ES2602620B1 - IN VITRO METHOD FOR PREDICTING AND / OR FORECASTING THE COMPATIBILITY OF BIOMATERIALS IN A SUBJECT - Google Patents

Abstract

In vitro method for predicting and / or predicting the compatibility of biomaterials in a subject. # The present invention relates to a set of markers, preferably proteins, all related to the complement activation system pathway, which are particularly useful for predict and / or predict the in vitro compatibility of a biomaterial, such as, for example, joint prostheses, dentures, valves, stens, etc., in your subject. Additionally, the present invention comprises an in vitro method for predicting and / or forecasting the compatibility of said materials by determining the peptide profile described in the invention, as well as a kit and / or device for carrying out said method.

Description

The present invention relates to a set of proteins particularly useful for predicting and / or predicting the in vitro compatibility of a biomaterial, used as an implantable medical device, such as any type of prosthesis, such as s / ents, cannulas, step marks , dental implants etc. Additionally, the present invention further comprises an in vitro method for predicting and / or predicting the compatibility of said biomaterials by determining and quantifying the peptide profile adhered to said biomaterials, as well as a kit and / or device for carrying out said method. .

STATE OF THE TECHNIQUE

The success of an implant depends on its biological acceptability, that is, that it produces a minimum damage and a minimum immune reaction in the implanted subject, being necessary for that the first requirement that said implant must meet is its compatibility, and of course, the non-existence of reaction to a foreign body. In this sense, the tests that the biomaterial has to pass to be used as an implant are multiple and complicated, including from in vitro tests against different cell lines (those of the tissue that will be in contact with the implant), to determine its cytotoxicity, cell proliferation, etc., until in vivo tests with those prototypes that have demonstrated good in vitro properties. In addition, preclinical studies and clinical trials in humans are necessary. This whole 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 risks for patients who give their consent to participate in them.

At present, in clinical practice, there is no type of personalized trial that allows to predict and / or predict the success or failure of an implant, whatever type it is, (for example, dental implant, knee prosthesis, hip or various screws in contact with bone, such as spinal stabilizers or junction plates for broken bones, stents, heart valves, dermal implants ...). Of the

Similarly, there is currently no accelerated trial that is capable of predicting and / or predicting the compatibility of a biomaterial, which is being developed by the manufacturers producing implants and / or biomaterials, having to carry out all the described trials previously 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 making biomaterial selection through in vitro experimentation is complex, due to the lack of a good correlation between both types of experimentation, in vitre vs in vivo.

It must be borne in mind that in order to carry out a regeneration process, preferably bone regeneration, certain conditions are required, such as cell signaling, the presence of progenitor cells, as well as the processes of vascularization, scarring, and in the case of tissue Bone osteoinduction. The interaction of the biomaterial-implant surface with the 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 individual's body fluids. 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 resulting in chronic and acute inflammation, high production of macrophages that constitute a response called a foreign body reaction, which in most cases involves implantation failure. Understanding the sequence of biological events that take place after implantation, such as blood clotting processes, the development of infections, the immune response to the foreign body, and finally implant rejection, is crucial in determining the compatibility of a biomaterial, and therefore, of prostheses per se. In this sense, the first layer of proteins adsorbed on the biomaterial and / or implant is responsible for triggering the cellular response to it.

In the literature, different in vitro assays can be found in which the implant is contacted with a sample obtained from an individual and the pattern of adsorbed proteins on the surface of the implants is analyzed to determine their compatibility in the subject. However, apparently in the literature, it has been shown that there is a minimal and very poor correlation between the results obtained in vi / ro with those obtained in vivo, which indicates a clear need for further development of in vitro tests relevant.

In JP4710006B2 a method is described in vitro for evaluating the compatibility of a biomaterial, such as a prosthesis, which comprises bringing said biomaterial into contact with the serum of a subject to which the prosthesis is implanted. The supernatant is then recovered and the presence / expression / differential activity of the lactate dehydrogenase enzyme is analyzed in said supernatant to predict the compatibility of said biomaterial. But this document does not determine or analyze the existence of correlation between the results obtained in vitro with compatibility results in vivo.

In the same way, in vitro patent application US2005170445 describes an in vitro method for determining the compatibility of implant biomaterials by determining and identifying protein expression profiles, specifically cytokines and growth factors, that are associated with the response. from the host organism to the implanted biomaterials. Among the biological samples used to determine the cytokine profile and growth factors that determine the compatibility of the analyzed material are: (i) any fluid collected after the biomaterial comes into contact with tissue of the host organism where such biomaterial has been implanted, or (ii) the supernatant of a cell culture to which the biomaterial has been exposed. The authors of the document also do not analyze the existence of a correlation between the results shown in vitro, with the in vivo compatibility of said material, analyzing the peptide profile they describe.

Müller R. et al. they refer to a method to evaluate the adsorption of proteins on the surface of biomaterials, and assess their compatibility (Müller R. et al. Analytical Biochemistry. 2006. Vol. 359 (2): 194-202). They employ a method based on amino-labeled groups, where the method is coupled to a chemiluminescence reaction for the direct determination of adsorbed proteins on the surface of the analyzed materials. Specifically, they evaluate the adsorption of albumin and fibronectin from human serum and saliva samples. As previously mentioned, in the results shown, there is no indication of any correlation between the results in vitro and the measurement of compatibility of a biomaterial and / or prosthesis in vivo.

The Weber N. et al. (Weber N. the al. Journal of Biomedical Materials Research. 2004, 68 (3): 496-503) describes a rapid technique for determining the compatibility of materials based on the analysis of changes in the polymeric structure of the material of covering of the prosthesis and its influence on the adsorption of fibrinogen on the surface of said biomaterial, concluding that the selective adsorption of plasma proteins in materials in contact with blood, or tissue, affects all subsequent interactions related to compatibility of artificial surfaces. In this study, using fluorescence, a screening technique has been developed using a 384-well format to analyze polymer-fibrinogen interactions and thus predict and / or predict biomaterial compatibility, but as in the cases mentioned previously there is no correlation established between the results shown and the biocompatibility measurement in vivo.

Taking into account the above, there is a need in the state of the art to develop reliable, fast and comparable in v methods, with the results obtained at the in vivo level, which allow to determine the compatibility of biomaterials and / or prostheses covered by said biomaterials, for faster transfer to the clinic.

DESCRIPTION OF THE INVENTION

The inventors of the present invention have identified a profile of markers, preferably protein markers, most of them related to the complement activation system pathway, where their determination and / or quantification is able to predict and / or predict the biocompatibility of biomaterials, preferably implantable biomaterials, and more preferably such as, for example, joint and / or dental prostheses, catheters, etc.

In particular, the inventors have identified and quantified the markers selected from the list consisting of: C-reactive protein (CRP), serum P-amyloid component (SAMP), subunit C of the Clq subcomponent of the complement (C1QC), subunit B of the Clq subcomponent of complement (C1QB), component of complement (COl) and subcomponent C1s of complement (C1S), vitronectin (VTN), Lambda-2 immunoglobulin C chain region (LAC 2) and variable Kappa immunoglobulin 4-1 (KV402), preferably as markers

proteins associated with the prediction and / or prognosis of the compatibility of biomaterials. These proteins are therefore markers of the compatibility of

Biomaterials in a subject. Therefore, the identification and quantification in vitro of the presence of this set of markers, represents a substantial improvement of the current state of the art, since so far no type of protein pattern that can be determined and quantified has been determined in vitro and that predict what result will be obtained in vivo.

Thus, a first object of the invention is the in vitro use of the identification and quantification of the presence of at least one of the markers selected from any of the list consisting of: CRP (SEO ID NO: 1), SAMP (SEO ID NO: 2), C10C (SEO ID NO: 3), C10B (SEO ID NO: 4), C07 (SEO ID NO: 5), C1S (SEO ID NO: 6), VTN (SEO ID NO: 7), LAC 2 (SEO ID NO: 8), KV402 (SEO ID NO: 9) and / or any combination thereof, to predict and / or predict the compatibility of a biomaterial that has been in contact with a biological sample isolated from a subject.

In a particular embodiment of the first object of the invention, this is characterized in that the (105) marker (s) that are identified and quantified are adsorbed to the biomaterial, once it has been in contact with the isolated biological sample of a subject, particularly of a subject to whom 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 said markers adsorbed to the surface of the analyzed biomaterials will determine the compatibility or incompatibility of said biomaterials with the subject to which the biological sample belongs.

For the purposes of the present 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 formed by a biomaterial and the tissue with which it is in contact, includes a large number of different in vitro and in vivo assays that are collected in 18010993.

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 union with bone tissue by forming 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 terms "biomaterial", "medical material», "medical device", "implant" or "prosthesis", are used interchangeably throughout this document, and refer to any biomaterial and / or device which is surgically implantable Generally, implants are used in combination with body tissues or organs temporarily, permanently or semi-permanently, to remedy a problem and can optionally be surgically removed or disappear by biodegradation within the body Examples of implants include bandages, sutures, staples, clamps, fabric, meshes, nets, artificial bones, screws, bone plates, orthopedic rods, nails, hip implants, knee implants, artificial hearts, teeth, dental implants, catheters and the like.

For the purposes of the present invention, the terms "marker", "markers", "biomarker" or "biomarkers" are used interchangeably throughout this document, and refer to a molecule, or the gene expression product , preferably proteins, or fragments and variants thereof that show substantial changes in a given condition and that can be used both for detection, and for the diagnosis and / or prognosis of a state and / or condition of an object or individual. For the purposes of the present invention, said terms refer particularly to the detection and quantification of said biomarkers, to predict and / or predict the compatibility of biomaterials, as described herein.

In a preferred embodiment, changes in the biomarker are changes in the amount thereof, as well as, alternatively, changes in their expression levels, relative to a reference value. The term "expression", as used herein, refers particularly to the determination of the amount of each biomarker with respect to a reference value. It will be appreciated that the

amount and / or levels of a biomarker can be established by determining the levels of the corresponding pOlipypeptide, or of pOlipypeptides

generated after digestion in case proteomic means are used for its determination. Alternatively, peptide biomarkers may be variants resulting from posttranslational modifications, including fragments thereof.

The term "Protein and reactive" or "CRP" refers to a protein encoded by the human "crp" gene that encodes a protein comprising the SEO ID NO: 1 sequence and / or collected in the UniProtKB database with the number P02741.

The term "serum P amyloid component" or uSAMP "refers to a protein encoded by the human" apcs "gene that encodes a protein comprising the SEO ID NO: 2 sequence and / or collected in the UniProtKB database with the P02743.

The term "subunit C of the C1q subcomponent of complement" or "C10C" refers to a protein encoded by the human "c1qc" gene that encodes a protein comprising the SEO ID NO: 3 sequence and collected in the UniProtKB database with the number P02747.

The term "subunit B of subcomponent C1q of complement" or "C10B" refers to a protein encoded by the human "c1qb" gene that encodes a protein comprising the SEO ID NO: 4 sequence and collected in the UniProtKB database with the number P02746.

The term "complement component C7" or "CO?" refers to a protein encoded by the human "cor 'gene that codes for a protein comprising the SEO ID NO: 5 sequence and collected in the UniProtKB database with the number

P10643.

The term "complement C1s subcomponent" or "C1S" refers to a protein encoded by the human "c1s" gene that encodes a protein comprising the SEO ID NO: 6 sequence and collected in the UniProtKB database with the number

P09871.

The term "Vitronectin" refers to a protein encoded by the human "vtn" gene that encodes a protein comprising the SEO ID NO: 7 sequence and / or collected in the UniProtKB database with the number P04004.

The term ~ Lambda-2 immunoglobulin region C chain "or" LAC2 ", refers to a protein encoded by the human" / ac2 "gene that codes for a protein comprising the SEO ID NO: 8 sequence and / or collected in the UniProtK8 database with the number POCG05.

The term "Kappa immunoglobulin variable 4-1" or "KV402" refers to a protein encoded by the human "kv402" gene that encodes a protein comprising the sequence SEa ID NO: 9 and / or collected on the basis of UniProtKB data with the number

P06312.

In a particular embodiment of the first aspect of the invention, this refers to the in vitro use of the identification and quantification of quantity of the set of markers that are selected from the list consisting of: CRP (SEQ ID NO: 1), SAMP ( SEQ ID NO: 2), C1QC (SEQ ID NO: 3), C1QB (SEQ ID NO: 4), COl (SEQ ID NO: 5), C1S (SEQ ID NO: 6), VTN (SEQ ID NO: 7 ), LAC 2 (SEQ ID NO: 8) and KV402 (SEQ ID NO: 9), which have been adsorbed to the biomaterial placed in contact with a biological sample of a subject, to predict and / or predict the compatibility of said biomaterial.

In another particular embodiment, the first aspect of the invention further comprises the identification and quantification of the amount of at least one marker selected from lysia consisting of: alpha chain of the C4b binding protein (C4BPA) (SEQ ID NO: 10), Kininogen-1 (KNG1) (SEQ ID NO: 11), complement C5 (C05) (SEQ ID NO: 12), Plasma protease C1 inhibitor (IC1) (SEO ID NO: 13), H factor complement (CFAH) (SEO ID NO: 14), component C3 of the complement (C03) (SEQ ID NO: 15), subcomponent C1r of the complement (C1R) (SEQ ID NO: 16),

Ficolin-2 (SEQ ID NO: 17), Mannose-binding lectin serine protease 1 (MASP1) (SEO ID NO: 18), and / or any combination thereof.

The term "alpha chain of the C4b binding protein" or "C4BPA" refers to a protein encoded by the human "c4bpa" gene that encodes a protein comprising the SEO ID NO: 1 O sequence and / or collected in the UniProtKB database with the number with sequence P04003.

The term "kinoinogen-1" or "(KNG1)" refers to a protein encoded by the human "kng1" gene that codes for a protein comprising the SEO ID sequence.

NO: 11 and / or collected in the UniProtKB database with the number P01042.

The term "eS complement" or "COS" refers to a protein encoded by the human "cOS" gene that encodes a protein comprising the SEO ID NO: 12 sequence and / or collected in the UniProtKB database with the number P01031.

The term "C1 plasma protease" or "IC1" refers to a protein encoded by the human "serping1" gene that encodes a protein comprising the sequence SEa ID NO: 13 and / or collected on the basis of UniProtKB data with the number P05155.

The term "complement H factor ~ or" CFAH ~ refers to a protein encoded by the human "CFH 'gene encoding a protein comprising the SEO ID NO: 14 sequence and / or collected in the UniProtKB database with The number P08603.

The term "complement component C3" or "C03 ~" refers to a protein encoded by the human "c3" gene that encodes a protein comprising the SEO ID sequence NO: 15 and / or collected in the UniProtKB database with the number

P01024.

The term "C1r subcomponent of complement ~ or" C1R "refers to a protein encoded by the human" c1r "gene that encodes a protein comprising the SEO ID NO: 16 sequence and / or collected in the UniProtKB database with the number

P00736.

The term "Ficolin-2" refers to a protein encoded by the human "FCN2" gene that encodes a protein comprising the SEO ID NO: 17 sequence and / or collected in the UniProtKB database with the number 015485.

The term "mannose binding lectin serine protease 1" or "MASP-1" refers to a protein encoded by the human "MAPS1" gene that encodes a protein comprising the SEO ID NO: 18 sequence and / or collected in the UniProtKB database with the number P48740.

In a preferred embodiment, the first aspect of the invention is characterized by the in vitro use of the identification and quantity quantification of the set of markers that are selected from the list consisting of: CRP (SEa ID NO: 1), SAMP ( SEQ ID NO: 2), C1QC (SEQ ID NO: 3), C1QB (SEQ ID NO: 4), C07 (SEQ ID NO: 5), C1 S (SEQ ID NO: 6), VTN (SEQ ID NO: 7), LAC 2 (SEQ ID NO: 8), KV402 (SEQ ID NO: 9), C4BPA (SEQ ID NO: 10), KNG1 (SEQ ID NO: 11), C05 (SEQ ID NO: 12), IC1 (SEQ ID NO: 13), CFAH (SEQ ID NO: 14), C03 (SEQ ID NO: 15), C1R (SEQ ID NO: 16), Ficolin-2 (SEQ ID NO: 17) and MASP1 (SEQ ID NO: 18).

A second aspect of the invention relates to an in vitro method for predicting and / or predicting the compatibility of a biomaterial, preferably a problem biomaterial, as described herein, in an isolated biological sample obtained from a susceptible subject. of using said biomaterial, where the method comprises the following stages:

to.

Incubate the biomaterial, preferably a biomaterial problem, with the isolated biological sample,

b.

Determine and quantify the presence of at least one of the biomarkers that are selected from the list consisting of: CRP, SAMP, C10C, C10B, C07, C1S, VTN, LAC 2 and KV402, and

C.

Compare the value obtained in step (b) with a reference value obtained

for each biomarker, in a reference biomaterial, where a value of at least twice higher for each of the biomarkers with respect to the value obtained in the reference biomaterial, is indicative that the biomaterial is not compatible with the subject.

In a first stage (step a.), The in vitro method of the invention comprises the incubation, in a biological sample, of the biomaterial problem of which it is intended to determine whether it is compatible or not, with the subject from which the sample was obtained biological

Any biological sample can be used to practice the method of the invention. The term "biological sample" refers to any biological material from a subject that comprises cells. The sample is preferably a sample of tissue and / or a biological fluid. Such a tissue sample may comprise, for example, renal tissue, joint tissue, vascular tissue and / or heart tissue, among others. Biological fluid includes, without limitation, lymph, cerebrospinal fluid (FeS), amniotic fluid, pleural effusion, bone marrow, lymph node, lymph fluid, synovial fluid, a single cell suspension prepared from solid tissue or urine cell lines, saliva , sweat, tears, blood, serum and blood plasma. The sample may be, but is preferably peripheral blood, blood, plasma or serum and more preferably the sample is serum.

Typically, the sample is human in origin, but alternatively, it can come from any other mammalian animal such as farm animals such as horses, cattle, sheep or pigs or they can alternatively be pets such as cats or dogs.

For the purposes of the present invention, the terms "subject", "individual" or "patient", used interchangeably throughout this document, refers to any mammalian animal and includes, but is not restricted to domestic, farm animals, Primates and humans, preferably humans. These terms are not intended to be limiting in any aspect, and these may be of any age, sex and physical condition.

In a particular embodiment, the subject is a subject suffering from a pathology that is susceptible to being 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 at a later stage of the method [step b.J, the markers that adhere to said biomaterial. In a preferred embodiment, the problem biomaterial is incubated with the isolated biological sample of 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.

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 biomarkers described herein, which have adhered to the problem biomaterial are identified or determined. in contact with the isolated biological sample.

In a particular embodiment, step b) of the method of the invention is characterized in that it comprises determining and quantifying the simultaneous presence of the set of

markers comprising: CRP, SAMP, C1QC, C1QB, COl, C1S, VTN, LAC 2 and KV402.

In another more particular embodiment of step b), the method of the invention is characterized in that it further comprises the determination and quantification of at least one of the biomarkers selected from the list consisting of: C4BPA, KNG1, C05, IC1, CFAH , C03, C1R, ficolin-2, MASP-1, and / or any combination thereof.

In another more particular embodiment of step b) the method of the invention is characterized in that it comprises the simultaneous determination and quantification of the set of biomarkers selected from the list consisting of: CRP, SAMP, C1QC, C1QB, COl, C1S, VTN , LAC 2, KV402, C4BPA, KNG1, C05, IC1, CFAH, C03, C1 R, ficolin-2 and MASP-1.

In another alternative embodiment of step b) 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 C4BPA, CFAH and VTN biomarkers with respect to the CRP and SAMP biomarkers. In a more particular embodiment, at least one of the ratios selected from any of the following list is determined: CFAH / CRP; CFAH / SAMP; C4BPAlCRP; C4BPAlSAMP; VTNC / CRP and VTNC / SAMP. In a more preferred embodiment, the set of ratios that are selected from the list consisting of: CFAH / CRP is determined; CFAH / SAMP; C4BPAlCRP; C4BPAlSAMP; VTNC / CRP and VTNC / SAMP. Values of the CFAH / CRP, C4BPAlCRP and VTN / CRP ratios below 25, 7 and 30, respectively, would indicate that the biomaterial does not have biocompatibility characteristics with the patient.

As mentioned above, the determination of the biomarker profile described in the present invention is carried out by identifying and determining the values of said biomarkers that have adhered to the biomaterial problem once it has been in contact with the biological sample of the subject to which said biomaterial will be implanted.

After the period of incubation of the biological sample of the subject with the problem biomaterial, it is preferably subjected to a series of washes to eliminate biomarkers that are not strongly adhered to the biomaterial. In a preferred embodiment said washes are preferably carried out with double distilled water and more preferably, a final wash with a buffer solution preferably comprising sodium chloride at a concentration of between 50-500 mM.

In another more particular embodiment, the markers attached to the problem biomaterial are collected or eluted from said biomaterial, preferably by treating 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 the person skilled in the art and useful to obtain the proteins or biomarkers adhered to said biomaterial.

Then, in another particular embodiment, in said eluates comprising the biomarkers adhered to the biomaterial, the biomarkers that form the protein pertile 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 biomarker 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 semiquantitative or quantitative manner allows differentiating between a compatible biomaterial and an incompatible one. The presence, as well as the amount of the biomarker detected, establishes a differential pertile between compatible and incompatible biomaterials.

The measurement of the quantity or concentration, preferably semiquantitatively or quantitatively, can be carried out directly or indirectly. Direct measurement refers to the measure of the amount and the concentration of the proteins or biomarkers of the invention. Said signal to 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 biomarkers. The indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

The term "quantity" as used in the description refers to, but is not limited to, the absolute or relative amount of the biomarkers of the invention, but also refers to any other value or parameter related thereto or that can be derived from these. Said values or parameters comprise values of signal intensity obtained from any of the physical or chemical properties of said biomarkers, 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 biomarkers of the present invention, associated with the determination of the biocompatibility of a biomaterial, can be carried out by quantifying the levels of the proteins or peptides by conventional methods, known to those skilled in the art, including, but not limited to, luminescent chemo methods, immunohistochemical staining or biochemical detection (eg, immunohistochemical tests), immunological techniques, flow cytometry, immunoprecipitation (or their equivalents for reagents other than antibodies), high efficiency liquid chromatography (HPLC ), mass spectrometry (MS), measurement of plasma resonance absorbance, etc. In a particular embodiment, the quantification of the levels of biomarkers identified in the present invention associated with the compatibility of a biomaterial is performed by immunological techniques, such as ELlSA ("EnzymeLinked Immunosorbent Assay"), Western-blot, RIA (radioimmunoassay) , Competitive EIA (competitive enzyme immunoassay), DAS-ELlSA ("Double Antibody Sandwich ELlSA"), mass spectroscopy, immunocytochemical and immunohistochemical techniques, techniques based on the use of protein or microarray biochips that include specific antibodies or assays based on colloidal precipitation in formats such as dipsticks.

Preferably, the proteins are detected by Western-blot, ELlSA, 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 ELlSA. Likewise, the person skilled in the art will recognize that the present invention relates not only to the biomarkers described in the invention, but also to fragments thereof that could be generated, for example, by alternative processing or splicing phenomena, proteolytic breakdown of said peptides or proteins, etc. Such fragments retain the ability to be used as markers for the determination of the biocompatibility of a medical material as described herein. The biomarkers of the invention, as well as their fragments, may include post-translational modifications, such as phosphorylation, glycosylation, acetylation, isoprenylation, myristoylation, proteolytic processing, etc.

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 values obtained for said markers in a reference biomaterial, which has also been contacted with a biological sample isolated from the subject, where a value of at least twice higher for each of the biomarkers obtained in step b) with respect to the value obtained for said biomarkers in the reference biomaterial, It is indicative that the biomaterial problem is not compatible with the subject.

For the purposes of the present invention the term "biomaterial problem" refers to a biomalerial whose compatibility or incompatibility with a subject is not known.

For the purposes of the present invention the term "reference biomaterial" refers to a biomaterial of which its compatibility with a subject is known, particularly with a mammal and more particularly with a human being. The levels of the markers identified in this invention associated with the compatibility of a biomaterial in the reference sample can be determined in a manner similar to how the levels of said markers are determined in the problem 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 Its compatibility with a subject is known. For the purposes of the present invention, and by way of example, a biocompatible material from which to obtain the reference amount for the biomarkers of the invention may be titanium, preferably of medical grade.

In another preferred embodiment of this aspect of the present invention, the reference amount or value is obtained from a reference biomaterial.

The term "comparison," as used in the description, refers to, but is not limited to, the comparison of the amount of biomarkers as described in the invention, adhered to the problem biomaterial that is grown or incubated together. with the biological sample of a subject, with respect to the amount of the same biomarkers that have adhered to a reference biomaterial also grown 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 biomalerial. The comparison described in step e) of the method of the present invention can be performed manually or assisted by a computer.

In another more preferred embodiment of step e) of the method of the invention, this is characterized in that 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, C10B, COl, C1S, VTN, LAC2 and KV402, preferably the value obtained for the set of said markers is preferably compared with the reference value obtained for said markers in the reference biomaterial.

In another more preferred embodiment of step e) of the method of the invention, this is characterized in that the value obtained in the biomaterial problem for the set of CRP, SAMP, C10C, C10B, COl, C1S, VTN, LAC2 markers is compared , KV402, and also for at least one of the markers selected from any of the list consisting of: C4BPA, KNG1, COS, IC1, CFAH, C03, C1 R, Ficolin-2, MASP-1, more preferably for the set of the markers CRP, SAMP, C10C, C10B, COl, C1S, VTN, LAC2, KV402, C4BPA, KNG1, COS, IC1, CFAH, C03, C1R, Ficolin-2 and MASP-1, with the reference value obtained for said markers in the reference biomaterial.

According to the method of the invention, as described herein, if the level of at least one of the markers described in the invention in the problem biomaterial is increased, preferably at least twice with respect to the level of said marker in the reference biomaterial, then, the biomaterial problem will not be compatible with said subject; and / or if the level of at least one of the markers described in the invention, in the biomaterial problem under study is maintained and / or decreased with respect to the reference level of said marker in the reference biomaterial, then, the material Problem will be supported.

In the context of the present invention, it is said that the amount and / or level of the biomarker or biomarkers of the invention "is (n) increased" with respect to the amount and / or the level of said (s) biomarker (s) in the reference biomaterial », when the quantity and / or level of expression of said biomarker (s) is high (or higher) in the biomaterial problem with respect to the quantity and / or level of expression of said biomarker (s) in the reference biomaterial. In accordance with the present invention, the amount and / or level of the biomarker (s) in a problem biomaterial is considered to be increased with respect to the amount and / or level of said biomarker (s) in the biomaterial. of reference when the quantity and / or level of said biomarker (s) in the biomaterial problem is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30,35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, (n) times, with respect to the quantity and / or level of expression of said (s) biomarker (s) in the reference biomaterial.

Also, in the context of the present invention, it is said that the amount and / or level of a biomarker (s) "is decreased" with respect to the amount and / or level of said biomarker (s) ( is) in the reference biomaterial when the quantity and / or level of said biomarker (s) is reduced (or is lower) with respect to the quantity and / or level of said biomarker (s) in the reference biomaterial. In accordance with the present invention, it is considered that the amount and / or level of a biomarker (s) in a biomaterial problem under study is decreased with respect to the amount and / or level of said biomarker (s) in the biomaterial. of reference when the quantity and / or level of said biomarker (s) in the biomaterial problem is decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30.35, 40, 45, 50.55, 60, 65, 70, 75, 80, 85, 90, 95, 100, (n) times, with respect to the quantity and / or level of expression of said biomarker (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 compatible with respect to the subject to be implanted with said biomaterial.

As those skilled in the art will understand, such an assessment, although preferred, may not be correct for 100% of the biomaterials that are predicted to be compatible. The term, however, requires that a statistically significant part of the biomalerials can be identified as being compatible or available to be. If a part is statistically significant, it can be determined by the person skilled in the art using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test , elc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The values of p are preferably 0.2, 0.1, 0.05.

The third aspect of the present invention relates to a kit for predicting and / or predicting the compatibility of a biomaterial, hereinafter kit of the invention, useful for carrying out the described in vitro method of the invention.

In a preferred embodiment, the kit of the invention comprises the elements necessary to carry out the determination and quantification of at least one of the markers described in the present invention. In a more preferred embodiment of the kit of the invention, it is characterized in that the elements necessary to determine at least one of the markers of the invention are selected from the list consisting of: oligonucleotides, probes, oligo waves, antibodies, reagents, or any combination of the previous ones, which allow to detect and quantify the presence of at least one of the markers selected from the list consisting of: CRP, SAMP, C1QC, C1QB, COl, C1S, VTN, LAC2, KV402, C4BPA, KNG1, C05, IC1, CFAH, C03, C1 R, Ficolin-2, MASP-1, and / or any combination thereof.

In another more preferred embodiment, the kit of the invention comprises the elements necessary to detect and quantify the presence of the set of markers comprising: CRP, SAMP, C1QC, C1QB, COl, C1S, VTN, LAC2 and KV402. In a more preferred embodiment, the kit further comprises the elements necessary to detect and quantify the presence of the markers of the invention that are selected from the list consisting of: C4BPA, KNG1, C05, IC1, CFAH, C03, C1 R, Ficolina-2, MASP-1, and / or any combination thereof.

In another more preferred embodiment, the kit of the invention comprises the elements necessary to detect and quantify the presence of the set of markers comprising: CRP, SAMP, C1QC, C1QB, COl, C1S, C4BPA, KNG1, C05, IC1, CFAH, C03, C1 R, Fieolina-2 and MASP-1.

In yet another more preferred embodiment, the determination and quantification of the markers of the invention, by means of the kit of the invention is carried out after contacting the biomaterial problem with a biological sample isolated from a subject, as described herein, preferably the isolated biological sample being a serum sample.

In another preferred embodiment, the kit of the invention can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention.

Another aspect relates to the use of the kit of the invention, to predict and / or predict the compatibility of a biomaterial with a subject.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Micrographs obtained by scanning electron microscopy (SEM) of the sol-gel hybrid coatings applied on SAE titanium discs: (a) 70% methyltrimethoxysilane (MTMOS) -30% tetraethyl orthosilicate (TEOS) [70M30T], (b) 35% MTMOS -35% 3-glieidoxypropyl-trimethoxysilane (GPTMS) -30% TE OS [35M35G30T],

(e) 50% MTMOS -50% GPTMS [50M50Gl and (d) 50% triethoxyvinylsilane (VTES) -50% GPTMS [50V50Gl. 1 O ~ m calibration bar.

Figure 2. AFM images of the sol-gel hybrid coatings on titanium discs: 70M30T (a), 35M35G30T (b), 50M50G (e) and 50V50G (d). Figure 3. Mechanical profilometry measurements of Ra. The bars indicate the standard deviations. Figure 4. Contact angle results for hybrid sol-gel coatings applied on titanium discs. The bars indicate the standard deviations. Figure 5. Cell viability and in vitro mineralization of mouse osteosarcoma cells (MC3T3-E1). (a) Percentage of cell survival by analyzing the activity of alkaline phosphatase (ALP) (PNP mM / h) normalized with respect to the levels of the amount of tolal protein (1-19 / 1-11) of cultured MC3T3-E1 cells on titanium discs coated with the formulations 70M30T, 35M35G30T, 50M50G and 50V50G. As a positive control, cells grown in the absence of a disc were used and as a negative control, cells grown in the presence of uncoated titanium disc. There were no statistically significant differences between the different formulations in the measured times (ANOVA, p s 0.05). Figure 6. Representative optical microscopy images of histological sections obtained from the in vivo test (EXAKT® Gomori trichrome and cut) after two weeks of implantation of the coated implants: a) 70M30T, b) 35M35G30T, c) 50M50G and d) 50V50G. The area corresponding to fibrous connective tissue (FCT) is delimited by dashed lines. Abbreviations: MAT = material; FCT = fibrous connective tissue; NB = new huesolosteoid. 20x magnification. Figure 7. Area (mm2) occupied by connective fibrous tissue for the implants coated by each of the four coatings. Significant differences were found between the 70M30 / 35M35G30T and 50M50G / 50V50G coatings (p 0.05 ANOVA with a Kruskal-Wallis post-test).

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.

Materials and methods

Titanium discs Titanium discs (12 mm in diameter and 1 mm thick) were manufactured from a commercial grade pure titanium bar of grade 4 (1lerimplant SL). Titanium discs

Shot blasting and with acid treatment (Sandblasted Acid Etching-SAE) were subjected to abrasion with 4 IJm aluminum oxide particles and given the treatment

acid by immersion in sulfuric acid for 1 h to simulate a moderately rough implant surface. The discs were cleaned in acetone, ethanol and purified water of 18.2 O (20 min in each liquid) in an ultrasonic bath and vacuum dried. Finally, all titanium discs were sterilized using UV radiation.

Sol gel synthesis and sample preparation Hybrid silicon coatings were obtained through the sol-gel method. The precursors used were: methyltrimethoxysilane (MTMOS), 3-glycidoxypropyltrimethoxysilane (GPTMS), tetraethyl orthosilicate (TEaS) and triethoxyvinylsilane (VTES) (Sigma-Aldrich). Four different compositions with different molar percentages were synthesized: 70% MTMOS -30% TEOS (70M30T), 35% MTMOS -35% GPTMS -30% TEOS (35M35G30T), 50% MTMOS -50% GPTMS (50M05G) and 50% VTES -50% GPTMS (50V50G). As solvent 2-propanol was used in a volume ratio of 1: 1 alcohol / siloxane. Hydrolysis of the alkoxysilanes was carried out by adding the corresponding stoichiometric amount of acidified aqueous solution at a rate of one drop per second. The solution was kept 1 h under stirring and then 1 h at rest. Samples were prepared immediately after this time. SAE titanium discs were used as a substrate. The coating was obtained using a dip coater (KSV Instrument-KSV DC). The disks were immersed in the corresponding sol-gel solution at a speed of 60 cm / min, for 1 min, extracted at a speed of 100 cm / min. Finally, the samples were cured for 2 h, at a temperature of 80 ° C for 70M30T and 35M35G30T coatings, and at a temperature of 140 ° C for 50M50G and 50V50G coatings.

Physical-chemical characterization of coated titanium discs. The contact angle was measured using an automatic contact angle meter (Dataphysics OCA 20). 10 JI of ultrapure water were deposited on the sol gel coated surface at a dosing rate of 27.5 IJUs at room temperature. Contact angles were determined with SeA software

20. Five discs of each biomaterial were studied by depositing 2 drops in each of them. The surface topography of the coated titanium discs was characterized using atomic force microscopy (AFM) under dry conditions. The measurements were carried out at a scan size of 60 IJm with a scan speed of 1 Hz. To determine the roughness a mechanical perkillometer Dektack 6M (Veeco) was used. Two coated discs of each composition were tested. Three measurements were made for each disk, to obtain the average value of the Ra parameter. The coatings were studied by SEM with a Leica-Zeiss device under vacuum. A platinum coating was applied to make the samples more conductive for SEM observation.

Rehearsals in vi / ro

Cell cultures

MC3T3-E1 cells (mouse osteosarcoma) were grown on sol-gel coated titanium discs, at a concentration of 1x104 cell / well in DMEM medium (Dulbecco's modified Eagle's medium) with red phenol (Gibco-Life Technologies, Grand Island, NY , USA) with 1% 100x penicillin / streptomycin (Biowest Inc., USA) and 10% Fetal Bovine Serum (FBS) (Gibco-Life Technologies, Grand Island, NY, USA). After an incubation period of 24 h at 3rC in a humid atmosphere of 95% and 5% CO2, the medium was replaced by an osteogenic medium consisting of DMEM with 1x penicillin / streptomycin red phenol, 10% FBS, 1% ascorbic acid (5mg mL -1) and 0.21% j3-glycerol phosphate and incubated again under the same conditions. The culture medium was changed every 48 h. In each plate, cells at the same concentration mentioned above were used as control of the culture conditions.

Cytotoxicity

The cytotoxicity of the biomaterial was determined using ISO 10993-5 by spectrophotometry, by indirect contact of the material extract with the cell line. The 96-Cell Titter Proliferation Assay (Promega®) cell assay was used to measure cell viability after 24 hours of incubation of the cells with the extract. A negative control (an empty well) and a positive control (latex, known to be toxic to cells) were used. 70% was considered as the limit below which a biomaterial is considered cytotoxic.

ALP activity

The activity of ALP (alkaline phosphatase) was determined following the conversion protocol of p-nitrophenyl phosphate (p-NPP) to p-nitrophenol. 0.1 mL aliquots were used to carry out the test. 100 ~ L of p-NPP (1mg mL-1) in buffer (50 mM glycine; 1mM MgCb pH 10.5) was added to 100 µL of the supernatant resulting from the lysate. After 2 h of incubation in the dark (37 ° C, 5% CO2), absorbance was measured spectrophotometrically in a microplate reader at a wavelength of 405 nm. The activity of the ALP was determined from a standard curve obtained from different solutions of p-nitrophenol and 0.02 mM sodium hydroxide. The results were presented as mmol of p-nitrophenol / hour (mM PNP h-1), and the data were expressed as normalized ALP activity for the total protein content (1-I9 / 1 ..1I) with the kit Pieree SeA (Thermofisher) test after 7 and 14 days.

Analysis of the adsorbed or adhered biomarkers on the surface of the problem biomalerials to determine their compatibility Titanium discs coated with the different sol-gel compositions analyzed in the examples included herein (4 discs of each biomaterial) were incubated on a plate 24 wells for 180 minutes in a humidified atmosphere (37 oC, 5% C02), after adding 2 mL of human serum (male serum, serotype AB, Sigma-Aldrich). After this time, the serum was removed in order to determine the adsorbed / adhered proteins to the analyzed biomaterials. To do this, the discs were washed five times with double distilled water; after which a final wash was carried out with 100 mM NaCI, 50 mM Tris-HCI pH 7.0. After performing such washes, markers that were not well adhered will be removed from the discs. On the other hand, the proteins that remained adhered to the analyzed biomalerial, forming a film on said biomaterial, were collected by means of a treatment of the biomaterial superstition with a solution of 4% 100 mM SDS DTT 0.5M in TEAB (Triethylammonium bicarbonate buffer solution ). Four elutions were performed for each of the biomaterials analyzed and the four elutions of each biomaterial were mixed to give a replica of each biomaterial, which was repeated three more times to obtain a total of four samples per biomaterial (quadrupled). The total protein content for each of the replicas was quantified colorimetrically (Pierce BCA assay kit (Thermofisher® », the representative value being 51 mg / mL.

Proteomic Analysis The eluted samples obtained above were resolved on a 10% polyacrylamide gel, using a Mini-Protean 11 electrophoresis cell (Bio-Rad®). A constant voltage of 150 V was applied for 45 minutes. The gel was fixed and stained with SYPRO Ruby stain (Bio-Rad®) following the manufacturer's instructions. The gels were washed with water and each street was cut into four bands. Each band was

digested following a standard digestion protocol (Anitua E. el al., J. Tissue Eng. Regen. Med .; 2015; 9: E1-12). The resulting peptides were resuspended in 0.1% by volume of toric acid and analyzed in a nano-scale liquid chromatography system coupled online to tandem mass spectrometry (LC-MS). The separation of the peptides was performed on an ACQUITY UPLC nano chromatograph (Waters) connected to a SYNAPT G2S spectrometer (Waters). The samples were desalted on a Symmetry 300 C18 UPLC Trap column (5 IJm, 180¡1m x 20mm, Waters) connected to an analytical column at BEH130 C18 column (1.7¡Jm, 75 IJm) (200mm, Waters) The column was equilibrated in 3% acetonitrile in 0.1% formic acid. The peptides were eluted at a flow of 300 nLlmin in a linear gradient of 3% -50% acetonitrile.

The Synapt G2S (Waters) spectrometer was used for the analysis of biomarkers or proteins. All acquisitions were made in electro spray positively. The data were corrected with an internal control called 'lock-spraY' using the monoisotopic value of the double-charged form of [Glu1] -fibrinopeptide B. The data acquisition was performed with high mass accuracy and in HDDA mode, which allows increase in signals due to the use of ionic mobility separation.

Progenesis LC-MS (Nonlinear Dynamics) software was used for differential proteomic analysis. The raw raw data files were imported into the program, and one of the samples was selected as reference chromatography against which the other chromatographic strokes were aligned. The abundance relationships between runs were calculated for each ~ value "at a certain retention time. The peak lisr containing the peptides detected in all samples was confronted by the Mascot search engine (Matrix Science). The tolerance for the searches was as follows: 10 ppm for the precursor, and 0.2 Da for the fragments. Cysteine carbadomethylation was selected as a fixed modification, and the oxidation of methionines as a variable modification. Proteins identified with at least two peptides and an FDR (Fa / se Discovery Rafe) <1% were considered for further analysis. Finally, the differentially eluted protein data in each of the biomaterials of analysis were included in the DAVID database, a bioinformatics tool for data annotation, visualization and integration in order to obtain the information of the "related protein nodes" .

In vivo essays

In order to evaluate the histological response of the biomalerials or selected coatings tested, implants coated with said biomaterials were surgically placed in the tibia of New Zealand rabbits (Oryctolagus cunicuJus). This implant model in rabbit tibia is widely described in literature for osseointegration of implants (Mori H, al., J. Oral Maxillofac. Surg. 1997; 55: 351-61). All these studies were carried out according to the protocols of the Ethical Committee of the University of Murcia in Spain and according to the European guidelines and the legal conditions reflected in the R.D. 223/1988 of March 14 and the Order of October 13, 1988 of the Spanish law on the protection of animals used for experimentation and other scientific purposes. Specifically, the rabbits were kept for 12 hours in cyclic light-dark conditions at room temperature of 20 ° C and a relative humidity between 45 and 65%. The animals were caged individually and fed a standard diet and filtered water ad libidum. The implants were supplied by Ilerimplant SL (Spain) The implants were internally connected in grade IV titanium of the GMI dental implants brand, 3.75 mm in diameter and 8 mm long Frontier model. A total amount of 40 of these implants was used, 20 uncoated as a control and 5 coated with each of the aforementioned biomaterials to test their compatibility. Both control and test samples were implanted under the same conditions and the results were compared.

A total of 20 rabbits between 2,000 and 3,000 9 in weight were used, corresponding to ages close to the physical closure, indicative of a bone volume suitable for the study. The period of implantation of the experimental model was 2 weeks. This time was chosen in order to obtain a complete osseointegration process. The implants were inserted in the proximal region of the left tibia. and right, so that each animal had a total of 2 implants, one as a control and the other as a biomaterial problem to be tested. The animals were sedated by intramuscular administration (1M) of Clopromazine Hydrochloride (10 mg / kg). Antibiotic prophylaxis (Amoxicillin) was administered and prepared for the intervention. In the operating room, anesthetic induction was performed by 1M injection of Ketamine Hydrochloride 50 mg / kg (lmalgene®, Merial, Georgia, USA), the maintenance schedule was "on demand" by administration of 20 mg / kg, via 1M at the slightest sign of agitation of the animal. After the 15 mm skin incision, the tibia was de-periostized and the implantation area was marked with a graphite tip. Then the bone defect was performed by successive milling (2.0, 2.8 and 3.2 mm in diameter), with low speed micromotor and continuous irrigation of physiological serum. After washing, the root prostheses were implanted by press-fit (hand pressure placement). The wound was closed by planes, cleaned with saline solution and covered with plastic spray dressing (Nobecutan, Inibsa Laboratories, Barcelona, Spain). After the different test periods, the animals were euthanized by inhalation of carbon monoxide, to extract the screws and study the surrounding tissues.

Histological quantification

Samples for histological examination were processed following the methodology described by Peris JL. et al. (Peris JL, et al. Rev. Spanish Osteoartic Surgery. 1993; 28: 231-8). Briefly, the samples were embedded in methyl methacrylate and by means of the EXAKT cutting technique (EXAKT Technologies, Inc., Oklahoma, USA) 25-30 mm thick samples were obtained. For the examination by optical microscopy, all sections were stained with a Gomori Trichrome solution. The ROl (Regían of Interest) was delimited with the ImageJ software, expressing the area occupied by the fibrous capsule (Fe) in mm2, for n = 4.

Statistic analysis

Data were analyzed by one-factor analysis of variance (ANOVA) and Newman-Keuls multiple comparison, if appropriate. Differences with p s 0.05 were considered statistically significant.

Example 1. Synthesis and physical-chemical characterization of the biomaterials used as implant coatings

Once the coatings were synthesized with the biomaterials analyzed in the present invention, the titanium discs were coated and their physical-chemical analysis and characterization proceeded. Four different and homogeneous sol-gel coating biomaterials have been analyzed: 70M30T, 35M35G30T, 50M05G and 50V50G, as can be seen in the SEM micrographs (Fig. 1). Figure 1 shows the partial roughness coverage of the SAE treated titanium, provided by the different coatings. The 70M30T biomaterial (Fig. 1a) shows a higher conservation level of the initial topography. In the AFM images it is clearly observed that the coating with the 70M30T biomaterial has a greater roughness (Fig. 2a). However, the coatings with the biomaterials 50M50G and SOV50G show a smoother topography surface (Fig.2c and 3d) since the initial irregularity of the substrate is mostly covered by the coatings. In contrast, biomaterial 35M35G30T shows an intermediate aspect (Fig. 2b).

The measurements with the mechanical profilometer (Fig. 3) Dektack 6M (Veeco) reveal that the 70M30T coating has a significantly higher Ra value compared to the biomaterials 35M35T30G, 50M50G and 50V50G. This result may be due to the presence of GPTMS in the last biomaterials that could finally affect the thickness of the coating and therefore the roughness. The contact angle measurements (FigA) obtained with the automatic Dataphysics OCA 20 contact angle meter show that the coatings with the TEaS precursor {organically unmodified precursor), 70M30T and 35M35G30T have the most hydrophilic surfaces, while 50M50G and 50V50G they show a greater contact angle, probably, as a result of the higher organic content in their compositions. All these variations should affect the biological response and therefore the compatibility of said biomaterials.

Example 2. Analysis of the compatibility of biomaterials in in vitro cultures.

Said analysis was carried out in in vitro cultures of MC3T3-E1 cells (mouse osteosarcoma) that were cultured in the presence of titanium discs coated with the biomaterials analyzed in the present invention, 70M30T, 35M35G30T, 50M50G and 50V50G. Regarding cell viability, none of the biomaterials tested showed cytotoxicity (Fig. 5a). Nor do measures of ALP enzyme activity show significant differences between the materials tested (Fig. 5b). However, it is clear that while the ALP activity of the empty well decreases between day 7 and day 14, all biomaterials and even control titanium appear to increase or maintain differentiation and metabolic processes of osteoblast cells.

The results in vi / ro show that, a priori, all the materials analyzed in the present invention would be combatible and would not show an inflammatory or immune response.

Example 3. Analysis of the in vivo compatibility of the biomaterials of the invention.

To corroborate whether there was a correlation to determine the compatibility of a biomaterial between the results obtained in vitro versus the results obtained in vivo, the inventors implanted in the tibia of a rabbit animal model the implants coated with the biomaterials analyzed in the present invention (see materials and methods).

The formation of the fibrous capsule surrounding the implant is contemplated as a natural immune response to the presence of the biomaterial. Therefore, the first layer of proteins that adheres can induce the immediate response of the host organism to the biomalerial.

The results have shown that there is a zone, in the samples obtained from the implants coated with the 50V50G and 50M50G biomaterials clearly covered by fibrous connective tissue (FCT) (Fig. 6c and 6d). Said fibrous tissue is found between the biomaterial (MAT) and the new bone or osteoid (NB) in said biomaterials 50V50G and 50M50G (Fig. 6c and 6d). These results show that said biomaterials are not biocompatible with the subject since they induce a rather potent inflammatory and immune response in the subject that has been implanted with them.

Analyzing the area (mm2) of the fibrous capsule (FCT) obtained in the samples of the rabbits analyzed, no significant differences were observed between the biomaterial 35M35G30T and 70M30T, also showing that the area values of the fibrous capsule are greatly reduced for said biomaterials, while in the case of the 50M50G and 50V50G biomaterials the area values shown are very high with respect to the 35M35G30T and 70M30T biomaterials (Fig. 7). Additionally, and as observed in said Fig. 7, there are statistically significant differences between the fibrous capsule formed by biomaterials 35M35G30T and 70M30T with respect to that formed by biomaterials 50M50G and 50V50G.

These in vivo results show that, contrary to what was observed in the in vitro experiments, the coatings with the biomaterials 50M50G and 50V50G proved not to be compatible with the subject, while the coatings with the biomaterials 35M35G30T and 70M30T, yes that They showed positive results in terms of compatibility. These results show the poor correlation between the results obtained in vitre vs in vivo to determine the compatibility of a biomaterial in a subject.

Example 4. Determination by proteomic analysis of the biomarker pertyl that determines the compatibility of biomaterials.

Once the poor correlation between in vitro vs in vivo results was demonstrated to determine the compatibility of a biomaterial, the proteins, particularly the peptide pattern, adhered to the surface of in vitro cultured biomaterials in the presence of human serum were analyzed. . Since the cellular response to contact with the biomaterial depends on the first layer of proteins adsorbed on it, it is intended to establish a correlation between the pattern of proteins detected and the in vivo results obtained.

Proteomic analysis reveals a correlation between the composition of the protein layer adhered to biomaterials or coatings and their response in vivo. After culturing the titanium discs with each of the coatings analyzed in the present invention in the presence of human serum, and isolating the eluates comprising the markers adhered to each disc (see materials and methods), said eluates were resolved on a gel of 10% polyacrylamide to determine the different whey proteins that had adhered to each biomaterial. Elution of the proteins obtained from the four coatings 70M30T, 35M35G30T, 50M05G and 50V50G was analyzed by LC-MSIMS, and 171 different proteins were detected.

Based on the results obtained by LC-MS / MS, the materials were classified into two groups according to the results obtained for each of them in the in vivo model. The first of the groups includes the biomaterials 70M30T and 35M35G30T, which according to the results in vivo showed a good osseointegration or compatibility with the subject (positive result), while the

second group is formed by the biomaterials 50M50G and 50V50G, which according to the results in vivo results in the formation of fibrous connective tissue around the implant area, so that these results show a poor compatibility of said biomalerials with the subject (negative result) (Fig. 7).

For the treatment of data obtained by LC-MS / MS, Progenesis 01 software was used, with a differential analysis (n = 4), in order to identify which of these proteins are predominantly differential among the different biomaterials. In addition, the "Database for Annotation, Visualization and Integrated Discovery was used

10 (DA VID) "to classify proteins in relation to their functions.

The comparison between materials 70M30T and 35M35G30T, on the one hand, and 50M50G and 50V50G on the other hand, shows small differences in the profile of proteins attached to these biomaterials (Tables 1 and 2). However, when compared

15 Biomaterials or coatings that show different results in vivo (70M30T and 50M50G, on the one hand and 35M35G30T and 50M50G, on the other hand) detect a greater number of proteins that are observed differentially and significantly present in compatible biomaterials with respect to non compatible (Tables 3 and 4).

Table 1. Analysis of the presence of statistically predominant proteins (p <O.05) in the coatings with the compatible biomaterials 70M30T vs 35M35G30T (Progenesis method) according to the present invention.

Presence Normalized

70M30T vs 35M35G30T

Protein

UniProtKB C.S. 70M30T average Average 35M35G30T Anova (p) Ratio 35M35G30T f70M30T DAVID

hMYH1

P12882 130.71 5.71E + 02 9.81E + 03 4, OOE-02 17.19 10

hLDHB

P07195 316.92 1, OOE + 04 1.22E + 05 1.84E-05 12.18 -

hDHE3

POO367 64.65 8.24E + 02 7.13E + 03 1.33E-03 8.65 -

hFCN2

Q15485 377.38 7.05E + 03 5.84E + 04 5.44E-04 8.28 1-2-7-5-10

hC1QA

P02745 178.51 3.17E + 04 9.68E + 04 2.15E-03 3.06 1-2

hHBA

P69905 57.48 3.75E + 04 6.50E + 04 3.37E-03 1.73 5-10

hCLUS

P10909 629.78 6.81E + 05 4.15E + 05 3.31E-02 0.61 1-4-9

hFA12

POO748 158.46 1.34E + 05 7.80E + 04 3.92E-02 0.58 3-5-8

Presence Normalized

70M30T vs 35M35G30T

Ratio

Half

Half

Protein

UniProtKB C.S. 70M30T 35M35G30T Anova (p) 35M35G30T DAVID

f70M30T

hAPOA5

NQ6Q788 168.81 5.41E + 03 2.85E + 03 3.16E-02 0.53 7-9

. ,

"

c.s .. Confidence score. The functions of DAVID claslficaclon were. (1) inflammatory / immune response, (2) hydroxylation, (3) blood coagulation, (4) regulation of apoptosis, (5) metal binding, (6) phosphorylation, (7) carbohydrate binding, (8) activity peptidase, (9) lipid transport and (10) cytoskeleton integrity.

Table 1 shows nine proteins that have been identified and that appear differentially present when the two biomaterials are compared with positive results in vivo (35M35G30T and 70M30T). Of the nine proteins 10 identified above, six were detected that showed a greater presence, statistically significant in the biomaterial or 35M35G30T coating, the MYH1 protein (17.19-fold) being the most predominant, and related to the integrity of the cytoskeleton. Proteins related to the immune system (C1QA and FCN2) or to the metal binding function such as the HBA protein were also found.

15 In addition, three other proteins were found, present in a large proportion in the 70M30T coating: the CLUS protein that could play a protective role against the establishment of chronic inflammatory disorders, the FAXII that is capable of triggering coagulation and the APOA5 that is related with lipid transport and carbohydrate binding function.

20 On the other hand, a comparative study of adhered proteins in biomaterials with a negative response in the in vivo model (50M50G vs 50V50G) was also carried out. Results are shown in table 2.

Table 2. Analysis of the presence of statistically predominant proteins (p <O.05) in coatings with 105 non-compatible biomaterials 50M50G vs 50V50G (Progenesis method) according to the present invention.

HHORN hDSC1 hLCN1 protein

UniProtKB Q86YZ3 Q08554 P31025 C.S. 165.54 236.90 121.78 Presence Normalized Medium Average SOM50G 50V50G 1.36E + 03 4.66E + 03 1.29E + 04 2.77E + 04 5.51E + 04 7.54E + 03 70M30T vs 35M35G30T Ratio Anova (p) 50V50G / 50 M50G 1.35E-02 3.41 3.23E-02 2.14 3.61 E-03 0.14 DAVID 5-8-10 5 8

5 C.S .. Confidence score. The DAVID classification functions were. (1) inflammatory / immune response, (2) hydroxylation, (3) blood coagulation, (4) regulation of apoptosis, (5) metal binding, (6) phosphorylation, (7) carbohydrate binding, (8) activity peptidase, (9) lipid transport and (10) cytoskeleton integrity.

10 Only three proteins were predominantly detected in biomaterials 50M50G vs 50V50G. Although both biomaterials have different chemical compositions, they have a very similar protein profile composition. HORN and DSC1 proteins are clearly more present in the

15 50V50G biomaterial, and both have the function of metallic binding, and the LCN1 protein, with endopeptidase activity, was more present in 50V50G biomaterial.

On the other hand, the proteins detected in biomaterials 70M30T and 35M35G30T (positive in vivo compatibility results) were compared with proteins

20 detected in the biomaterial 50M50G (negative result of in vivo compatibility). Twenty serum proteins with differential presence in these biomaterials were identified (Table 3).

Table 3. Analysis of the presence of statistically predominant proteins (p <O.05) between the coatings with compatible vs. non-compatible biomaterials: 70M30T lIS SOMSOG and 35M35G30T vs SOMSOG.

~

Normalized abundance

70M30T lIS 50M5QG 35M35G30T vs 5OM5OG

Protein

UniProt is 70M30T average 3SM35G media 5OMSOG media Anova (p) Ratio 5OM50GnO M30T Anova (p) Ratio 5OM5OG1 3SM35G30T DAVID

hCRP

P02741 122.50 4.46E + 03 6.62E + 03 3, 49E + 04 1.68E-02 7.83 3.65E-02 5.28 1-5-8

hLCN1

P31025 121.78 1.29E + 04 1.03E + 04 5.51 E + 04 1.81 E-02 4.28 1.09E-02 5.37 8

hSAMP

P02743 465.85 1, B4E + 05 2.97E + 05 5.72E + 05 3,29E-02 3.48 3,22E-02 1.93 1-5-7-8

HAPOE

P02649 1435.34 1.44E + OB 1.12E + 06 3,4BE + 06 3.02E-02 2.40 2,09E-03 3.09 3-4-5-67-9

hC10C

P02747 358.67 1.11 E + 06 1.18E + 06 2.51E + OB 8,12E-04 2.26 5,20E-03 2, 12 1-2

hC1QB

P02746 487.43 6.74E + 05 7.38E + 05 1.51E + OB 5.46E-03 2.24 4.43E-03 2.05 1-2

hCQ7

P10643 144.73 2.39E + 04 2.67E + 04 5.31 E + 04 1.76E-02 2.22 4.25E-02 1.99 one

hVTNC

P04004 327.47 3.39E + 05 4.24E + 05 7.50E + 05 1.71E-04 2.21 1.99E-03 1.77 7

hKV402

P06312 215.21 3.21E + 05 3.46E + 05 6.68E + 05 2,40E-03 2.08 1,15E-04 1.93 -

hC1S

P098? 1 566.82 2.51E + 05 2.37E + 05 5.19E + 05 B, 14E-04 2.07 6.43E-03 2, 19 1-2-5-8

hC: 4BPA

P04003 209.98 8.27E + 04 7.60E + 04 1.71E + 05 5.74E-03 2.06 1.61E-02 2.25 one

hKNG1

P01042 325.69 2.23E + 05 2.48E + 05 4.51E + 05 4.12E-03 2.02 2.27E-02 1.82 1-2-3-45-7

hLAC2

POCG05 353.61 4.80E + OB 5.27E + 06 8.28E + 06 1,3BE-02 1.73 3.07E-02 1.57 -

hAPOA4

P06727 1952.03 5.98E + 05 4.48E + 05 1.00E + 06 1.88E-02 1.67 4,56E-03 2.23 5

hKV201

P01614 143.61 6.28E + 05 5.71 E + 05 1.02E + 06 2.96E-02 1.63 1.87E-02 1.79 -

hK22E

P35908 4811, 54 4.42E + OB 4,54E + 06 2.80E + 06 3,52E-02 0.63 2.17E-02 0.62 10

hK1C10

P13645 4566.75 1.06E + 07 1.19E + 07 6.48E + 06 1.56E-02 0.61 1,60E-02 0.55 10

hK2C78

Q8N1N4 669.51 5.02E + 04 5.35E + 04 3.00E + 04 3.48E-02 0.60 2.39E-02 0.56 10

hHORN

Q86YZ3 165.54 3.44E + 03 8.79E + 03 1.36E + 03 2.48E-02 0.40 1,80E-02 0, 16 5-8-10

hFILA2

Q50862 430.49 5.59E + 04 5.61E + 04 1.98E + 04 4.67E-02 0.35 9,82E-03 0.35 5-8

m

[/) N

or ~

N

~

N

or P

CS .: Confidence score. The DAVID classification functions were: (1) high inflammatory / immune response, (2) hydroxylation, (3) healthy coagulation, (4) regulation of apoptosis, (5) metal bonding, (B ) phosphorylation, (7) carbohydrate binding, (8) peptidase activity, (9) transport of IIpids and (10) integrity of the cytoskeleton.

A group of fifteen proteins were found differentially more absorbed on the surface of the 50M50G biomaterial than in the in vivo positive response materials (lOM30T and 35M35G30T). VTNC, APOE and KNG1, have a role in bone metabolism and could intervene in the process of regeneration of said tissue. In addition, VTNC could be involved in the complement regulation cascade. However, some immunoglobulins and many proteins related to the immune system were also present. In fact, the proteins CRP, SAMP, C10B, C10C, COl, C1S and C4BPA were classified with the DAVID database within the group related to the acute inflammatory response.

10 All these proteins are involved in the activation of the complement cascade with the exception of C4BPA, which is capable of regulating this process. Keratins, HORN and FILA2 were absorbed more, differentially, in 70M30T and 35M35G30T coatings than in 50M50G. These proteins play a role in the integrity of the cytoskeleton and have peptidase activity.

15 The proteomic results of the biomaterials or coatings 70M30T and 35M35G30T (positive in vivo compatibility result) were compared with the proteins adsorbed and detected in the biomaterial or 50V50G coating (negative in vivo compatibility result). Table 4 presents the sixteen

20 proteins that were identified with differential presence, including fifteen were with greater presence in the biomaterial or 50V50G coating.

Table 4. Analysis of the presence of statistically predominant proteins (p <O.OS) between 70M30T vs coatings

SOV50G and 35M35G30T vs SOV5OG (Progenesis method).

m

[/) N

or ~

N

'"~

N

or P

c.s .: Confidence score. The DAVID classification functions were: (1) inflammatory / immune response, (2) hydroxylation, (3) coagulation

blood, (4) regulation of apoptosis, (5) metal binding, (6) phosphorylation, (7) carbohydrate binding, (8) peptidase activity, (9) lipid transport and (10) cltoskeleton integrity.

Except for vitronectin (VTNC), all the characteristic proteins of the biomaterial or 50M50V coating are directly involved in the immune response, they belong to the DAVID group of proteins related to the acute inflammatory response (CRP, C05, SAMP, C1QB, IC1, CFAH, C07 , C1S, C03, C1R and C1QC). It should be noted that most of the proteins in this group are involved in the activation of the complement cascade, with the exception of CFAH, which is an important regulatory / repressor protein for the activation of the alternative complement pathway. With respect to the most differentially present proteins on biomaterials or positive result coatings; n alive, the SPB3 protein was the

10 only detected. Said SPB3 protein has endopeptidase activity.

It should be noted that a greater presence was detected with a significant difference in the proteins related to the immune / inflammatory response adhered to the surface of biomaterials 50V50G and 50M50G, compared with the other two

15 biomaterials, 70M30T and 35M35G30T.

Table 5 shows the proteins that are attached statistically significantly at the same time in the 50M50G and 50V50G biomaterials (no compatibility shown in the in vivo model), with respect to the coatings

20 35M35G30T and 70M30T (compatibility shown in the in vivo model).

Table 5. Differentially attached proteins at the same time in 50M50G and 50V50G

with respect to sol-gel coatings 35M35G30T and 50V50G (method

Progenesis). ANOVA (p-value <0.05).

Description hCRP hSAMP hC1QC hC1QB hC07 hVTNC hKV402 hSAMP hC1QC

SEO ID NO: 1 2 3 4 5 7 9 2 3 UniProtKB P02741 P02743 P02747 P02746 P10643 P04004 P06312 P02743 P02747

Table 5 shows nine proteins, of which six of them are related to the immune system. These immune system proteins are identified as CRP, SAMP, C1QB, C1QC, C1S, LAC2, KV402, VTNC and C07. As it has been

5 mentioned above, all of them are involved in the acute inflammatory response process. Notably, CRP is particularly more present in the worst response materials in vivo and its role in the complement cascade is well known, it is an activator of the classical route.

10 The results described so far establish that the proteins related to the activation / inhibition complement cascade process are differentially more present in biomaterials or coatings 50M50G and 50V50G (worse in vivo behavior). Although proteins with complement inhibitory properties such as VTNC, are predominantly adhered to

15 50M50G and 50V50G coatings, both induce the formation of a connective tissue

or dense fibrous when tested in vivo. The explanation to this question could be related to the ratio of inhibitory activator proteins present in each surface. Thus, in Table 6 you can see how the biomaterials 70M30T and 35M35G30T show a greater proportion of the inhibitory proteins of the

20 complement with respect to other biomaterials.

Table 6. Relation (rati o) of biomarkers inhibitors / activators of the

complement detected and adhered to biomaterials 70M30T, 35M35G30T,

50M50G and 50V50G.

Relationship

70M30T 35M35G30T 50M50G 50V50G

CFAHICRP

75.19 61, 30 17.44 10.32

CFAHISAMP

2.04 1.37 1.07 1.28

C4BPAlCRP

18.53 11.48 4.88 1.89

C4BPAlSAMP

0.50 0.26 0.30 0.24

VTNCICRP

76.05 64.09 21.47 9.87

VTNCISAMP

2.06 1.43 1.31 1.23

Table 6 shows the ratios of absorbed immune system proteins (C4BPA, CFAH and VTNC) and absorbed activator proteins (CRP and 30 SAMP), selected for being more differentially adhered to the different

materials. It can be seen that the 70M30T and 35M35G30T biomaterials show the highest values of the ratio of absorbed complement inhibitor proteins vs. complement activating proteins, compared with the other biomaterials with poor behavior in vivo.

In conclusion, the results shown in the present invention show the existence of a specific and specific peptide profile of proteins that remain attached to the analyzed biomaterial once it has previously been contacted with a serum sample of a subject, where the identification and

Quantification of said peptide pattern is able to determine the compatibility of said biomaterial in vivo.

Claims (31)

1. In vitro method for predicting and / or predicting the compatibility of a biomaterial in an isolated biological sample obtained from a subject, comprising the following stages: 8. Incubate the biomaterial with the isolated biological sample,

b.

Determine and quantify the joint presence of the markers attached to the biomaterial that are selected from the list consisting of: protein and reactive (CRP), serum P amyloid component (SAMP), subunit e of subcomponent C1q of complement (C1QC), subunit B of subcomponent C1q of complement (C1QB), component e7 of complement (CO?), subcomponent C1s of complement (C1S), vitronectin (VTN), Lambda-2 immunoglobulin C chain region (LAC2) and Kappa variable immunoglobulin 4-1 (KV402 ).

C.

Compare the value obtained in step (b) with a reference value

obtained from a reference biomaterial, where a value of at least twice higher for each of the markers with respect to the reference value obtained for the reference biomaterial, is indicative that the biomaterial is not compatible with the subject.

2.

The method according to claim 1 wherein the CRP marker comprises SEO ID NO: 1, the SAMP marker comprises SEO ID NO: 2, the C10C marker comprises SEO ID NO: 3, the C10B marker comprises SEO ID NO: 4 , marker C07 comprises SEO ID NO: 5, marker C1S comprises SEO ID NO: 6, marker VTN comprises SEO ID NO: 7, marker LAC2 comprises SEO ID NO: 8 and marker KV402 comprises SEO ID NO: 9.

3.

The method according to any one of claims 1 to 2, wherein step b) further comprises determining and quantifying at least one of the markers selected from the list consisting of: alpha chain of the C4b binding protein (C4BPA), Kininogen-1 (KNG1), complement C5 (C05), plasma protease inhibitor C1 (IC1), complement factor H (CFAH), component C3 of complement (C03), subcomponent C1 r of complement (C1R), ficolin- 2, Mannose binding lectin serine protease 1 (MASP-1), and / or any combination thereof.

4. The method according to claim 3 wherein the C48PA marker comprises SEO ID NO: 10, the KNG1 marker includes SEO ID NO: 11, the C05 marker comprises SEO ID NO: 12, marker IC1 comprises SEO ID NO: 13, marker CFAH comprises SEO ID NO: 14, marker C03 comprises SEO ID NO: 15, the C1R marker comprises SEO ID NO: 16, the Ficolina-2 marker comprises SEO ID NO: 17 and the MASP-1 marker comprises SEO ID NO: 18. 5. The method according to any one of claims 1 to 4 characterized in that it comprises determining and quantifying the set of markers selected from the list consisting of CRP comprising SEO ID NO: 1, SAMP comprising SEO ID NO: 2 , C10C comprising SEO ID NO: 3, C10B comprising SEO ID NO: 4, C07 comprising SEO ID NO: 5, C1 S comprising SEO ID NO: 6, VTN comprising SEO ID NO: 7, LAC2 comprising SEO ID NO: 8, KV402 comprising SEO ID NO: 9, C4BPA comprising SEO ID NO: 10, KNG1 comprising SEO ID NO: 11, C05 comprising SEO ID NO: 12, IC1 comprising SEO ID NO: 13, CFAH comprising SEO ID NO: 14, C03 comprising SEO ID NO: 15, C1R comprising SEO ID NO: 16, Ficolina-2 comprising SEO ID NO: 17 and MASP-1 comprising SEO ID NO: 18.

6.

The method according to any one of claims 1 to 5, wherein step (b) is carried out by any of the methodologies selected from the group comprising: mass spectroscopy, immunohistochemistry, ELlSA and its variants, immunofluorescence, immunocytochemistry and immunoprecipitation.

7.

The method according to any one of claims 1 to 6 wherein step (b) is carried out by EUSA.

8.

The method according to any one of claims 1 to 7 wherein the isolated biological sample is selected from the list consisting of: blood, serum and blood plasma.

9.

The method according to any of claims 1 to 8, wherein the isolated biological sample is serum.

10.

The method according to any one of claims 1 to 9, wherein the subject is a mammal.

eleven.

The method according to any one of claims 1 to 10, wherein the subject is a human.

12.

In vitro use of the determination and quantification of the joint presence of the markers that are selected from the list consisting of: CRP, SAMP, C1QC, C1QB, C07, C1S, VTN, LAC2 and KV402. to predict and / or predict the compatibility of a medical biomaterial in an isolated biological sample obtained from a subject.

13.

In vitro use according to claim 12 wherein the CRP marker comprises SEO ID NO: 1, the SAMP marker comprises SEQ ID NO: 2, the C1QC marker comprises SEQ ID NO: 3, the C1QB marker comprises SEQ ID NO: 4, the COl marker comprises SEO ID NO: 5, the C1S marker comprises SEO ID NO: 6, the VTN marker comprises SEQ ID NO: 7, the LAC2 marker comprises SEO ID NO: 8 and the KV402 marker comprises SEO ID NO: 9.

14.

In vitro use according to any of claims 12 to 13 which further comprises determining and quantifying the presence of at least one marker selected from the list consisting of: C4BPA, KNG1, C05, IC1, CFAH, 03, C1 R, Ficolin -2, MASP-1 and / or any combination thereof.

fifteen.

In vitro use according to claim 14 wherein the C4BPA marker comprises SEQ ID NO: 10, the KNG1 marker comprises SEQ ID NO: 11, the C05 marker comprises SEO ID NO: 12, the IC1 marker comprises SEQ ID NO: 13, the CFAH marker comprises SEO ID NO: 14, the C03 marker comprises SEQ ID NO: 15, the C1R marker comprises SEQ ID NO: 16, the Ficolina-2 marker comprises SEO ID NO: 17 and the marker MASP-1 includes SEO ID NO: 18.

16.

Use in vi / ro according to any of claims 12 to 15 characterized in that it comprises determining the joint presence of the markers selected from the list consisting of: CRP comprising SEO ID NO: 1, SAMP comprising SEQ ID NO: 2, C1QC comprising SEQ ID NO: 3,

C1QB comprising SEO ID NO: 4, C07 comprising SEO ID NO: 5, C1S comprising SEO ID NO: 6, VTN comprising SEO ID NO: 7, LAC2 comprising SEO ID NO: 8, KV402 comprising SEO ID NO: 9, C4BPA comprising SEO ID NO: 10, KNG1 comprising SEO ID NO: 11, C05 comprising SEO ID NO: 12, IC1 comprising SEO ID NO: 13, CFAH comprising SEO ID NO: 14, C03 comprising SEO ID NO:  15, C1R comprising SEO ID NO: 16, Ficolina-2 comprising SEO ID NO: 17 and MASP-1 comprising SEO ID NO: 18.

17.

In vitro use according to any of claims 12 to 16 wherein the isolated biological sample is selected from the list consisting of: blood, serum and blood plasma.

18.

In vitro use according to any of claims 12 to 17, wherein the isolated biological sample is serum.

19.

In vitro use according to any of claims 12 to 18, wherein the subject is a mammal.

twenty.

In vitro use according to any of claims 12 to 19, wherein the subject is a human.

21. Kit to predict and / or predict the compatibility of a biomaterial comprising antibodies, reagents, or any combination of the above, capable of detecting and quantifying the presence of markers that are selected from the list consisting of: CRP , SAMP, C10C, C10B, COl, C1S, VTN, LAC2 and KV402. 22. Kit according to claim 21 characterized in that the CRP marker comprises SEO ID NO: 1, the SAMP marker comprises SEO ID NO: 2, the C10C marker comprises SEO ID NO: 3, the C10B marker comprises SEO ID NO: 4, the COl marker comprises SEO ID NO: 5, the C1S marker comprises SEO ID NO: 6, the VTN marker comprises SEO ID NO:?, The LAC2 marker comprises SEO ID NO: 8 and the marker KV402 includes SEO ID NO: 9. 23. Kit according to any of claims 21 to 22 characterized in that it further comprises the antibodies, reagents, or any combination of the foregoing, capable of detecting and quantifying the presence of at least one of the markers selected from the list consisting of : C4BPA, KNG1, COS, IC1, CFAH, C03, C1 R, Ficolian-2, MASP-1, and / or any combination thereof. 24. Kit according to claim 23 wherein the C4BPA marker comprises SEO ID NO: 10, the KNG1 marker comprises SEO ID NO: 11, the COS marker comprises SEO ID NO: 12, the IC1 marker comprises SEO ID NO: 13, the CFAH marker comprises SEO ID NO: 14, the C03 marker comprises SEO ID NO: 15, the C1R marker comprises SEO ID NO: 16, the Ficolina-2 marker comprises SEO ID NO: 17 and the MASP- marker 1 includes SEO ID NO: 18. 25. Kit according to any of claims 21 to 24 characterized in that it comprises the antibodies, the reagents, or any combination of the foregoing, capable of detecting and quantifying the joint presence of the markers that is selected from the list consisting of: CRP comprising SEO ID NO: 1, SAMP comprising SEO ID NO: 2, C10C comprising SEO ID NO: 3, C10B comprising SEO ID NO: 4, C07 comprising SEO ID NO: 5, C1S comprising SEO ID NO: 6, VTN comprising SEO ID NO: 7, LAC2 comprising SEO ID NO: 8, KV402 comprising SEO ID NO: 9, C4BPA comprising SEO ID NO: 10, KNG1 comprising SEO ID NO: 11, C05 comprising SEO ID NO: 12, IC1 comprising SEO ID NO: 13, CFAH comprising SEO ID NO: 14, C03 comprising SEO ID NO: 15, C1R that includes SEO ID NO: 16, Ficolina-2 that includes SEO ID NO: 17 and MASP-1 that includes SEO ID NO: 18.

26.

In vitro use of the kit according to any of claims 21 to 25 to predict and / or predict the compatibility of a biomaterial in contact with a biological sample isolated from a subject.

27.

Use in vi / ro according to claim 26 wherein the isolated biological sample is selected from the list consisting of: blood, serum and blood plasma.

28.

Use according to claim 27, wherein the isolated biological sample is serum.

29.

Use according to any one of claims 26 to 28, wherein the subject is a mammal.

30

Use according to any of claims 26 to 2, wherein the subject is a human.