WO2010004066A1 - Matrices tridimensionales de monetita porosa estructurada para ingeniería tisular y regeneración ósea, y método de preparación de las mismas - Google Patents
Matrices tridimensionales de monetita porosa estructurada para ingeniería tisular y regeneración ósea, y método de preparación de las mismas Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/28—Bones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3847—Bones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/42—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
- C01B25/26—Phosphates
- C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T428/00—Stock material or miscellaneous articles
- Y10T428/24—Structurally defined web or sheet [e.g., overall dimension, etc.]
- Y10T428/24744—Longitudinal or transverse tubular cavity or cell
Definitions
- the present invention is framed within tissue engineering and, in particular, bone regeneration.
- the invention relates to a porous three-dimensional matrix of biocompatible monetite, structured porosity, predefined and reabsorbable, as well as the method of synthesis capable of producing said material and its applications.
- These matrices constitute a perfect base for cell colonization and proliferation allowing its application in tissue engineering and bone regeneration thanks to its advantageous properties of biocompatibility, reabsorption, osteoinduction, revascularization, etc.
- Biomaterials have been used for almost a century to repair or replace bone segments of the musculoskeletal system.
- a bone-like material that is biocompatible, has no adverse biological reactions, is reabsorbable and gradually degrades as the new tissue forms, thus gradually transferring the loads to the new one. bone, avoiding a second surgical intervention for implant removal.
- the degradation and reabsorption of the bone Ia carry out the osteoclasts. These are cells derived from monocytes, which are fixed to the surface of the bone. Once fixed, they start releasing protons outside, in order to lower the pH of the external medium. With this acidic environment, hydroxyapatite crystals that are part of the bone mineral component are solubilized. Bone hydroxyapatite is solubilized in amorphous calcium phosphate particles, which are eliminated by macrophages, or in Ca 2+ and PO 4 3 ions that accumulate in the extracellular fluid.
- osteoclasts are also responsible for the degradation of the organic phase of the bone through enzymatic processes.
- the mechanical properties of the bone substitute should be as similar as possible to those of the spongy bone.
- the material must also help the stability of the fracture and be sufficiently resistant to reduce the necessary time of immobilization or external support. Said material must be reabsorbable, biocompatible and osteoinductive, that is, it must attract mesenchin cells and other cell types located near the implant and favor its differentiation into osteoblasts, and also osteoconductors, that is, it must act as a template for the formation of new bone.
- the non-absorbable materials used so far in bone implants are being replaced by the absorbable ones.
- These biomaterials do not interfere with the development and growth of the newly formed bone, since they are gradually replaced by host tissue. In addition, they have a greater biocompatibility, participate in a natural way in bone reconstruction and do not need to eliminate them, by surgery, after bone regeneration. These materials have to be maintained long enough for the correct regeneration of the bone and gradually disintegrate without causing damage to the patient and without intervening in the proper development and growth of the bone.
- the biomaterials that form forming a mineral calcium phosphate have special interest in bone regeneration since they resemble the mineral phase of the natural bone and are susceptible to bone remodeling and reabsorption due to their metastable crystalline structure.
- calcium phosphates stand out; hydroxyapatite (PAH), tricalcium phosphate (B-TCP) and dicalcium phosphate dihydrate (DCPD) (Stubbs et al, 2004; Schnettler et al 2004).
- PAH hydroxyapatite
- B-TCP tricalcium phosphate
- DCPD dicalcium phosphate dihydrate
- Hydroxyapatite has been one of the most interesting. This material is per se the inorganic phase of which the bones are formed and that is why it has been widely used in bone regeneration. Examples of this are some commercial products such as Interpore 200® Interpore 500®, Cerasorb® and Collagraft®. However, and because it has one of the most stable crystalline structures, the material suffers from a slow reabsorption.
- PAH is the material with the highest biocompatibility, as it is the closest to the crystals formed by the bone, but it is not reabsorbable in vivo.
- the degradation of this material is produced by contact with solutions with a low pH and by phagocytosis. Through the solution, amorphous calcium phosphate particles are released that can be eliminated by macrophages by phagocytosis or be embedded in the new bone formed. Macrophages can dissolve these particles and restore Ca and P to the organism pool (Frayssinet et al 1999; Be Spotifyd et al 1996). However, it has not been observed that these particles give rise to osteoclastic activation (Frayssinet et al 1999).
- B-TCP has more osteoconductivity and better reabsorption than PAH (Franco et al 2006). It is considered as a moderately reabsorbable material, in vivo studies it has been observed that it takes at least one year for its reabsorption in animals and 6 to 8 months in humans (Wiltfang et al 2003; Suba et al 2004). Its degradation increases calcium deposits and this is associated with increased alkaline phosphatase activity, an enzyme that is involved in bone formation (Trisi et al 2003; Sugawara et al 2004).
- the DCPD is also biocompatible, osteoconductive and the most reabsorbable because it is the most soluble at physiological pH. This allows new bone to form faster. It biodegrades in physiological environments and is reabsorbed by adjacent cells (Tris et al 2003). It is proven that it is reabsorbed in vivo, up to three times faster than PAH and B-TCP (Herrón et al 2003; Chow et al 2003; Tas & Bhaduri 2004; Tamini et al 2006;).
- the Monetite is reabsorbed in vivo in a similar manner and time. It dissolves at physiological pH, gradually in the extracellular tissues that surround the implant and the cells that colonize it, (endothelial cells, osteoclasts, osteoblasts, macrophages %) would be responsible for its elimination or reuse as it happens in the bone.
- the biomaterial has an adequate porosity that allows the colonization and cell proliferation, vascularization, increase of the contact surface and therefore increase of the surface of interaction with the host tissue that allows the acceleration of bone regeneration. These characteristics must be accompanied by a correct rate of resorption that gives the cells the time necessary for regeneration.
- Gbureck, Uwe et al 2007, refer to Brusita and Moneti ⁇ a implants prepared by the three-dimensional printing technique. To achieve these implants, firstly, Brusite matrices are obtained which are hydrothermally dehydrated by being transformed into Monetite.
- US6605516 presents bone substitutes with a controlled anatomical shape that fit exactly to the morphology of the lesion.
- Said substitutes are composed of chemically consolidated calcium phosphate cement materials.
- the invention also relates to porogenic molds and phases that allow obtaining calcium phosphates with external geometries and macroporous architectures by using said molds.
- the invention presents Brusite materials, not presenting Monetite materials and neither being the macroporous structures presented therein valid for the purpose of the present invention.
- the present invention provides monetite matrices (metastable phase of calcium phosphate of monetite), with a high thermal stability that allows the sterilization of the material by autoclaving, thus simplifying the sterilization processes and also, due to its specific structural arrangement of pores, disposition that is obtained thanks to a specific design of the material , supposes an improvement of the osteoinductive capacity of materials proposed by the state of the art since it is synthesized in the form of a porous block with defined macroporosity characteristics defined by increasing the specific surface, as well as the area of contact with the osteoblasts and facilitating the work processes Nutrient support for cells, a crucial factor for osteogeneration. All this together with its high resorption capacity in the appropriate period of time so that the adjacent cells colonic the material and can replace the reabsorbed material by physiological bone matrix.
- the cells of the implant area, osteoblasts of the adjacent bone, mesenchymal stem cells of the bone marrow and endothelial cells of the systemic circulation must be able to colonize simultaneously and homogeneous biomaterial. This will allow the formation of a new physiological bone matrix as the biomaterial is resorbed and the development of a new vascular system, which will be the one that supplies the blood supply necessary for the survival of the new tissue.
- porous structure An important property to take into account in relation to this aspect is the porous structure, because it influences both the biodegradability, the greater the porosity degree, the better the resorption, as in the cellular colonization.
- the materials must have pore sizes and interconnections that allow colonization of both endothelial cells (for the formation of new blood vessels) and bone cells.
- microporous and interconnected character which allows the diffusion of nutrients and gases and also of the metabolites of the cellular activity.
- Bone is not a compact material but has different porosities that intercommunicate. Interconnected pore systems communicate the solid (cortical) bone with the spongy (trabecular) bone ( Figure 16). These porosities range from 100-150 ⁇ m in the cortex to 500-600 ⁇ m in the spongy.
- the present invention presents a new tissue engineering system, intended to regenerate the bone structure by addressing a healing strategy rather than merely repair. Said regeneration has application against osteoporosis.
- Tissue engineering is considered as a discipline that improves, maintains and repairs pathologies in organs and tissues.
- the creation of a system based on tissue engineering involves the integration of viable cells, a biocompatible material specially designed for a biomedical application and signaling molecules that regulate the cellular activities that are required at each moment of treatment.
- the present invention provides matrices with non-random porosity geometry, that is, ordered or predefined, composed of monetite, in whose design the porosities of the bone have been taken into account, so that it is of neovascularization and cellular colonization.
- Said material is presented sterilized, ready for use and thanks to its specific design, it achieves a specific structural arrangement of pores, that is, a spatial distribution and spatial configuration of ordered porosity induced and previously established, which implies an improvement of the osteoinductive capacity against other calcium phosphates, including other combinations of calcium phosphate that include monetite.
- Said matrices are obtained in the form of a porous block with defined macro, meso and microporosity characteristics that increase the specific surface, as well as the zone of contact with the osteoblasts, facilitating the processes of nutrient transport for the cells, a crucial factor for the osteogeneration
- monetite matrices of the invention has taken into account the characteristic porosities of natural bone, porosities that allow neovascularization and cell colonization.
- the new matrices of the invention are composed of the Monetita biomaterial, a dehydrated DPCD (DPC), ideal for bone regeneration.
- Said matrices are made up of at least 95% ⁇ 5% monetite, preferably 95% monetite and more preferably 100% monetite.
- Traces of material correspond to beta tetracalcium phosphate. In vitro degradation of this material does not affect cell proliferation and is also bioactive, non-cytotoxic, non-mutagenic and hemocompatible as shown in example 4.
- the matrices of the invention are reabsorbed in the appropriate period of time so that the adjacent cells colonize the material and can replace the reabsorbed material by physiological bone matrix.
- Matrix refers to any three-dimensional structure useful in bone regeneration that allows cell growth and proliferation of the invading cells.
- Ratimal stem cells preferably obtained from adipose tissue, but can also be from bone marrow or any other location that has been shown to be the source of these cells.
- These cells can be used differentiated towards the osteoblastic or endothelial lineage.
- Endothelial cells - Combinations of adult mesenchymal stem cells not differentiated or differentiated towards osteoblastic or endothelial lineage, osteoblasts, osteoclasts, bone osteocytes and endothelial cells.
- Macropores when the pores have diameters greater than or equal to 100 microns.
- Wlesoporos when the pores have diameters less than 100 microns but greater than or equal to 10 microns.
- Micropores When the pores have a diameter less than 10 microns.
- Amorphous matrix That which presents a random porosity geometry, not ordered or predefined, that does not follow a spatial distribution and spatial configuration of ordered and previously established porosity, regardless of whether said porosity is natural (intrinsic to the material) or induced.
- Structured or structured porosity matrix The one that presents a non-random, ordered or predefined porosity geometry, presenting a spatial distribution and spatial configuration of induced and previously established ordered porosity.
- the matrices of the present invention are matrices of structured porosity with a predefined porosity that gives them a series of ideal properties for use in bone regeneration.
- Osteoinduction bone neoformation by apposition to the material, forming a framework for cell proliferation with osteoblastic activity, forming new bone. It is the act or process of stimulating osteogenesis.
- Osteogenesis generation or development of bone tissue, through the differentiation of mesenchymal cells to osteoblasts.
- Bone regeneration formation of new bone that, after a remodeling process, is identical to the existing one. Bone regeneration causes a response in which blood vessels, cells and extracellular matrix are involved.
- the biomaterial of the invention finds application in tissue engineering and bone regeneration and, therefore, can be used in the treatment of the following bone pathologies:
- Cell colonization capacity of cell expansion over the biomaterial, being able to proliferate and increase the cell population until invading the entire matrix.
- a measure of the ability to colonize a matrix is the analysis of the number of cells on the biomaterial over time (data from the proliferation graph).
- Cell adhesion ability of cells to bind to other cells or to a matrix. Adhesion can be produced by specific interactions such as electrostatic forces and is regulated by specific proteins called adhesion molecules. The ability to adhere to a biomaterial can be analyzed by microscopic visualization of the cells arranged on the biomaterial. The contact surface between the cells and biomaterial will be a representative measure of the affinity that the cells have for that biomaterial.
- the present invention refers to biocompatible three-dimensional matrices, of structured porosity composed of porous monetite, hereinafter matrices of the invention, comprising three-dimensional matrices of structured porosity monetite, corresponding to cylindrical macropores of between 350-650 ⁇ m in diameter , uniformly separated between 0.4-0.6mm from each other.
- Said monetite presents the intrinsic porosity of the material, on which the indicated structured macroporosity is induced.
- said structured porosity is distributed in the maximum area of the matrix that allows said matrix to maintain its mechanical stability in a stable manner.
- said maximum area is the remainder of removing the outer perimeter zone of the matrix, which ranges between 0.1 and 0.9 mm in width, preferably 0.5 mm in width.
- the materials used in osteogenesis must mimic the morphology, structure and function of the bone to achieve a correct integration into the host tissue.
- the structure determined by the porosity and the pore diameter of the materials used in bone regeneration influences bone formation both in vivo and in vivo.
- the pores are necessary for the formation of bone tissue, since they allow the migration and proliferation of osteoblasts and mesenchymal cells and also vascularization.
- the material of the invention provides the necessary conditions to achieve the correct regeneration of the bone thanks to its porosity characteristics that allow the colonization and proliferation of the cell types necessary for such effect.
- the minimum diameter required for bone formation was considered around 100 ⁇ m, so that the processes of migration and cell transport could be carried out.
- diameters greater than 300 ⁇ m are proposed since the presence of these macropores increases bone formation because they allow the formation of capillaries inside.
- Vascularization affects the development of osteogenesis. The pores with small diameters favor hypoxia conditions and do not induce osteogenesis but chondrogenesis.
- the long and large tunnel-shaped pores of the matrix of the invention allow its vascularization and the development of osteogenesis.
- pores with high diameters increase the contact surface, which also increases the surface of interaction with the host tissue, which will accelerate the degradation carried out by macrophages.
- the vascular network that can be formed is irregular in the biomaterial structure and cannot connect to the vascular network of the bone, so that the implant cannot be integrated effectively with the receptor tissue.
- the porosity structure adopted by the matrices of the present invention takes into account the incorporation of pores with the appropriate size so that the required cell species coexist and a bone and vascular network can be formed throughout the implant and also the connection with The receiving area, so that tissue integration can occur.
- the new design incorporates macropores of 350 ⁇ m -650 ⁇ m in cylindrical form (in the form of a tunnel), which completely cross the structure of the material, for a suitable cellular colonization (in terms of different cell types and a sufficient number of each type) of the cells of adjacent tissues, as well as an integration with the recipient tissue.
- macropores of 350 ⁇ m -650 ⁇ m in cylindrical form (in the form of a tunnel), which completely cross the structure of the material, for a suitable cellular colonization (in terms of different cell types and a sufficient number of each type) of the cells of adjacent tissues, as well as an integration with the recipient tissue.
- a micropore network for a sufficient diffusion of nutrients, gases and waste products of cellular metabolism.
- amorphous biomaterials which show a distribution of unstructured and non-predefined macroporos, produced in the process of obtaining the cement of the present invention, have pores that do not connect the internal structure. That is, the number of macropores is insufficient and its distribution inappropriate so that adequate colonization of the cells can occur, these being mostly relegated to the surface of the material.
- the success in the process of formation of a new bone is directly related to the amount of bone-forming cells that intervene in the process, as well as in the formation of a consistent vascular network throughout the biomaterial.
- the matrices of structured porosity material of the invention which have a spatial distribution and spatial configuration of ordered, induced and previously established macropores, allow a wide cellular colonization throughout the biomaterial, a greater diffusion of nutrients and signaling molecules that will determine cell behavior.
- the matrices of the invention with a high percentage of porosity, especially macroporosity, in which there are pores with high diameters (> 300 ⁇ m, specifically between 350 and 650 ⁇ m, and preferably 500 ⁇ 60 ⁇ m) and in shape of continuous tunnels, the osseointegration of the implant will increase after surgery.
- the present invention refers to the method of synthesis of the matrices of the invention, which comprises the formation of a monetite matrix of structured porosity comprising:
- the product obtained in stage 1 results in a solid phase that is mixed with distilled water to give rise to a liquid phase.
- the invention proposes the use of tricalcium-beta phosphate as basic calcium phosphate, and calcium monophosphate as acid phosphate.
- the molar ratio of basic phosphate / acid phosphate is 1.6-1.8 for a time of approximately 10 minutes, the concentration of porogen 1-20% by weight and Ia retardant between 0.4-0.6% by weight; preferably base phosphate / acid phosphate molar ratio of 1,785, porogen concentration 3-
- the molar ratio of basic phosphate / acid phosphate to carry out the mixture is 1.6-1.8, preferably 1.785, for a time of approximately 10 minutes.
- Calcium carbonate is added in concentrations between 1-20% by weight, preferably between 3-10%.
- the invention proposes the use of pyrophosphate in a proportion of 0.4-0.6% by weight, with 0.54% being the preferred option.
- the mold of the invention used for the development of the biomaterial refers to any mold that has cylindrical punches, whose base has a diameter of between 350- and 650 ⁇ m, and that are separated from each other between 0.4 and 0.6 mm, .
- Said mold can be constructed of silicone, metal, resistant plastic material or any type of material that allows it to be applied in its use.
- the mold can have any desired shape, depending on the shape and size of the biomaterial that is required to repair a particular bone defect for each patient, always maintaining the obtained biomaterial the characteristic porosity characteristics of the biomaterial of the invention, that is, cylindrical macroporos with a diameter between 350- and 650 ⁇ m, more preferably 500 ⁇ m ⁇ 60 ⁇ m in diameter, uniformly separated between 0.4 and 0.6 mm, more preferably 0.5mm ⁇ 60 ⁇ m, in addition to the intrinsic porosity of the biomaterial.
- Said molds allow obtaining the matrices of the invention, in which the structured porosity is distributed in the maximum area of the matrix that allows said matrix to maintain its mechanical stability in a stable manner.
- said molds allow the obtaining of matrices in which the maximum area in which the structured porosity is distributed is the remaining area of eliminating the outer perimeter zone of the matrix, between 0.1 and 0.9mm of width, preferably 0.5 mm width.
- the invention also contemplates the use of more than one mold:
- a first mold that allows obtaining monetite matrices, in the desired form but without structured porosity
- a second mold that, on a flat surface, has cylindrical punches, with a diameter of between 350- and 650 ⁇ m, preferably 500 ⁇ m ⁇ 60 ⁇ m, and that are separated from each other between 0.4 and 0.6 mm, preferably 500 ⁇ m ⁇ 60 ⁇ m. Said second mold must be applied, after removing the first mold, introducing into it the pieces obtained with the first mold. The second mold is covered with a lid as shown in Figure 1c.
- the biomaterial of the invention can be presented in the form of tablets, sheets, cylinders, etc., and any other form that is useful for repairing a particular bone defect of a patient.
- the mold has the shape of a tablet or cylinder with a diameter between 2 and 50 mm, preferably between 2 and 15 mm and a height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:
- the punches are cylindrical with a diameter of 500 ⁇ m ⁇ 60 ⁇ m, separated
- a first mold is made of silicone and has cylindrical holes for pads or cylinders of the size of the matrices of the invention to be manufactured.
- said gaps have a diameter between 2 and 50 mm, preferably between 2 and 15 mm and height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:
- Said molds do not intervene in the formation of macropores.
- the second mold is metallic, it has the dimension of each of the previous pieces, and at its base, evenly distributed, it has cylindrical punches of 500 microns ⁇ 60 ⁇ m, separated from each other 500micras ⁇ 60 ⁇ m, which give place to the macroporous component of the monetite matrices, distributed respecting a minimum perimeter zone of 0.5mm (taken from the edge of the tablet) free of punches.
- said metal molds have a diameter between 2 and 50 mm, preferably between 2 and 15 mm and height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:
- the procedure is the same as the previous one, with the difference that immediately after making the mixture of the solid and the liquid phase, the first silicone mold is filled.
- the silicone mold pieces are removed.
- the pieces are introduced into the metal mold with punches (covering with the metal lid according to Figure 1c), until the setting ends in a water bath at 37 0 C for 30 minutes.
- they are removed from the metal mold obtaining the cylindrical pieces with the determined porosity.
- the formed matrices are subjected to autoclaving between 120 and 13O 0 C for 24-25 minutes, producing their conversion to Monetita, completely sterilized and suitable for use.
- the use of these molds gives rise to monetite tablets of structured porosity.
- said pads have a diameter between 2 and 50 mm, preferably between 2 and 15 mm and height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:
- diameter 10mm and height 3 to 5mm preferably 3mm or 5mm, which have a uniform distribution of 64 macropores with a diameter of 500 ⁇ m ⁇ 60 ⁇ m, separated 500 ⁇ m ⁇ 60 ⁇ m from each other.
- the monetite tablets have a minimum perimeter zone of
- the final distribution of macropores in said tablets respects both the minimum perimeter zone of 0.5mm free of macropores, as well as the size and distance between pores (as described above).
- the products of the present invention find application in the field of tissue engineering, and bone regeneration.
- the monetite matrices of the invention obtained through the defined molds, are applicable for the support and growth of cells and the previously defined applications.
- the tablets of the invention are applied in the form of several units (as a set of pieces), being arranged so that they adapt completely to the bone defect space, facilitating the homogeneous entry of nutrients, gases and cells throughout the entire area to be repaired, facilitating its recovery thanks to this provision and preventing necrotic areas from occurring.
- the invention refers to the use of the matrices of the invention as growth support for mesenchymal cells of different origins, including adipose origin, osteoblasts, endothelial cells and combinations of undifferentiated or differentiated adult mesenchymal stem cells towards osteoblastic lineage. or endothelial, osteoblasts, osteoclasts, bone osteocytes and endothelial cells, for use in bone regeneration.
- the Monetite matrices of structured porosity of the invention are reabsorbed in vivo in a longer time and similar way than the DCPD, avoiding the inconvenience of its transformation into HA (as example 10 shows where the structured porosity matrices of the invention are compared against brushite matrices made with the structured porosity of the matrices of the present invention).
- these matrices will dissolve at physiological pH, gradually in the extracellular tissues that surround the implant and the cells that colonize it, (endothelial cells, osteoclasts, osteoblasts, macrophages ...) will be responsible for their elimination or reuse As happens in the bone.
- its combination with calcium carbonate in the process of obtaining it, prevents its transformation to PAH.
- DPCD reabsorption Ia begins between 4 and 8 week time period which is suitable for the adjacent cells to colonize the material and can replace material reabsorbed by physiological bone matrix. This biodegradability is adjusted to what happens in the organism, where bone growth in defects can take place in a period of time between 2 and 6 months, depending on the type of bone and the size of the defect (Francone V. 2004 ).
- the monetite can show very low resistance and elasticity with respect to that of the trabecular bone (elasticity 50-100 MPa and compression 5-10 MPa). However, it would be almost impossible to match the mechanical properties of the bone. And, it has been shown that it is sufficient for the material to achieve sufficient mechanical properties to support cell growth, since the cells, upon invading the material will form the organic phase of the implant and improve the mechanical properties.
- the porous monetite matrices of the invention meet this requirement.
- Monetite material is absorbable, reabsorbable, bioactive, has characteristics similar to bone. This material allows cell growth both on its surface and inside, once in the bone defect, it will allow the cells (endothelial, osteoblasts, osteoclasts ...) to form the necessary scaffold that will connect to the healthy bone. Subsequently, the monetite will be eliminated little by little, without undergoing transformation to hydroxy apatite, by the action of the osteoclasts, and the osteoblasts will synthesize the new mineral phase that will replace the monetite, completely eliminating the initial defect.
- a first object of the invention relates to a three-dimensional matrix of monetite of structured porosity characterized by presenting in its structure vertical cylindrical macroporos between 350 and 650 ⁇ m in diameter, which longitudinally cross the matrix from one end to the other, there being a separation between 0.4-0.6 mm between each macropore.
- the diameter of the macropores is preferably 500 ⁇ m ⁇ 60 ⁇ m.
- the separation between macropores is preferably 500 ⁇ m ⁇ 60 ⁇ m.
- Another object of the invention relates to the monetite matrix of structured porosity whose content in monetite is at least 90%, preferably 95% and more preferably 100%.
- a following object of the invention constitutes the monetite matrices of structured porosity characterized by being obtained by thermal transformation of a precursor material.
- said precursor material that is thermally transformed to monetite consists of a mixture of a solid phase composed of basic calcium phosphates, acidic calcium phosphates, a porogen and a retardant that is set by the addition of distilled water.
- the basic phosphate / acid phosphate molar ratio is 1, 6 -1, 8, the concentration of porogens 1-20% by weight, that of retarder between 0.4-0.6% by weight and The proportion (P / L) is 3.
- the basic phosphate / acid phosphate molar ratio is 1785, the concentration of porogens is 3-10% by weight and the retardant is 0.54% in weight.
- the acidic calcium phosphate is calcium monophosphate, the basic calcium phosphate is beta tricalcium phosphate, the porogenic agent calcium carbonate and the retardant is sodium pyrophosphate.
- the precursor material is Brushite.
- Another object of the invention constitutes the three-dimensional matrices of monetite of structured porosity according to previous claims characterized in that they can adopt any type of form required for the repair of a particular bone or tissue defect.
- said die consists of a cylinder with a base diameter between 2 and 50 mm, and a height between 1 and 50 mm.
- said cylinder has a base diameter between 2 and 15 mm, and a height between 1 and 5 mm.
- said cylinder has a minimum perimeter zone of 0.5mm free of macropores.
- the cylinder has:
- Another object of the invention relates to the mold for the preparation of a three-dimensional matrix according to the objects of the previous invention, characterized by presenting a homogeneous distribution of punches of 360-660 ⁇ m in diameter uniformly separated between 0.4-0.6 mm from each other .
- Said mold can be composed of silicone, metal, resistant plastic or any other material that allows its application, being able to adopt any type of required form.
- the mold has a cylinder shape with a base diameter between 2 and ⁇ mm and a height between 1 and 60 mm.
- said cylinder has a base diameter between 2 and 1 ⁇ mm and a height between 1 and ⁇ mm.
- said cylinder has:
- the molar ratio of basic phosphate / acid phosphate is 1.6-1.8, the concentration of porogen is 1-20% by weight, that of retardant between 0.4-0.6% by weight and the proportion (P / L) is 3.
- the molar ratio of basic phosphate / acid phosphate is 1,785, the concentration of porogens is 3-10 % by weight and the retarder is 0.54% by weight.
- the acidic calcium phosphate is calcium monophosphate
- the basic calcium phosphate is beta tricalcium phosphate
- the porogenic agent calcium carbonate and the retardant is sodium pyrophosphate.
- the product of phase 1 is Brushite.
- thermal sterilization is carried out by autoclaving.
- said autoclaving is carried out at 120-130 0 C and for 24-25 minutes.
- the mold used is the mold described in the preceding objects of invention.
- a silicone mold having a cylinder shape with a base diameter between 2 and 50mm, and a height between 1 and 50mm is used prior to the use of said molds.
- said silicone mold has a base diameter between 2 and 15 mm and a height between 1 and 5 mm.
- Another object of the invention constitutes the use of the mold described in the objects of the previous invention, for obtaining calcium phosphates that take their form.
- said calcium phosphate consists of monetite.
- Another object of the invention relates to the use of three-dimensional matrices of monetite of structured porosity as a support for cell cultures.
- Another object of the invention relates to the three dimensional matrices of monetite of structured porosity characterized in that they additionally comprise cells.
- said cells are mesenchymal cells, osteoblasts, osteoclasts, osteocytes, endothelial cells or combinations thereof.
- Another object of the invention relates to the use of three-dimensional monetite matrices of structured porosity with or without cells, for the preparation of a therapeutic agent for the regeneration of bone structure.
- said bone structure regeneration is carried out to combat osteoporosis. DESCRIPTION OF THE FIGURES.
- Figure 1 a) Metal pieces fixed on a glass plate of the same size as the Monetita cylinders to be synthesized b) Silicone molds obtained from the pieces of Figure 1a), with the holes of the size of the pieces that are going to be manufactured, without taking into account for the moment the formation of the macropores c) Metallic mold with metallic punches that will give rise to a homogeneous controlled macroporosity in the monetite matrix.
- Figure 2 Design of an example of a mold used to obtain the monetite matrix, with a homogeneous distribution of vertical pores of 500 ⁇ 60 mm in diameter, regularly spaced and reproducible.
- Figure 3 Photograph of one of the porous monetite matrix shapes seen in elevation (a) and in profile (b). In this image the cylindrical pores of equal size can be seen, distributed regularly by the structure of the matrix and how these pores completely cross the structure.
- Figure 4 Particular embodiments of the monomers / tablets of the invention and their dimensions a) tablet of 5 mm in diameter ( ⁇ ) and 3 mm in height (h) with a total of 12 macropores of 0.5 mm spaced diameters ( ⁇ .m) to each other for 0.5mm (dm) b) 10mm diameter ( ⁇ ) and 3 or 5mm high (h) pickup, with a total of 64 macropores of 0.5mm diameter ( ⁇ .m) spaced apart from each other by 0.5mm (dm) c) 8mm diameter ( ⁇ ) and 3 or 5mm high (h) pickup with a total of 39 macropores of 0.5mm diamete ( ⁇ .
- Figure 5 X-ray diffraction of the precursor porous brushite (before heat treatment) and porous monetite (after heat treatment) obtained after the material transformation and sterilization process.
- the structural analysis of the samples (Rietvel analysis) after the autoclave sterilization shows that the material consists mainly of monetite 95 ⁇ 5% and the rest is ⁇ tricalcium phosphate (also called ⁇ -TCP).
- ⁇ -TCP tricalcium phosphate
- Figure 6 Front (a) and lateral (b) images of the amorphous monetite matrix, that is, without the structured porosity.
- the porosity that is appraised is inherent to the process of obtaining, the majority of the porosity of the biomaterial is composed of micropores, in which the cell colonization cannot be carried out,
- Figure 7 Scanning electron microscopy images at different magnifications of the monetite biomaterial without controlled porosity. These images show a fundamentally microporous biomaterial (c) and with the minimum presence of some macropores (b) arranged randomly, by way of cavities, which in no case get through the matrix (a, b).
- Figure 8 Scanning electron microscopy image in which the monetite biomaterial of the invention is observed with pores of 500 ⁇ im distributed by the matrix.
- Figure 9 Graph of the cytotoxicity study of the Monetite biomaterial of the invention in L929 cells. From the MTT test it is observed that there are no significant differences in the proliferation of L929 cells between those that have been in contact with the monetite and those that have not, which makes it possible to conclude that the structured porosity monetite of the invention is not cytotoxic.
- Figures 10 Images of inverted phase contrast microscopy obtained from the "Mouse Lymphoma assay". As a result of the test, representative images of wells considered as (a) and (b) positive (mutant cells, colony growth) or (c) and (d) as negative (non-mutant cells, absence of colonies) are shown.
- Figure 11 Histogram of the frequencies of mutation of the Monetite of structured porosity of the invention in the presence (Monetite + S9) and absence (Monetite) of metabolic activation. Said frequencies compared to the negative and positive controls used in the presence and absence of metabolic activation allow us to conclude that the monetite of structured porosity of the invention is not a mutagenic biomaterial.
- Figure 12 Determination of the hemocompatibility of the monetite biomaterial of the invention. Osteoblast and AMSC culture media that were for 24 hours in Contact with the monetite of the invention were used to determine the percentage of hemolysis against positive and negative controls. From the graph it can be concluded that the Monetite of the invention is a hemocompatible biomaterial.
- FIG. 13 Scanning electron microscopy images at different magnifications of a structured macroporosity monetite matrix according to the invention.
- the macropores allow mesenchymal stem cells to colonize the surface of the biomaterial (a) and be introduced by said macropores (b, d).
- (c) we observe the longitudinal section of a macropore.
- Cells interact with each other by emitting cytoplasmic prolongations, just as occurs in a physiological tissue.
- Figure 14 Scanning electron microscopy images of mesenchymal stem cells arranged in the monetite biomaterial of uncontrolled porosity. It can be seen that the cells are arranged on the surface of the matrix, without the possibility of colonizing its interior, since they have a size significantly larger than the microporosity that characterizes the biomaterial.
- Figure 15 Proliferation of mesenchymal stem cells arranged on the monetite material with uncontrolled porosity (gray) compared to those arranged on the monetite biomaterial of structured porosity of the invention (black).
- Figure 16 Bone tissue morphological scheme: 1. Cortical bone. 2. Trabecular bone. 3 Haver system. 4 Blood vessel. 5 Havers Channel. 6 Volkmann Channel. 7 Periosteum. 8 Bone lining. 9 Glasses of the periosteum. 10 Osteoclasts 11 Osteoblast. 12 Osteocytes.
- Figures 17 and 18 SEM images in which it can be observed at x40 magnification and at x80 magnification as different concentrations of AMSC predifferentiated to bone are arranged on the same surface of the biomaterial of the invention.
- Figures 17a and b refer to the biomaterial without cells
- Figures 17 c-h are relative to the different cell concentrations used from 0.5x106 to 2x106 cells.
- Figures 18 ah refer to the cell concentrations employed from 3x106 to 6x106 cells.
- Figure 19 Images of confocal micorcopy of the cells on (a) the surface of the structured porosity monetite biomaterial of the invention and (b) inside the macropore channels of said biomaterial after several days in culture.
- the AMSC nuclei predifferentiated inside the pores of the biomaterial are observed (the reconstruction of the pore in its entirety is carried out by mounting serial images). From these images it is observed how an increase of cells occurs in the surface of the biomaterial as well as the walls of the macropores as the growing time increases.
- Figures 20 and 21 Zenith images of SEM at different magnifications of the AMSC cells predifferentiated in the biomaterial at different association times (1, 4, 7, 10 and 15 days on the surface of the biomaterial ( Figures 20 ae respectively) and on the inside the channels of the biomaterial macropores (figures 21 ae respectively).
- Figure 22 and 23 Analysis of the expression of the genes involved in osteogenesis in AMSC such as osteonectin (OTN), osteocalcin (OCA), osteopontin (OPN), type 1 collagen (COL-1), TGF- ⁇ 1 and alkaline phosphatase ( FA), using RT-PCR in undifferentiated AMSC cells ( Figure 22) and predifferentiated ( Figure 23) alone and associated with the biomaterial for 4, 7, 10 and 15 days.
- OTN osteonectin
- OCA osteocalcin
- OPN osteopontin
- COL-1 type 1 collagen
- FA alkaline phosphatase
- Figure 24 Images of confocal microscopy of the unmarked.
- Figure 24 indicates the observations that must be made in the reading of each of the following figures 25 to 31.
- Figure 24 is divided into 4 quadrants: the upper left quadrant (i) refers To the staining of the nuclei of the cells, the upper right quadrant (H) refers to the marking of only the protein, the lower left quadrant (iii) refers to the double staining of cell nuclei + protein and the lower right quadrant (iv) refers to the triple staining in which the cell nuclei + protein + biomaterial are observed.
- the figures a-f are also subdivided with the aforementioned quadrants, the indicated information must be interpreted in each of them.
- Figures 25-26 Image of confocal microscopy of the imununmark of COL-1 of the AMSC predifferentiated on the surface (topview, figure 25) and inside the channels (sideview, figure 26) of the biomaterial at different times of cultivation.
- Figure 27-28 Image of confocal microscopy of the osteocalcin imnunmark in the AMSC predifferentiated on the surface (topview, figure 27) and inside the channels (sideview, figure 28) of the biomaterial at different times of cultivation.
- Figures 29-30 Image of confocal microscopy of the osteopontin mnunmark in the AMSC predifferentiated on the surface (topview, figure 29) and inside the channels (sideview, figure 30) of the biomaterial at different times of cultivation.
- Figure 31 Image of confocal microscopy of the imununmark of type-1 collagen, osteocalcin and osteopontin in the pre-differentiated AMSCs, growing on the surface of the biomaterial (topview figure 31 ac) and inside the channels (sideview, figure 31 df) during 4 days. These results indicate that the predifferentiated MSCs found in the biomaterial are capable of synthesizing and secreting proteins related to bone synthesis.
- Figure 32 and 33 Analysis of fundamental elements by SEM-EDX of the biomaterial and the AMSC associated with the Monetite of structured porosity of the invention for 4 and 7 days (figure 32) and 10 and 15 days (figure 33).
- the images of the left column refer to the specific areas in the center of the channels from which the analyzes of the elements present in the cells (images of the right column) have been performed.
- the graphs indicate a different distribution of elements than the one found in the biomaterial.
- FIG 34 Image of SEM-EDX in which the distribution of the basic elements in an area in which only AMSC are found is shown. In the images of Calcium and Phosphorus, you can see the electrodense particles, formed by the two elements (they are in the same location of the area).
- FIG. 35 Secretion of TGF- ⁇ 1 (pg / ml), obtained from different concentrations of predifferentiated cells growing without monetite for 7 days in culture. A gradual increase in the concentration of TGF- ⁇ 1 (pg / ml) is observed for lower cell concentrations and a slight decrease or destabilization for higher cell concentrations due to the negative feed-back mechanism of TGF- ⁇ 1.
- FIG. 36 Secretion of TGF- ⁇ 1 (pg / ml) obtained from pre-differentiated cells over time in culture. 2x10 6 cells were seeded on a 6cm 2 surface, analyzed
- the secretion at different times in culture observing a typical behavior of feedback mechanisms that consists of an increase in the synthesis and secretion of the mechanism followed by a decrease in secretion until a new increase in secretion begins.
- FIG. 37 Secretion of TGF- ⁇ 1 (pg / ml), obtained from different concentrations of predifferentiated cells growing on the biomaterial for 7 days in culture. From this graph it can be seen how the presence of the factor in the medium correlates with the increase in the number of cells in the biomaterial.
- FIG 38 Secretion of TGF- ⁇ 1 (pg / ml) obtained from predifferentiated cells growing on the biomaterial over time in culture. 2x10 6 cells were seeded on the biomaterials, the secretion is analyzed at different times in culture. The graph shows how there is an increase in secretion from day 1 to day 10 of cultivation, at which point it begins to stabilize and descend moderately.
- Example 1 Method of synthesis of the matrices of the invention
- the solid phase comprises but is not limited to an acidic calcium phosphate, a basic calcium phosphate, a porogen such as calcium carbonate and a setting retardant such as sodium pyrophosphate.
- the solid phase of the calcium cement consists of a basic calcium phosphate and acidic calcium phosphate.
- the basic calcium phosphate is tricalcium-beta phosphate ( ⁇ -TCP) and the acidic calcium phosphate is calcium monophosphate.
- ⁇ -TCP tricalcium-beta phosphate
- the two components are mixed in a molar ratio of 1,785 in mortar with hand for 10 minutes.
- Calcium carbonate is added in concentrations between 1-20% (weight / weight) preferably between 3-10%.
- Sodium pyrophosphate 0.54% (weight / weight) is used as a retarder of the setting reaction.
- ⁇ -TCP tricalcium-beta phosphate
- 34.42g of DCPD and 10.01 g CC in 2: 1 molar ratio
- the mixture is heated in the oven (Veckstar) at 900 0 C for 14 hours.
- the synthesis of ⁇ -TCP occurs according to the reaction: 2CaHPO4 -2H2O + CaCO3 ⁇ Ca3 (PO4) 2 + 5H2O + CO2
- the powder is then screened and the powder having a particle size smaller than 322 ⁇ m is used.
- the liquid phase consists of distilled or double distilled water.
- the solid phase formed by 0.8 g of anhydrous calcium monophosphate, 1.4 g of beta tricalcium phosphate, 12 mg of sodium pyrophosphate and 110 mg of carbonate is weighed and 0.77 ml of the liquid phase is mixed in a ratio.
- the cement is set for 30 minutes in a water bath at 37 0 C.
- the bicarbonate reacts with the hydrogenations of the medium, decomposing into carbon dioxide, forming holes and thus generating a spongy brushite matrix.
- the biomaterial is then washed several times in distilled water to remove acid residues in the medium until it reaches a pH close to 7, which is optimal for cell growth to be carried out in later stages.
- sterilization is carried out.
- the process used for said sterilization comprises autoclaving the set material in a temperature range 120-130 0 C for 24-25 minutes. During this process the brushite is transformed into monetite.
- the resulting cement, brushite is arranged on a surface shaped of interest for setting and subsequent sterilization, thus obtaining a matrix amorphous, with little presence of macropores and irregular distribution thereof, as can be seen in figures 6 a and b,
- Figure 3 shows an example of a structured porosity matrix of monetite produced by the process described in the invention.
- the resulting material shows a spongy appearance with a given pore distribution.
- Figure 5 shows the diffraction diagram of the samples before and after heat treatment in the autoclave. It can be seen in Figure 4 that the heat treatment in addition to sterilizing the material causes the crystalline transformation of the brushite to monetite structure.
- Example 2 Concrete realization of concrete monetite tablets with structured porosity.
- the powder component formed by 0.8 g of anhydrous calcium monophosphate, 1.4 g of beta tricalcium phosphate, 12 mg of sodium pyrophosphate and 110 mg of calcium carbonate was mixed for 30 seconds with 0.77 ml of water.
- the molds described below were applied to the cement for 30 seconds.
- silicone molds with the following dimensions and number of punches were used:
- the punches are cylindrical, with a diameter between 500 ⁇ m ⁇ 60 ⁇ m, separated 500 ⁇ m ⁇ 60 ⁇ m from each other, and distributed respecting a perimeter of 0.5mm (taken from the edge into the mode) free of punches.
- the structure of said punches is that of those represented in Figure 2.
- the bicarbonate reacts with the hydrogenations of the medium decomposing into carbon dioxide forming holes and thus generating a spongy brushite matrix.
- the biomaterial is then washed several times in distilled water to remove acid residues in the medium until it reaches a pH close to 7, which is the optimum for cell growth.
- the resulting material consists of the specified spongy cylindrical pads, constituted by the structured porosity biomatrix of the invention, of the dimensions indicated in each case, with macroporos homogeneously distributed in said pads.
- the indicated molds allowed to obtain the following matrices with homogeneously distributed cylindrical pores, with an average pore size of 500 ⁇ m ⁇ 60 ⁇ m, separated 0.5mm ⁇ 60 ⁇ m from each other, which allow to connect the micro and macropores generated by the porogen:
- these monetite tablets of the invention obtained, have a perimeter of 0.5mm (taken from the edge of the tablet into the same) free of macropores, allowing them to maintain the conditions of mechanical stability and strength necessary to be used in its applications.
- the silicone mold is used to obtain Monetite cylinders of adequate size (without intervening in this phase in the formation of mcaroporosity).
- liquid siliciona was added on the glass plate with the metal parts, and it was expected to polymerize. Once polymerized, it was removed from the glass plate.
- the silicone molds obtained have cylindrical holes of the size of the Monetite units to be manufactured ( Figure 1b). These silicone molds with the holes of the The size of the pieces to be manufactured do not have punches and, therefore, do not yet contemplate the formation of macropores.
- metal molds were manufactured with the dimension of each piece of Monetite obtained with each of the indicated silicone molds.
- Said metal molds consist of two parts, a first one that presents the punches that give rise to the reproducible macroporous component and a lid ( Figure 1c).
- the dimensions of the metal molds manufactured were as follows:
- the punches are cylindrical, with a diameter between 500 ⁇ m ⁇ 60 ⁇ m, separated 500 ⁇ m ⁇ 60 ⁇ m from each other, and distributed respecting a perimeter of 0.5mm (taken from the edge into the mold) free of punches.
- the silicone molds were filled with the product immediately resulting from mixing the solid phase and the liquid phase.
- the silicone mold pieces were removed. The process is simple since the mold is like a very flexible rubber. - Thirdly, the pieces were introduced into the metal mold with the punches, and covered. Said mold is introduced into a water bath at 37 0 C for 30 minutes until the setting ends.
- the formed matrices are subjected to autoclaving between 120 and 13O 0 C for 24-25 minutes, producing their conversion to Monetita, completely sterilized and suitable for use.
- the pieces obtained presented the same porosity and dimensions as the pieces obtained in example 1a (figure 4).
- Example 3 Comparative studies between Monetita matrices of structured porosity and Amorphous Monetita
- the biomaterial arranged in the form of an amorphous matrix obtains an uncontrolled porosity. That is, they show an irregular macropore distribution, produced during the process of obtaining cement, described in examples 1.1 to 1-6.
- the macropores of the amorphous matrix are hollow in the biomaterial and do not connect the internal structure (Figure 7).
- FIGS 6c and 8 show a monetite matrix with structured macropores.
- the homogeneous distribution of the macropores can be seen.
- the monetite matrix of structured porosity will favor a correct bone regeneration by providing adequate conditions for the correct colonization and cell proliferation.
- the macropores (100 to 500 ⁇ M) allow an optimal means for the integral colonization of the cells provided in the matrix, as well as the neovascularization and migration of osteoblasts and oatebclasts of the implant area and the formation of new bone homogeneously throughout the structure provided.
- the structured porosity biomaterial developed in the present invention has a characteristic macroporous structure, which will allow a complete and homogeneous distribution of the osteogenic cells provided in the matrix and also the entry of cells from the recipient tissue, which will colonize and integrate The new structure, to begin its resorption process as well as form a new bone matrix that will be deposited on the implant to give rise to new bone, with mechanical and physiological characteristics very similar to the original tissue.
- sheep were used to which a critical defect was made in the tibia and a stabilization. by osteosynthesis techniques.
- the unstructured Monetita biomaterial was applied in 3 of them and the structured one in 3 others, leaving the adjacent leg in all of them as a control (with formation of the critical defect and stabilization of the fracture but without biomaterial filling) .
- Prior to the implantation of the biomaterials they were seeded with an equal number of mesenchymal stem cells from the adipose tissue obtained from the sheep.
- the formation of new bone tissue is observed restricted to the peripheral area to the implant, leaving the rest of the matrix without cell colonization, nor by the previously seeded cells, or by those of the recipient tissue, and also the formation of a new vascularization is not induced.
- the in vitro tests performed referred to cytotoxicity, genotoxicity (mutagenecity) and hemocompatibility, taking into account that the biomaterial of structured porosity monetite of the invention can be considered as an implantable product that will be in permanent contact with the bone, being The duration of the contact exceeding 30 days.
- these assays determine cell lysis (cell death), cell growth inhibition and other effects on cells caused by medical devices, materials and / or their extracts.
- the extraction conditions being the thickness of the materials> 0.5 mm, 3 cm2 of the material have been put in contact with 1 ml of the culture medium that acts as an extracting agent.
- the cell line used, to test the cytotoxicity of the material was the fibroblastic line of mouse L929 grown in DMEM culture medium with 10% fetal bovine serum.
- the cytotoxicity and proliferation of the monetite of structured porosity was determined by the MTT test.
- This test is based on the metabolic reduction of MTT by the mitochondrial enzyme succinate dehydrogenase in a colored compound (formazan) and determines the mitochondrial functionality of the cells that have been in contact with the monetite of the invention, based on positive and negative controls established.
- the amount of live cells in the culture is proportional to the amount of formazan produced and therefore to the amount of absorbance recorded by a spectrophotometer.
- the in vivo mutagenic potential of the Monetite of structured porosity of the invention was determined by the test called "Mouse Lymphoma Assay". That essay is based on the quantification of thymidine kinase gene mutations in L5178TK +/- mouse lymphoma cells, induced or not after treatment of these cells with the Monetite biomaterial of structured porosity.
- Cells deficient in the Thymidine Kinase (TK) gene due to the TK - / - mutation are resistant to the cytotoxic effects of trifluorothymidine (TFT).
- TFT trifluorothymidine
- the cells capable of producing TK are sensitive to TFT, which inhibits metabolism and stops cell division.
- mutant cells are capable of proliferating in the presence of TFT, while normal cells containing at least one allele of the TK gene are not.
- the test was carried out in 96-well plates and the final result was obtained after visually counting the positive wells (figures 10 a and b), where the growth of a cell colony is observed) and the negative ones (figures 10 c and d, where it is not observe any growth). Once the positive and negative wells of each 96 plate are counted, a series of formulas established for the test are applied and the results are expressed in terms of mutation frequencies.
- the cells were exposed to the product to be tested in the presence and absence of an adequate metabolic activation system, since sometimes it may happen that a product to be tested is not mutagenic, but that the metabolites generated are in vivo from that product.
- the system most commonly used to simulate in vitro liver metabolism is a postmitochondrial fraction called S9 to which cofactors are added and obtained from rat livers treated with enzyme inducers such as Arochlor 1254.
- S9 postmitochondrial fraction
- Arochlor 1254 enzyme inducers
- MMS Methylmethanesulfonate
- the hemolysis tests evaluate the effects produced on the blood or its components by medical devices or materials that come into contact with the blood, using an appropriate model or system.
- the hemolysis tests determine the degree of lysis of the red blood cells and the release of hemoglobin caused by medical devices, materials and / or their in vivo extracts.
- the hemocompatibility of the structured porosity monetite of the invention was determined by a colorimetric assay for the determination of hemoglobin in whole blood and of hemoglobin released to the plasma when the blood is exposed to the monetite. Since the biomaterial is in the solid phase, cell culture media (osteoblasts and AMSC) that were in contact for 24 hours with the monetite were tested. The results show that the coefficient of variation of the calibration lines, samples and quality controls (% CV) is ⁇ 20% in all cases (except in the case of calibrator 6) and 2/3 of the straight line values Quality control shows a percentage of difference from the theoretical (% PVDF) ⁇ 20%, so that the test results are within the established acceptance criteria.
- the hemolysis percentages of the compounds used were the following, considering the hemoglobin concentration value of 10.19 mg / ml as 100% of hemolysis as presented by the blood used:
- Example 5 Comparative study of bioactivity between the amorphous porous monetite matrix and the structured porosity monetite matrix
- bioactivity of a material will depend on both its chemical-physical composition and its structure.
- a study is carried out to determine the effect of the use of the amorphous matrix or of the structured porosity matrix indicated on the proliferation capacity of mesenchymal stem cells, one of the cell lines involved in the process of bone regeneration along with osteoblasts of the recipient tissue.
- the porous biomatrix was obtained, as described above, it was washed with pH 7.4 culture medium for one or two hours to hydrate and neutralize the pH (changing the culture medium 2 or 3 times). Subsequently, adult mesenchymal stem cells of adipose tissue (ATMC) were seeded directly on the material, at a concentration of 0.5.10 6 -6.10 6 cells per cm 2 . After two hours of planting, culture medium was added to cover all the material, renewing it every two or three days.
- ATMC adipose tissue
- the cells were cultured in the biomaterial for 7 days, after which, the biomatrix on whose surface the cells had adhered by electron microscopy was analyzed. scanning (SEM), to observe the ability of adhesion and colonization of said cells on the biomaterial of porous monetite.
- the images obtained by SEM show that mesenchymal stem cells are able to adhere perfectly to the biomaterial, adopting an adequate morphology and that they also establish intercellular contacts, as occurs in a physiological tissue (figure 13 c and d) .
- the cells expand perfectly with the biomaterial, interacting maximally with it and emitting cytoplasmic extensions (filipodes), which increase the contact surface and increase the level of intercellular contact.
- the biomaterial of structured porosity provides a greater surface in which cells can adhere, proliferate and begin to perform their functions in the process of bone regeneration. That is, they can initiate the creation of a new bone matrix that will replace the biomaterial and express signaling molecules that will enhance and direct bone remodeling and neovascularization.
- the cells over time give proliferation values lower than the number of cells arranged in time 0 hours.
- These cells have no place to distribute and are compacted in the macropores without continuity of the surface, inhibiting their proliferation and being located only on the surface of the material without the possibility of colonizing its interior, they could only be introduced into the few randomly arranged macropores.
- These macropores are found by way of cavities that, in no case penetrate the entire structure, which would make it difficult to interact with the surrounding tissue in vivo and the arrival of nutrients and oxygen to all cells.
- These cells can only be distributed on the surface of the biomaterial. These cells they are compacted due to lack of space, inhibiting their proliferation and locating most of them only on the surface of the material.
- the cells arranged in the matrix of monetite with structured porosity are distributed throughout all the pores, inside these and by the surface of the material giving growth values greater than time 0 hours. These cells are not compacted by having a larger surface area of contact with the material and therefore do not inhibit their growth.
- the implant For bone regeneration to succeed, the implant must be integrated into the bone structure of the organism.
- the patient's cells endothelial, osteoblasts, osteoclasts, macrophages, etc.
- the patient's cells endothelial, osteoblasts, osteoclasts, macrophages, etc.
- a sufficient quantity of cells in the product is necessary so that a potent trophic effect is created, which activates the area and triggers the regenerative process.
- the biomaterial must provide a high number of cells, but without said cells reaching the porous structure of the biomaterial.
- the cellular contribution must be important since as the biomaterial is degraded, it has to be replaced by matrix synthesized by the cells themselves.
- the ideal amount of cells is that which occupies practically the entire surface of the biomaterial but does not produce the clogging of the porous structure, for the following reasons:
- the procedure used consisted in sowing 1 cm diameter, 0.5 cm high monetite discs, and 64 macropores with a diameter of 500 ⁇ m, with increasing cell concentrations ranging from half a million cells to 6 million per biomaterial (0, 5x10 6 -1x10 6 -2x10 6 -3x10 6 -4x10 6 -5x10 6 -6x10 6).
- the cells are kept in contact with the biomaterial for 8 days, to allow their adaptation and settlement.
- the results are analyzed by SEM.
- Example 7 Analysis of the evolution of the cells in the matrix. Analysis of the cellular state in the matrix at different times. Once the appropriate cell dose range was selected for disposal in the biomaterial, the evolution of the cells in the biomaterial of structured porosity over time was studied. For this, an in vitro cell behavior analysis was carried out at different times. 7.1 Observation of predifferentiated cells in the structured porosity matrix over time:
- SEM SEM and also a visualization of the cells with nuclear Hoechts staining by confocal microscopy.
- the visualization by SEM provides data on the affinity and interaction capacity of the cells with the biomaterial, through the observation of the contact surface.
- the processing of the samples for SEM eliminates cells from the biomaterial, which can be visualized by fluorescent techniques.
- the SIDEVIEW image ( Figure 19b) is an assembly of several serial images to be able to observe the cells throughout the length of the macropore.
- the cells colonize the inside of the channels from day 1 of association. As time goes by, a greater cellular upholstery and large aggregates are observed at 10 and 15 days of culture.
- the images of the results of observation by SEM also show images of the surface of the biomaterial (TOPVIEW) and of the interior of the pore as a whole (SIDEVIEW).
- the AMSC interacts adequately and homogeneously with the biomaterial of the structured porosity monetite of the invention, the majority of its surface is invaded, without the pore filling, which will allow the passage of nutrients and host cells that will go to the trophic call of the AMSC.
- the Monetite biomaterial of structured porosity has a macroporous distribution that favors the homogeneous distribution of cells throughout the matrix.
- this porous arrangement allows the arrival of nutrients, gases and signaling molecules produced by the same cells. All this determines that the cells are in better conditions and that they can intercommunicate more effectively to express their osteogenic phenotype.
- the new biomaterial structure potentiates the osteoinductive effect of the nature of the matrix (derived from calcium phosphate, like bone), and induces the expression of genes related to osteogenic differentiation.
- analyzes of the expression of genes related to bone differentiation are performed, by RT-PCR, comparing the structure of the amorphous Monetite matrix with respect to the structured porous.
- RNA from the cells that are on the biomaterials and analysis of the expression of the following genes by RT-PCR: alkaline phosphatase, osteopontin, osteonectin and osteocalcin. These genes are directly related to the bone differentiation process and are activated as mesenchymal stem cells and osteoblasts carry out their bone differentiation process.
- the results indicate an induction of the expression of osteoinductive genes in the cells that are in the Monetite biomaterial of structured porosity with respect to the amorphous.
- osteoblasts With respect to osteoblasts, an induction of late differentiation gene expression such as alkaline phosphatase and osteoclacin is observed.
- AMSC cells express all the genes studied, osteonectin, osteocalcin, osteopontin collagen type 1, TGF- ⁇ 1 and the enzyme alkaline phosphatase.
- osteonectin, osteocalcin, collagen Type 1 and TGF- ⁇ 1 maintain their expression at 4, 7, 10 and 15 days of the culture on the biomaterial. Osteopontin expression appears decreased at 4 and 7 days, but is recovered and maintained at 10 and 15 days of culture in the biomaterial.
- the expression of the alkaline phosphatase enzyme is very light in the AMSC, it is lost during cultivation in the biomaterial and begins its expression after 15 days of culture.
- Type 1 collagen, osteopontin and osteonectin are expressed early in osteoprogenitor cells. Osteocalcin appears when mineralization begins.
- the AMSC express both proteins involved in the beginning of osteoblast differentiation and in the final phase of said differentiation. In addition, they are capable of synthesizing collagen, which is part of the organic component of the bone matrix. These proteins once synthesized can be adsorbed and be trapped in the new matrix that is formed.
- the alkaline phosphatase is an enzyme that releases inorganic phosphorus from phosphoric esters, necessary for mineralization, that is, it participates in the mineralization of bone and in the maturation of the osteoid matrix and therefore its expression is very late in the process Give cell differentiation.
- TGF- ⁇ 1 is a potent stimulator of bone formation, potentiates osteoblast differentiation and bone matrix synthesis and inhibits the synthesis of proteases that degrade the matrix. In fact, it is being used as a prognostic serological marker of the consolidation capacity in the process of evolution of pseudoarthrosis.
- the predifferentiated cells still show the same pattern of expression of the genes related to bone regeneration as the AMSC without differentiating.
- the pre-differentiated AMSCs are arranged in the biomaterial, the expression of these genes is maintained, showing no signs of interaction that decrease the expression of genes involved in bone regeneration ( Figure 23).
- the low expression of the alkaline phosphatase enzyme may be due to the fact that in the initial stages of the formation of the osteoid matrix this enzyme does not intervene preferentially. In the beginning of the formation of the bone in the first place there is the synthesis and excretion of proteins to the matrix, these form an ordered structure in which calcium salts will be deposited. The alkaline phosphatase intervenes at the end of the process when the mineralization occurs. This enzyme generates phosphate ions (which in this case are already provided by the biomaterial) and the increase in the concentration of these ions in the matrix creates nucleation centers for the deposition of mineral salts.
- the structured Monetite biomaterial of the invention unlike that of amorphous Monetite, allows a complete colonization of both its external and internal structure by the cells, the arrival of nutrients and gases throughout its structure to maintain high viability profiles and an induction of proliferation, as well as a better expression of genes related to osteosynthesis and generation of new bone matrix.
- Example 8 Analysis of the secretion of extracellular matrix in the biomaterial of structured porosity by the cells over time. POWER.
- Bone is a highly vascularized mineralized connective tissue that contains specialized cells, organic matrix formed by proteins and mineral phase composed of calcium salts.
- the protein matrix Ie allows to be flexible and tolerate tension, while calcium salts Ie give firmness and resistance to pressure.
- the components of the protein matrix are first synthesized, forming an ordered structure in which the calcium salts will subsequently be deposited.
- the protein matrix represents one third of the bone weight. It is formed by proteins such as type-1 collagen (> 95%) and others that are involved in calcium fixation, such as osteocalcin.
- Collagen-1 and OPN are expressed early in osteoprogenitor cells.
- the OCA appears when the mineralization begins and is a useful marker for the final stages of osteoblastic differentiation.
- the predifferentiated cells synthesize in their cytoplasm type 1 collagen, osteopontin and osteocalcin as it happens in bone cells. It has also been shown that predifferentiated cells express the genes of OPN, OCA and type 1 collagen when they are arranged on the monetite matrices of structured porosity of the invention.
- TOPVIEW images (figures 25, 27, 29 and 31a) of the biomaterial surface are presented, and SIDEVIEW images (figures 26, 28, 30 and 31b), corresponding to reconstructions of longitudinal cuts inside the pore.
- the AMSC are capable of synthesizing calcium deposits to form the mineral phase of the bone.
- the osteoblasts participate in the mineralization of the organic matrix, producing 100 nm matrix vesicles surrounded by membrane, in which Ca 2+ and PO4 2 accumulate, rich in alkaline phosphatase and pyrophosphatase, enzymes capable of generating PO4 ions 2-.
- the increase in these ions causes nucleation centers to form, necessary for mineral salts to deposit.
- osteocalcin which according to the results obtained, is part of the organic matrix that synthesizes the predifferentiated cells on the biomaterial.
- the high expression of this protein suggests that cells can secrete calcium deposits to form the mineral of the new bone. Therefore, it is interesting to study whether these cells can release calcium deposits to the extracellular environment.
- This calcium could be part of the new matrix, either forming hydroxyapatite crystals or binding to proteins and being absorbed in the matrix as in the body. The procedure carried out was as follows:
- the images of the results obtained show specific areas in which the distribution of elementary chemical elements has been analyzed by means of SEM-EDX (figures 32 and 33). This technique allows to determine which elements and their proportion in a sample using a high definition. In this case, it is possible to determine if the cells are producing elements related to the mineralization of the bone matrix.
- the graphs of figures 32 and 33 indicate a different distribution of elements from that found in the biomaterial. A completely different distribution of elements appears, among which Silicon is a novelty, a distinctive element from cells, which does not appear in any sample taken in the biomaterial and a very significant increase in Carbon. That is to say in cells we can distinguish:
- electrodense particles appear increasingly whose main chemical composition is phosphorus and calcium ( Figure 34).
- Electronse particles of Calcium and Phosphorus are synthesized and excreted by the cells, since they appear associated with silicon (exclusive of the cells) and the points of Measurements have been taken in an area without biomaterial.
- These particles can be matrix vesicles that are found in the organism, in which Ca 2+ and PO 4 2 " accumulate. These elements are what initiate the formation of the new mineralized bone matrix.
- Silicon formed by the cells is very relevant as an indicator of new matrix formation and bone regeneration capacity. In the body, silicon is concentrated in osteoblasts and is involved in the production of the matrix and in the deposit of mineral salts.
- Silicon acts as an element that allows longitudinal links between proteins and polysaccharides or between polysaccharides. It is involved in the formation of the protein structure ordered in the matrix, so that the correct bone mineralization is carried out.
- Example 9 Analysis of the ability of autocrine secretion of growth factors related to bone regeneration by cells when they are arranged in the biomaterial of monetite with structured porosity. POWER.
- TGF- ⁇ 1 is an important factor in bone remodeling since it is synthesized by osteoblasts, enhancing their differentiation and favoring the synthesis of osteoid matrix (Riancho et Ia 2003). TGF- ⁇ 1 has chemotactic effects on osteoblast precursors, stimulating its proliferation and collagen synthesis (Fernandez-Tresguerres et al 2006).
- Figure 36 shows the secretion of the growth factor by the predifferentiated cells over time in culture. A peak is observed in the synthesis and secretion to the medium at 4 days after cultivation, then a decrease until day 10, after which a new increase in secretion begins.
- - 1 o synthesis and secretion are given to the medium.
- - 2nd it joins its specific receptor on the surface of the recipient cell to exercise its function, at which time a decrease in its presence in the culture medium can be observed.
- the presence of the factor in the medium correlates with the increase in the number of cells in the biomaterial, until a stabilization of the secretion occurs again, which may be due to the fact that it is not necessary to increase the levels for its performance.
- This increase may also not be related to an increase in the number of cells, but due to an induction to enhance the synthesis of extracellular matrix. As of day 10 of association, its synthesis decreases or the factor is mostly linked to receptors exerting their function, not being observed freely in the culture medium.
- the feed-back mechanism of the factor is regulated somewhat differently than that observed when the cells do not grow on the Monetite matrix of structured porosity of the invention, so that the increase in secretion is maintained until day 10, descending from this day.
- predifferentiated cells growing on the monetite biomaterial of structured porosity are capable of synthesizing and secreting the TGF- ⁇ 1 factor to the outside environment.
- the expression of the factor remains constant throughout the time in culture except for day 7 in which a slight lower expression
- the expression in predifferentiated cells growing with and without biomaterial it is similar. Therefore, it is assumed that the different quantification of the factor in both cases is due to a difference in the speed of binding to the receiver and transmission of the signal inside.
- the cells that grow on the biomaterial may also have more receptors and the factor is mostly linked to them, which would be an enhancement of the bone regeneration process, so the detection of the soluble factor in these cases is lower.
- the predifferentiated cells growing on the monetite biomaterial of structured porosity of the invention synthesize and secrete the TGF- ⁇ 1 culture medium. This factor may favor the synthesis of osteoid matrix.
- Example 10 In vivo comparison of the monetite matrices of structured porosity of the invention against structured porosity matrices of Brushita.
- the structured Monetite biomaterial of the present invention has advantages over Brushita, since it is more stable and has a more adequate resorption rate and adapted to bone remodeling.
- exposure to the rabbit skull was carried out by means of a sagittal incision of the scalp.
- the periosteum was carefully dissected, bicortical defects of 1 cm in diameter were prepared.
- the results showed that the implantation zone showed no signs of inflammation with any of the biomaterials used.
- the histological study evidenced the formation of new bone since week 4, as well as the first signs of resorption (perforations in the biomaterials, areas of osteoclast clustering).
- Monetite material can still be observed, which provides more stability to the bone regeneration process and more coupling with the bone remodeling phase.
- the increase in the resorption time of the Monetite biomaterial of the invention will lead to the formation of greater bone mass, since the osteoblasts will have more time for the formation and deposition of a new mineralized bone matrix.
- the resorption rate of the Monetite is more adjusted to bone remodeling, maintaining for longer the adequate scaffolding for the colonization of the osteoblasts and for the synthesis of new bone matrix, without risk of formation of Hydroxyapatite, due to a too high resorption rate, as can happen in the case of Brushita.
- Example 11 Comparison of a particular embodiment of the structured porosity monetite matrix of the invention against a monetite matrix with different porosity structure.
- the biomaterial developed in the present invention has characteristics that are of special relevance to achieve an effective bone regeneration, among which are a homogeneously distributed microporosity and macroporosity, and its application as a set of pieces, which will allow a better adaptation to the bone defect, a homogeneous entry of nutrients, gases and cells throughout the area to be repaired, so that they do not give rise to necrotic areas.
- the regenerative capacity of the tablets of the invention of 5 mm in diameter, 3 mm high and 12 macropores of 0.5 mm in diameter separated by 0.5 mm were compared between if with respect to a Monetita biomaterial that presents the porosity structure of example 1 of the patent application US6605516.
- Said matrix corresponds to a cylinder 10mm in diameter by 10mm high, which has a central channel 2mm in diameter and a hexagonal net of 0.5mm diameter cylindrical pores, parallel to the central 2mm macropore, and separated by a distance of 1mm from each other.
- said matrix does not have a homogeneous pore diameter control, and must be applied in a single piece, so that the full size conforms to the bone defect.
- the histomorphometric analysis confirmed 3 months after implantation a colonization of the osteblasts and osteoclasts of the bone throughout the biomaterial structure of the structured porosity monetite of the invention, and the formation of new bone in a homogeneous way, with a total integration the same at 6 months with an incipient vascular network that will allow the survival of the new tissue formed without the formation of necrotic areas.
- practically all of the new tissue formed is restricted to the peripheral area to the implant, leaving its inner area with a significantly lower colonization of adjacent tissue cells and with no signs of formation of new blood vessels .
- bone defects in patients do not form perfect forms, as occurs when these defects are induced in sheep, as part of an experimental study. Bone defects are very different and the edges of the fracture are often very irregular. In some cases the space that constitutes the bone defect is very limited, as occurs for example in hypertrophic pseudoarthrosis, so introducing a single preformed block that fits in the area is very complicated and is not able to conform to a deformed area .
- the use of the design of the invention a set of Monetita biomaterial pieces, of small size, with a homogeneous macroporic structure, allows its adaptation to complicated bone defects and of different shapes and dimensions, so that the affected area is completely exposed to the biomaterial and the cells provided to activate the healing process.
Abstract
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Priority Applications (10)
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MX2011000162A MX2011000162A (es) | 2008-07-08 | 2009-07-08 | Matrices tridimencionales de monetita porosa estructurada para ingenieria tisular y regeneracion osea, y metodo de preparacion de las mismas. |
ES09793974.8T ES2676070T3 (es) | 2008-07-08 | 2009-07-08 | Matrices tridimensionales de monetita porosa estructurada para ingeniería tisular y regeneración ósea, y método de preparación de las mismas |
EP09793974.8A EP2298696B1 (en) | 2008-07-08 | 2009-07-08 | Three-dimensional matrices of structured porous monetite for tissue engineering and osseous regeneration, and method for the preparation thereof |
JP2011517180A JP5759370B2 (ja) | 2008-07-08 | 2009-07-08 | 組織工学および骨の再生のための、構造化された多孔率を有するモネタイトの三次元マトリクス、および、当該三次元マトリクスの調製方法 |
CN200980126930.9A CN102089238B (zh) | 2008-07-08 | 2009-07-08 | 用于组织工程和骨再生的结构化多孔三斜磷钙石的三维基质及其制备方法 |
CA2729920A CA2729920C (en) | 2008-07-08 | 2009-07-08 | Three-dimensional matrices of structured porous monetite for tissue engineering and bone regeneration, and method of preparation thereof |
RU2010153515/15A RU2491960C9 (ru) | 2008-07-08 | 2009-07-08 | Трехмерные матрицы из структурированного пористого монетита для тканевой инженерии и регенерации кости и способ их получения |
US13/002,939 US9320828B2 (en) | 2008-07-08 | 2009-07-08 | Three-dimensional matrices of structured porous monetite for tissue engineering and bone regeneration, and method of the preparation thereof |
BRPI0910349-0A BRPI0910349B1 (pt) | 2008-07-08 | 2009-07-08 | Biomaterial na forma de um conjunto de partes e método de síntese de uma matriz tridimensional de monetita com porosidade estruturada |
AU2009267935A AU2009267935A1 (en) | 2008-07-08 | 2009-07-08 | Three-dimensional matrices of structured porous monetite for tissue engineering and osseous regeneration, and method for the preparation thereof |
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PCT/ES2009/000358 WO2010004066A1 (es) | 2008-07-08 | 2009-07-08 | Matrices tridimensionales de monetita porosa estructurada para ingeniería tisular y regeneración ósea, y método de preparación de las mismas |
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BR (1) | BRPI0910349B1 (es) |
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Cited By (3)
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US20110305736A1 (en) * | 2010-06-10 | 2011-12-15 | Dr. Suwelack Skin & Health Care Ag | Stratiform Perforated Biomatrices |
US8481802B2 (en) * | 2010-06-10 | 2013-07-09 | Medskin Solutions Dr. Suwelack Ag | Stratiform perforated biomatrices |
WO2012101428A1 (en) * | 2011-01-24 | 2012-08-02 | King's College London | Method of making monetite |
Also Published As
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BRPI0910349A2 (pt) | 2015-10-06 |
US20110158963A1 (en) | 2011-06-30 |
CN102089238A (zh) | 2011-06-08 |
EP2298696B1 (en) | 2018-03-21 |
EP2298696A4 (en) | 2015-08-12 |
PT2298696T (pt) | 2018-06-25 |
JP5759370B2 (ja) | 2015-08-05 |
WO2010004057A1 (es) | 2010-01-14 |
MX2011000162A (es) | 2011-05-24 |
RU2491960C9 (ru) | 2013-11-10 |
AU2009267935A1 (en) | 2010-01-14 |
CN102089238B (zh) | 2014-03-26 |
BRPI0910349B1 (pt) | 2019-03-06 |
CA2729920C (en) | 2013-11-19 |
KR101629041B1 (ko) | 2016-06-09 |
WO2010004066A8 (es) | 2011-03-24 |
CA2729920A1 (en) | 2010-01-14 |
ES2676070T3 (es) | 2018-07-16 |
KR20110061542A (ko) | 2011-06-09 |
RU2491960C2 (ru) | 2013-09-10 |
EP2298696A1 (en) | 2011-03-23 |
JP2011529429A (ja) | 2011-12-08 |
RU2010153515A (ru) | 2012-08-20 |
US9320828B2 (en) | 2016-04-26 |
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