estudio está destinado a regeneración de tejido blando; son indicativos de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. (ix) an ANT3 marker value, PROC; and PROS up to 1.5 times lower than the reference value obtained for the reference biomaterial when the biomaterial under study is intended for soft tissue regeneration; are indicative of a good ability to induce tissue regeneration by the biomaterial under study.
3. El método de las reivindicaciones 1 o 2, donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Filaggrin-2 (FILA2); Desmoplaquina (DESP); Fibronectina (FINC); Desmocolina 1 (DSC1); Tetranectina (TETN); y Placoglobina (PLAK); donde un valor de los marcadores DESP; FILA2; PLAK; DSC1; TETN; y FINC al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. 3. The method of claims 1 or 2, wherein step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Filaggrin-2 (FILA2); Desmoplakin (DESP); Fibronectin (FINC); Desmocholin 1 (DSC1); Tetranectin (TETN); and Placoglobin (PLAK); where a value of the markers DESP; ROW2; PLAK; DSC1; TETN; and FINC at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
4. El método de cualquiera de las reivindicaciones 1 a 3, donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Factor derivado del epitelio pigmentario (PEDF); Alfa-2-glicoproteína rica en leucina (A2GL); Angiopoietina-1 (ANG-1); y Factor inducible por hipoxia 1-α (HIF-α); donde un valor de los marcadores PEDF; A2GL; ANG-1; y HIF-α al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. 4. The method of any of claims 1 to 3, wherein step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Pigment epithelium-derived factor (PEDF); Leucine-rich alpha-2-glycoprotein (A2GL); Angiopoietin-1 (ANG-1); and Hypoxia Inducible Factor 1-α (HIF-α); where a value of the PEDF markers; A2GL; ANG-1; and HIF-α at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
5. El método de cualquiera de las reivindicaciones 1 a 4, donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Plasminógeno (PLMN); Cadena a del fibrinógeno (FIBA); Inhibidor del activador del plasminógeno- 1 (PAI2); Glicoproteina rica en histidina (HRG); y Calicreína (KLKB1); donde un valor de los marcadores PLMN; FIBA; PAI2; HRG; y KLKB1 al menos 1,5 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. 5. The method of any of claims 1 to 4, wherein step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of: Plasminogen (PLMN); Fibrinogen a-chain (FIBA); Plasminogen activator inhibitor-1 (PAI2); Histidine-rich glycoprotein (HRG); and Kallikrein (KLKB1); where a value of the PLMN markers; FIBA; PAI2; HRG; and KLKB1 at least 1.5 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
6. El método de cualquiera de las reivindicaciones 1 a 5, donde el biomaterial en estudio está destinado a regeneración de tejido óseo y donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Apolipoproteína E (APOE); Lactotransferrina (TRFL); Estaterina (STATH); y Peptidyl-prolyl cis-trans isomerasa B (PPIB); donde un valor de los marcadores APOE; TRFL; STATH; y PPIB al menos 2 veces superior al valor de referencia obtenido para el biomaterial de referencia es indicativo de una buena capacidad de inducción de regeneración de tejidos por parte del biomaterial en estudio. 6. The method of any of claims 1 to 5, where the biomaterial under study is intended for the regeneration of bone tissue and where step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of : Apolipoprotein E (APOE); Lactotransferrin (TRFL); Staterin (STATH); and Peptidyl-prolyl cis-trans isomerase B (PPIB); where a value of the APOE markers; TRFL; STATH; and PPIB at least 2 times higher than the reference value obtained for the reference biomaterial is indicative of a good ability to induce tissue regeneration by the biomaterial under study.
7. El método de cualquiera de las reivindicaciones 1 a 5, donde el biomaterial en estudio está destinado a regeneración de tejido blando y donde la etapa b comprende además la determinación y cuantificación de al menos uno de los marcadores seleccionados de la lista que consiste en: Catepsina B (CATB); Calistatina (SERPINA4); IGF y/o cualquiera de las proteínas de unión a IGF; A2AP; A1AT; CYTA; ITIH3; y PCOC1; donde un valor de IGF y/o cualquiera de las proteínas de unión a IGF; A2AP; A1AT; CYTA; ITIH3; y/o PCOC1 superior al valor de referencia obtenido para el biomaterial de referenda; un valor de CATB inferior al valor de referenda obtenido para el biomaterial de referenda, y un valor de SERPINA4 inferior al valor de referenda obtenido para el biomaterial de referenda, es indicativo de una buena capaddad de inducdón de regeneración de tejidos por parte del biomaterial en estudio. 7. The method of any of claims 1 to 5, where the biomaterial under study is intended for regeneration of soft tissue and where step b further comprises the determination and quantification of at least one of the markers selected from the list consisting of : Cathepsin B (CATB); Callistatin (SERPINA4); IGF and/or any of the IGF binding proteins; A2AP; A1AT; CYTA; ITIH3; and PCOC1; where a value of IGF and/or any of the IGF binding proteins; A2AP; A1AT; CYTA; ITIH3; and/or PCOC1 higher than the reference value obtained for the biomaterial of referendum; a CATB value lower than the reference value obtained for the reference biomaterial, and a SERPINA4 value lower than the reference value obtained for the reference biomaterial, is indicative of a good tissue regeneration induction capacity by the biomaterial in study.
8. El método de cualquiera de las reivindicadones 1 a 7, donde el biomaterial de referencia es titanio. 8. The method of any of claims 1 to 7, wherein the reference biomaterial is titanium.
9. El método de cualquiera de las reivindicadones 1 a 8, donde la muestra biológica es suero. 9. The method of any of claims 1 to 8, wherein the biological sample is serum.
10. Uso in vitro de la determinación y cuantificación de la presenda de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS, preferiblemente de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS y al menos un marcador que se selecciona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; VTNC; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF; A2AP; A1AT; CYTA; ITIH3; y PCOC1 en una muestra biológica aislada obtenida de un sujeto y adherida de manera diferendal a la superfide de un biomaterial médico, para prededr y/o pronosticar la capaddad de inducdón de regeneración de tejidos por parte de dicho biomaterial. 10. In vitro use of the determination and quantification of the presence of the markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS, preferably of the markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least a marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF binding proteins; A2AP; A1AT; CYTA; ITIH3; and PCOC1 in an isolated biological sample obtained from a subject and differentially adhered to the surface of a medical biomaterial, to predict and/or predict the ability of said biomaterial to induce tissue regeneration.
11. Kit para prededr y/o pronosticar la capaddad de inducdón de regeneración de tejidos por parte de un biomaterial que comprende los anticuerpos, los reactivos, o cualquier combinación de los anteriores, capaces de detectar y cuantificar la presenda de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS, preferiblemente de los marcadores CO5, IC1, CO3, CFAH, C1R, FCN2 y CLUS y al menos un marcador que se selecdona de la lista que consiste en: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; VTNC; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; FILA2; DESP; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF y/o cualquiera de las proteínas de unión a IGF; A2AP; A1AT; CYTA; ITIH3; y PCOC1. 11. Kit to predict and/or predict the ability to induce tissue regeneration by a biomaterial comprising antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of markers CO5, IC1 , CO3, CFAH, C1R, FCN2 and CLUS, preferably from the markers CO5, IC1, CO3, CFAH, C1R, FCN2 and CLUS and at least one marker that is selected from the list consisting of: CRP; SAMP; C1QC; C1QB; CO7; C1S; C4BPA; CO8A; NCTV; THRB; ANT3; PROC; PROS; KNG1; PF4V; FA10; FA12; ROW2; OFF; DSC1; TETN; FINC; PLAK; PEDF; A2GL; ANG-1; HIF-α; PLMN; FIBA; PAI2; HRG; KLKB1; APOE; TRFL; STATH; PPIB; CATB; SERPINA4; IGF and/or any of the IGF binding proteins; A2AP; A1AT; CYTA; ITIH3; and PCOC1.
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