CN1644221A

CN1644221A - Composite material for porous material and gel use thereof

Info

Publication number: CN1644221A
Application number: CN 200510023630
Authority: CN
Inventors: 徐小良; 刘爱红
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-01-26
Filing date: 2005-01-26
Publication date: 2005-07-27

Abstract

A composition of porous material and gel for preparing artificial bone is prepared from collagen and porous material chosen from alpha-tricalcium phosphate/hydroxy apatite, beta-tricalcium phosphate/hydroxy apatite-coral, calcium phosphate/hydroxy apatite, calcium carbonatel tricalcium phosphate, etc.

Description

Composite material of porous material and gel and application thereof

Technical Field

The invention relates to a composite material of a porous material and gel, a preparation method and application thereof in bone and cartilage tissue engineering. In particular to a method for preparing a simulated artificial bone which combines hydroxyapatite with ideal porous structure and collagen. The material of the invention can be widely used for scaffolds of bone tissue engineering and slow release carriers of cells and medicines.

Background

Hydroxyapatite (HAP) has good biocompatibility and osteoconductivity and can be directly bonded to new bone, so that HAPs of various structures and properties have been developed in different ways for repairing damaged or diseased parts of bone. HAP is too stable in vivo to be absorbed easily because it exhibits a crystal structure similar to bone mineral, tending to maintain chemical and biological equilibrium with bone tissue. Tricalcium phosphate (alpha-TCP or beta-TCP) is better absorbed and degraded than HAP, but researchers report that it degrades too quickly to facilitate new bone incorporation. Studies have shown that a certain proportion of β -TCP/HAP biphasic ceramics are more effective in repairing bone defects than the single ceramic.

The pores of the calcined bone are similar to the structure of the cancellous bone, and the calcined bone has good communication between pores, is beneficial to the growth of osteoblasts and blood vessels, and has good bone conduction effect. The calcined bone prepared by the traditional method mainly consists of HAP, although HAP can be directly combined with new bone, HAP is difficult to absorb in vivo, thereby influencing the later remodeling of the new bone and the biomechanical strength of the new bone. There is a need for partially or completely converting HAP in calcined bone to TCP, preparing alpha or beta-TCP/HAP biphasic calcined bone or alpha or beta-TCP calcined bone, and improving the method of preparing absorbable biphasic calcined bone. Chinese patent (application No. 02145493.0) added diammonium hydrogen phosphate and calcined at 900-1300 deg.c to convert part or all of the calcined bone into beta-TCP, but the calcination temperature is still too high and it is necessary to lower the calcination temperature. alpha-TCP is absorbed more rapidly than beta-TCP, and it is necessary to convert the calcined bone partially or completely to alpha-TCP.

The main component of the natural porous coral is calcium carbonate, which is absorbed too fast in vivo and is not beneficial to bone defect repair. U.S. Pat. No. 3929971 and Chinese patent No. 97103827 perform hydrothermal exchange reaction in a closed reaction vessel containing diammonium hydrogen phosphate solution to completely convert calcium carbonate into HAP, but the cost is high and HAP is difficult to absorb. U.S. Pat. No. 4,976,736 discloses the cost of high-cost process for converting the coral surface into HAP by autoclaving the coral surface in a high temperature (> 200 ℃ C.) autoclave after addition of ammonium phosphate. U.S. Pat. No. 4,976,733 converts coral hydroxyapatite partially to beta-TCP by high temperature calcination at 1000 deg.C-1250 deg.C after addition of ammonium phosphate. However, the temperature is too high, and the crystallinity of the material is too high to be absorbed. Reducing the calcination temperature, reducing the crystallinity, or converting it partially or completely to α -TCP, can facilitate its absorption in vivo. The calcium hydrophosphate/HAP, calcium carbonate/TCP and TCP/HAP double-phase coral are prepared by a simple and effective method, and the TCP, HAP, calcium hydrophosphate coral or calcium carbonate coral with high crystallinity can reduce time and cost and adjust the proper in vivo biodegradability of the coral.

At present, the artificial biological ceramics often affect the bone tissue to enter the deep part of the material due to insufficient communication between pores. The improvement of the traffic of the artificially synthesized biological ceramics is beneficial to the growth of new bone tissues and blood vessels.

The porous calcium-phosphorus material has the defects of being too brittle, low in compressive strength and fragile in vivo, so that the conductive osteogenesis capacity of the porous calcium-phosphorus material is influenced. U.S. Pat. No. 6,376,573 discloses a method for improving the compressive strength of a porous calcium-phosphorus material by polymerizing lactic acid and glycolic acid into polylactic acid and polyglycolic acid in the micropores of the porous calcium-phosphorus material, but the catalyst used is cytotoxic. The artificial bone prepared by combining hydroxyapatite and collagen has high compressive strength, but most artificial bones have non-porous structures or have non-ideal pore structures, so that the artificial bone prepared by combining hydroxyapatite and collagen with ideal porous structures needs to be prepared.

The gel can be used as a carrier of growth factors and cells for tissue engineering. Because of the poor osteoconductivity of the gel, osteogenesis is promoted in combination with a porous material that is highly osteoconductive. The fibrinogen and ceramic material are compounded to be used as bone marrow stromal cell carriers in the literature, and the polymer PLA/PGA and alginate are compounded to be used as cartilage cell carriers in the literature, but the effect is not satisfactory, and the compounding method of the material and the cells still needs to be improved. There is no report of using porous materials in combination with gels as carriers for mixtures of genetically transfected cells, cells and growth factors or immunosuppressants. The concentration of cells and growth factors in the porous material can be increased by adopting other gels and improving a composite method, and the osteogenesis inducing capacity is improved.

An ideal tissue engineered bone or cartilage should possess three elements: seed cells, osteoinductive or chondrogenic growth factors such as bone morphogenetic proteins, and scaffolds (or vectors). The compounding method of the material and the cells or the growth factors is a difficult point of tissue engineering, and the attaching efficiency and the density of the cells are difficult problems to be solved in compounding. The porous material has good osteoconductivity, but we have found that when cells are incorporated into the porous material, only a portion of the cells can adhere to the porous material and most of the cells cannot be seeded into the porous material. The porous material without surface modification is not beneficial to cell attachment, and the material needs to be subjected to surface modification to promote the attachment of cells and the material. Chinese patent (02145493.0) has a collagen coating on the surface of calcined bone, which can improve the cell adhesion ability obviously, but is easy to block the pore channels of the material.

Disclosure of Invention

The invention aims to: the gel is a good composite of porous materials and gel, and the application of the porous composite material.

In order to achieve the purpose, the technical scheme of the invention is as follows: the material is formed by compounding a porous material and collagen gel, wherein the porous material is one or a combination of alpha-tricalcium phosphate (alpha-TCP)/Hydroxyapatite (HAP) two-phase calcined bone and coral, beta-TCP/HAP two-phase calcined bone and coral, calcium hydrophosphate/HAP, calcium carbonate/TCP, calcium carbonate/calcium hydrophosphate two-phase coral, calcium hydrophosphate coral, HAP coral, alpha-TCP or beta-TCP calcined bone and coral, natural coral, allogeneic or xenogeneic cancellous bone, an artificially synthesized porous ceramic material and a porous HAP/collagen composite material; the gel is injectable gel, and is one of collagen, alginate, sodium hyaluronate, agarose, chitosan, Pluronic, polyvinylpyrrolidone, polypropylene fumarate gel, Fibronectin (FN) and fibrinogen gel. The porous material has good bone conductivity, and cells are more easily combined into the porous material through the composition of the porous material and the gel.

The porous material is modified in order to increase osteoconductivity. And the improved method is simple and easy to operate.

Wherein, the TCP/HAP biphase calcined bone is prepared by impregnating ammonium dihydrogen phosphate, triammonium phosphate or orthophosphoric acid with the concentration of 0.05M/L-2M/L, calcining at 800-1300 ℃ or 1200-1600 ℃, and combining quenching to partially or completely convert HAP in the calcined bone into beta-TCP or alpha-TCP.

The TCP/HAP diphasic calcined bone can also be prepared by soaking diammonium hydrogen phosphate solution with the concentration of 0.05-2M/L, calcining at 800-900 ℃ or 1200-1600 ℃ and quenching to partially or completely convert HAP in the calcined bone into beta-TCP or alpha-TCP.

The coral is an improved coral obtained by calcining natural coral at 400-600 deg.C to remove organic components and improve the crystallinity of calcium carbonate.

The preparation method of the calcium hydrophosphate/HAP biphasic coral comprises the following steps: the natural coral is prepared by soaking in 0.05-7M/L phosphate solution, performing hydrothermal exchange reaction in autoclave at open low temperature of less than 200 deg.C and low pressure of less than 100 bar, and partially or completely converting calcium carbonate in the coral into calcium hydrogen phosphate/HAP biphasic coral or hydroxyapatite by controlling time for 1-500 hr and concentration of phosphate, wherein the phosphate is one or combination of dipotassium hydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, triammonium phosphate, tripotassium phosphate, trisodium phosphate, calcium hydrogen phosphate, and calcium dihydrogen phosphate.

The preparation method of the calcium carbonate/calcium hydrophosphate double-phase coral comprises the following steps: the natural coral is subjected to exchange reaction at low temperature or normal temperature and normal pressure by dipping in a phosphate solution with the concentration of 0.05-4M/L, wherein the time is controlled to be 2-200 hours, the low temperature or normal temperature is 0-50 ℃, calcium carbonate in the coral is partially or completely converted into calcium hydrophosphate, and the phosphate is at least one of ammonium dihydrogen phosphate, orthophosphoric acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate and calcium dihydrogen phosphate.

The preparation method of the TCP/HAP biphasic coral comprises the following steps: the coral hydroxyapatite is calcined by dipping orthophosphoric acid or an ammonium phosphate solution thereof with the concentration of 0.05-2M/L, the calcination temperature is 800-1000 ℃ or 1200-1600 ℃, and the HAP in the coral is partially or completely converted into beta-TCP or alpha-TCP by combining quenching, wherein the ammonium phosphate is at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate and triammonium phosphate.

The preparation method of the calcium carbonate/TCP double-phase coral comprises the following steps: the natural coral is made through soaking in 1-7M/L phosphate solution, calcining at 800-1300 deg.c or 1200-1600 deg.c, quenching to form two-phase calcium oxide/beta-TCP or CaO/alpha-TCP coral, setting the CaO/TCp coral in saturated humidity carbon dioxide incubator or steam and high concentration carbon dioxide gas container for at least two days to form two-phase calcium carbonate/beta-TCP or calcium carbonate/alpha-TCP coral, and replacing phosphate with at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, calcium biphosphate and calcium biphosphate.

The porous biological ceramic is one of alpha-TCP/HAP ceramic, beta-TCP ceramic or alpha-TCP ceramic, and the preparation method comprises the following steps: mixing beta-TCP/HAP and beta-TCP calcium phosphorus powder with plastic wires with the diameter of 100 microns and the length of 1-10mm, adding 1-15% of gelatin solution for bonding, pressurizing, drying and sintering at 900-1600 ℃, wherein the plastic wires are one or the combination of nylon, polycarbonate, acrylic acid polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate and polyethylene.

The porous ceramic material is compounded with polyglycolic acid (PGA) and/or polylactic acid (PLA), and the compounding method comprises the following steps: PGA and/or PLA are dissolved in organic solvent, after heating properly, the porous material is put into the solution, vacuumized and taken out for drying, and the steps can be repeated twice or more, wherein the organic solvent is acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate and hexafluoroisopropanol (hexafluoroisopropanol).

The preparation method of the artificially synthesized porous HAP/collagen composite material comprises the following steps: dissolving dialyzed soluble collagen in 60mM/L-1.08M/L orthophosphoric acid solution to prepare 0.2% -5% collagen phosphoric acid solution, mixing calcium hydroxide powder or suspension (more than 100mM/L) with naphthalene granules with the diameter of 100 microns or plastic wires with the length of 1-10mM, wherein the volume ratio of the calcium hydroxide powder to the naphthalene granules or the plastic wires is 1: 0.3-3, then mixing the calcium hydroxide powder and the naphthalene granules or the plastic wires with the collagen phosphoric acid solution according to the calcium-phosphorus ratio of 5: 3, rapidly stirring, adjusting the pH value to be 6.5-9, placing the mixture in a 35-41 ℃ water bath box for 4 hours, carrying out heavy pressure ultrafiltration for not less than 24 hours, drying, cutting into small blocks, placing the small blocks in an organic solvent until the naphthalene granules or the plastic are completely dissolved, removing the organic solvent by drying, wherein the collagen accounts for 10-40% of the dry weight, wherein the plastic wires are polycarbonate and acrylic acid polystyrene, one or a combination of polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate, polyethylene or nylon, wherein the organic solvent is at least one of acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate and hexafluoroisopropanol (hexafluoroisopropanol).

The preparation method of the collagen gel solution comprises the following steps: digesting dermis or tendon of mammal with pepsin, extracting with 3% acetic acid or citric acid at 4-11 deg.C and pH2-4 at pH of above three days, adjusting pH to alkaline and pH of 10-11.5, inactivating pepsin, and refrigerating at 0-6 deg.C; finally, a low-salt neutralization method is adopted, namely the pH value is 6.5-7.5; acid salting-out method, namely NaCl concentration is 1-3M/L, and pH is 2-4; and a dialysis method for extracting and purifying the collagen solution by combining the dialysis method, wherein the dialysis method comprises the step of performing dialysis on dilute hydrochloric acid deionized water with the pH value of 1-4 under the aseptic condition to sterilize and remove salt and organic acid.

The method of compounding the porous material with the gel may be a coating method. Such as:

coating the surface of calcined bone and porous material with collagen gel, mixing collagen solution of 0.1-2mg/ml with PBS (with or without NaCl) of various concentrations, incubating at 37 deg.C to form gel, and air drying.

Coating the porous material with cellulose (FN) surface, soaking the porous material in FN 10-500 microgram/mL, incubating at 37 deg.C, and air drying.

On the basis of the technical scheme, the method for compounding the gel and the porous material can also be used for compounding the gel solution and a compound in vitro after mixing the gel solution and the compound to form a gel layer, wherein the compound is one of seed cells, gene transfected cells, growth factors, hormones, immunosuppressive agents and antibiotics or a mixture thereof (without xenogeneic serum). Wherein,

the seed cells can be autologous or allogeneic bone marrow-derived cells, including Bone Marrow Stromal Cells (BMSCs), myogenic cells, osteoblasts, bone precursor cells, periosteal cells, fibroblasts, mesenchymal cells, stem cells including embryonic stem cells and chondrocytes.

The gene transfected cells may be Bone Morphogenetic Proteins (BMPs), transforming growth factors, insulin-like growth factors, Fibroblast Growth Factors (FGF), platelet-derived growth factors, Vascular Endothelial Growth Factors (VEGF), transcription factors such as core binding factor (CBFA-1), calcitonin, parathyroid hormone or growth hormone; the method of gene transfection may be adenovirus, adeno-associated virus, retrovirus, herpes virus, tobacco virus, liposome, plasmid or electroporation.

The growth factors can be bone morphogenetic proteins (BMPs, such as BMP-1-BMP-16), transforming growth factors, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors and vascular endothelial growth factors, and the bone morphogenetic proteins comprise the BMPs, such as BMP-1-BMP-16.

The hormone can be calcitonin, parathyroid hormone, vitamin D, and growth hormone.

The immunosuppressant can be CTLA-4Ig, FK506, Anti-CD154, Anti-CD80, Anti-CD86 monoclonal antibody, and is combined with gene transfected cell or variant cell and loaded in the above vector.

When the compound is acellular, freeze-drying storage and transportation are adopted.

The complex comprises cells and a carrier complex, and is stored and transported at 1-8 ℃.

The alginate is calcium alginate, and the compounding method of the alginate and the porous material is as follows: mixing 0.3-3% sodium alginate with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, sucking into porous material, and dripping 0.05-0.2M/L calcium chloride, calcium gluconate or calcium sulfate solution to form calcium alginate gel.

The collagen and porous material compounding method comprises the following steps: mixing the collagen gel solution with concentration of 1-15mg/ml after dialysis with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or their mixture and Phosphate Buffered Saline (PBS) or culture solution, allowing pH to be neutral, rapidly absorbing into the porous material, and incubating at 37 deg.C to form gel.

The agarose is low-melting point agarose, and the compounding method of the low-melting point agarose and the porous material comprises the following steps: firstly, 0.3% -6% of agarose solution with 37-39 ℃ is mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixture suspension thereof at 37 ℃, and then is quickly absorbed into the porous material, and the porous material is cooled at 0-25 ℃ to form gel.

The compounding method of the fibrinogen and the porous material comprises the following steps: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, sucking into the porous material, and dripping 0.01-0.2M calcium chloride containing 100-3000IU/ml thrombin to form gel.

The compounding method of the sodium hyaluronate and the porous material comprises the following steps: the sodium hyaluronate gel solution can be mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, then compounded with a porous material, implanted into bone and cartilage defect sites, or mixed with the porous material in vivo bone or cartilage defects in open surgeries.

The porous material and the gel composite material are used as carriers for bone and cartilage tissue engineering.

The composite material is used as a carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is particularly suitable for cells transfected by Bone Morphogenetic Protein (BMP) genes, seed cells and a carrier of BMP, transforming growth factors, Vascular Endothelial Growth Factors (VEGF) or Fibroblast Growth Factor (FGF) mixture.

The porous material and gel composite material can be used as a carrier of cells or medicaments for constructing tissue engineering artificial bones, repairing bone and cartilage injuries, bone and cartilage defects, fracture, bone nonunion, periodontal defects, spinal fusion, enhancing the stability of artificial prostheses and enhancing the attachment of tendons or ligaments on bone surfaces.

Molecular tissue engineering combined with the transfection of growth factors such as BMP genes and tissue engineering for the repair of bone and cartilage defects is a new promising approach. Gene therapy for growth factors such as Bone Morphogenetic Proteins (BMP) is used to repair bone and cartilage damage. A disadvantage of adenovirus and the like mediated gene transfection is that it can elicit an immune response in the body and can damage the cells transfected with the gene. Reducing the damage of the body's immune response to gene transfected cells will improve the efficacy of gene therapy. Embedding these cells in a gel such as alginate or agarose reduces the body's exposure of immune cells and antibodies to these cells, thereby reducing damage to these cells. Therefore, the gel carrier can avoid cell diffusion and prevent cells transfected by growth factor genes mediated by adenovirus and the like from being attacked by autoimmune reaction, and plays a role of a slow release carrier of the growth factors.

Injectable vectors for tissue engineering and gene therapy can significantly reduce the trauma of open surgery. At present, collagen, calcium alginate, agarose and fibrin are used as carriers of cells by a non-injection method, calcium alginate is used as carriers for injection of bone marrow stromal cells and chondrocytes, or fibrin is used as carriers for injection of bone marrow stromal cells and periosteum cells, but the collagen, the calcium alginate, the agarose and the fibrin are used for forming gel in vitro and then injected, so that the injection is difficult. The method of forming gel in vivo after injection is favorable for injection and improves the curative effect. For the research of gel as an injectable vector for gene transfected cells, only sodium hyaluronate was reported as an injectable vector for bone marrow stromal cells transfected with BMP-2 gene. There are no reports of gels as injectable carriers for growth factors, immunosuppressants and seed cell mixtures. The selection of proper injectable carrier and injection method have important significance in repairing bone and cartilage defects.

In the invention, the porous material and the gel composite material are used, wherein, a collagen gel solution with the concentration of 1-15mg/ml after dialysis, a growth factor, a hormone solution or/and a cell suspension are mixed in PBS or a culture medium, the volume ratio is 1: 1, the pH value is neutral, and the mixture is immediately injected into a bone or cartilage defect part to form gel, or can be injected into the bone or cartilage defect part in open surgery to form gel.

The alginate solution can be used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof.

The porous material and the gel composite material are applied to bone and cartilage tissue engineering, wherein the method for injecting calcium alginate comprises the following steps: mixing 0.3-3% sodium alginate with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, and injecting sodium alginate mixed solution and 0.05-0.2M calcium chloride, calcium gluconate or calcium sulfate solution into the same part of the defect of bone or cartilage to form gel at the defect position of bone or cartilage.

The invention is applied in bone and cartilage tissue engineering, wherein a low melting point agarose solution is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof.

The agarose injection method comprises the following steps: firstly, 0.3% -3% agarose solution with temperature of 39 ℃ is mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixture thereof at the temperature of 37 ℃, and the mixture is injected into the bone or cartilage defect in vivo by a large-size needle to form gel.

The fibrinogen gel solution is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof.

The fibrinogen gel injection method comprises the following steps: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or their mixture, and injecting fibrinogen mixture and 10-3000IU/ml thrombin in 0.01-0.2M calcium chloride into the same part of bone or cartilage defect to form gel.

The sodium hyaluronate gel solution is independently used as an injectable carrier of seed cells, growth factors, hormones, immunosuppressants or a mixture thereof, is applied to bone and cartilage tissue engineering, and can also be injected into a bone or cartilage defect part in an open surgery to repair bone or cartilage injuries, and the seed cells do not comprise bone marrow stromal cells, gene transfected cells and a carrier of the bone marrow stromal cells transfected by BMP-2 genes.

The principle of the invention is as follows: hydroxyapatite (HAP) is the major component of bone, when ammonium phosphate solution (AP) infiltrates Calcined Bone (CB), where HPO₄ ^2-Ion heating and concentrating to obtain P₂O₇ ^4-、P₂O₇ ^4-OH of HAP capable of reacting with CB^-Reaction to produce PO₄ ^3-Ions. The concentration of ammonium phosphate solution and the number of times of calcination, temperature and time are important factors in determining the composition, content and crystallinity of TCP/HAP biphasic calcined bone. The invention reduces the crystallinity of beta-TCP/HAP dual-phase calcined bone or beta-TCP calcined bone by reducing the calcination temperature (800-900 ℃), which is beneficial to the absorption of materials. The calcination temperature is increased (1200-1600 ℃) and the alpha-TCP/HAP diphase calcined bone or the alpha-TCP calcined bone is synthesized by quenching, which is beneficial to the absorption of materials.

In the invention, besides diammonium hydrogen phosphate is used as a donor of phosphate radical, ammonium dihydrogen phosphate, triammonium phosphate, orthophosphoric acid and the like are also used as donors.

The invention solves the following technical problems:

the main component of the natural coral is calcium carbonate, which is absorbed too fast in vivo and is not beneficial to bone defect repair. The method is used for calcining natural coral at 400-600 ℃ for hours, removing organic components in the coral, improving the crystallinity of calcium carbonate, improving the biomechanical strength of the calcium carbonate and reducing the biodegradability of the calcium carbonate in vivo. Calcium hydrogen phosphate has good biocompatibility and biodegradability. The invention partially or completely converts the coral into calcium hydrogen phosphate/HAP or HAP by hydrothermal exchange reaction in a phosphate solution (such as disodium hydrogen phosphate) under the open condition of low temperature and low pressure, thereby reducing the manufacturing cost and adjusting the proper biodegradability of the coral. The method also comprises the step of adding ammonium phosphate into coral HAP to calcine at the low temperature of 800-1000 ℃, so that HAP is partially or completely converted into beta-TCP to improve the biodegradability. Or the calcination temperature is raised (1200-1600 ℃) and the alpha-TCP/HAP diphase coral or the alpha-TCP coral is synthesized by combining quenching, thereby improving the biodegradability. The direct drying and calcining method is adopted, namely the natural coral is immersed in ammonium phosphate solutions with different concentrations, and calcium carbonate in the natural coral is partially or completely converted into beta-TCP or alpha-TCP through calcination to form calcium carbonate/TCP double-phase coral, so that the cost can be reduced, and the proper biodegradability of the coral can be adjusted.

The communication between pores of the artificially synthesized porous bioceramic is improved, and the growth of new bone tissues and blood vessels is facilitated. Mixing the calcium-phosphorus powder and plastic wires with the diameter of 100-micron and the length of 1-10mm according to different proportions, adding gelatin solution for bonding and pressurizing, and sintering to form the porous ceramic material, thereby obviously improving the communication among pores of the porous ceramic.

In order to enhance the biomechanical strength of the porous calcium-phosphorus material, the polylactic acid (PLA) and/or polyglycolic acid (PGA) are dissolved in a volatile organic solvent and are permeated into micropores of the porous calcium-phosphorus material, and the solvent is solidified after being volatilized to enhance the mechanical strength of the porous calcium-phosphorus material, but the large pores of the material are not blocked. The artificial bone prepared by the method of the invention has the advantages of ideal porous structure of hydroxyapatite and collagen combined simulation artificial bone, good biomechanical strength, good biocompatibility, biodegradability and bone conductivity.

Solves the difficulty of tissue engineering of compounding materials and cells or growth factors. The invention improves the method for coating collagen on the surface of the calcined bone of Chinese patent (02145493.0), reduces the concentration of the collagen to 0.1-2mg/ml, reduces the possibility of blocking the pore channel of the material and improves the biocompatibility. Or the porous material is coated with the surface of cellulose (FN), which can obviously promote the cell adhesion and improve the surface coating effect.

The method adopts various gels such as collagen, alginate, low-melting-point agarose, fibrinogen, sodium hyaluronate, chitosan, polypropylene fumarate gel and the like, mixes the gels with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants or a mixture of the growth factors, the hormones and the seed cells, then compounds the gels with the porous material, forms the gel after treatment, avoids cell diffusion, improves the concentration in the porous material, plays a role of a slow-release carrier of the drugs such as the growth factors and the like, and improves the osteogenesis capacity.

Reducing the damage of the immune response of the body to the cells transfected by the gene will improve the efficacy of the gene therapy. Embedding these cells in a gel such as alginate or agarose prevents immune cells and antibodies from contacting these cells, thereby reducing damage to these cells, preventing adenovirus and other mediated growth factors such as BMP gene-transfected cells from being attacked by autoimmune reactions, and acting as a slow release carrier for growth factors such as BMP. The combination of immunosuppressive agents such as CTLA4Ig in the gel can further reduce the immune response of the body, and is particularly suitable for adenovirus-mediated gene therapy or allogeneic cell transplantation.

Injectable vectors for tissue engineering and gene therapy can significantly reduce the trauma of open surgery. The invention uses collagen, calcium alginate, agarose, hyaluronate, fibrinogen, etc. as the injectable carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or the mixture thereof, adopts the in vivo gel forming method after injection, is easy to inject, and improves the curative effect of repairing bone and cartilage defects.

The invention adopts a method of combining a low-salt neutralization method, an acid salting-out method and dialysis of dilute hydrochloric acid deionized water with pH of 1-4 to extract and purify a collagen solution, improves the purity of the collagen, can sterilize and remove harmful substances to cells such as salt, organic acid and the like, and is a good gel carrier for the cells.

The storage and transportation of tissue engineering products containing cells is a difficult point in tissue engineering. The invention adopts the method that the cells and the carrier are compounded and then are stored and transported at the temperature of 1-8 ℃ for 2-3 days.

The invention has the beneficial effects that:

1. the application is as follows: the porous material and the gel can be used independently or jointly as carriers of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixtures thereof, and are particularly suitable for the carriers of Bone Morphogenetic Protein (BMP) gene transfected cells, seed cells and BMP, transforming growth factors, Vascular Endothelial Growth Factors (VEGF) or fibroblast growth factors (bFGF) mixtures. The tissue engineered artificial bone is constructed, and can be used for repairing bone and cartilage injuries, bone and cartilage defects, bone nonunion, periodontal defects, spinal fusion, enhancing the stability of artificial prostheses or enhancing the attachment of tendons or ligaments on bone surfaces.

2. The biphasic calcined bone retains the structure of the cancellous bone, the pore diameter and the porosity of the biphasic calcined bone are similar to those of the cancellous bone, the pore diameter is about 400-600 mu m, and the porosity is about 85 percent, so that the biphasic calcined bone is beneficial to bone conduction. The observation of the ultramicro structure (2000-4000 times) shows that the material has honeycomb micropores. The biphasic calcined bone has a higher porosity and larger pores, about 0.5-5 μm, than HAP calcined bone. The converted coral such as coral poris still has the pore structure of natural coral and has excellent porosity and communication pore. The porosity of the porcellaran is 61.00 +/-3.44%, the pore diameter is about 200-300 mu m, and the traffic pore diameter is about 190-200 mu m, so that the porcellaran is conductive to bone formation and vascular ingrowth. The material is formed by stacking long spindle or needle-shaped fine particles, honeycomb micropores are formed among the fine particles, the diameter of each micropore is about 2-3 mu m, and the micropores can provide space for the growth factors and other medicaments to stay in the porous material.

3. And (3) biomechanical determination: the existence of beta-TCP has no obvious influence on the mechanical property of the calcined bone, and the maximum compressive strength is as follows: 3.188 ± 0.943MPa, modulus of elasticity: 85.327 +/-21.534 MPa. The maximum compressive strength of the calcium hydrophosphate/HAP biphasic coral is higher than that of natural coral and reaches 20.56 +/-3.121 MPa.

4. Various gels and porous materials have good cellular biocompatibility. After 4 days after the various gels and BMSCs mixtures were loaded on the porous material, scanning electron microscope observation showed that the cells grew well on the surfaces of calcium alginate, agarose, collagen, sodium hyaluronate gel and the porous material.

5. Intramuscular induction of osteogenesis in nude mice: after injecting the mixture of bone marrow stromal cells transfected by adenovirus-mediated human BMP-2 gene and gel into the muscles of nude mice, a large amount of new bone was formed in the muscles of nude mice of collagen group, hyaluronic acid salt group and calcium alginate group. In the agarose group there was a small amount of bone formation, but there were many growth plate-like cartilage formations. There was also growth plate-like cartilage formation in the calcium alginate group.

6. Nude mice subcutaneous induced osteogenesis test: the bone marrow stromal cells transfected by human BMP-2 gene mediated by adenovirus are loaded in calcium alginate, agarose, collagen gel and porous material and implanted into nude mice subcutaneously, and can form a large amount of new bones in BCB.

7. Animal experiments show that the adenovirus-mediated composite artificial bone of BMSCs transfected by human BMP-2 gene, collagen gel and coral or two-phase calcined bone can repair the defects of rabbit radius and sheep long bone.

8. In vivo absorption time: the ratio of the beta-TCP/HAP calcined bone, coral and porous ceramic calcined at 1000 ℃ is 4/6 in vivo absorption rate, about 40% at 6 months and 60% at 1 year. The beta-TCP/HAP ratio is about 70% of 8/2 in vivo absorption rate for 6 months, and is completely absorbed at 1 year, and the ratio meets the requirement of fracture healing.

Drawings

FIG. 1X-ray diffraction analysis: the crystalline phase of the calcined bone powder was analyzed for the composition of the material and the ratio of the components using an X-ray diffractometer (BRUKER-AXS D8 ADVANCE, USA). The unapplied liquid group (a) exhibited the typical HAP pattern (211), while the calcined bone prepared with the addition of 0.3m (b) and 1.0m (c) AP liquid groups exhibited the typical mixed pattern of β -TCP (217) and HAP, with β -TCP/HAP ratios of 4/6 and 8/2, respectively.

FIG. 2X-ray diffraction analysis: the crystal phase of the coral material powder was analyzed by an X-ray diffractometer (Inel XRG3000 USA) for the components of the material and the ratio of the components. a. The natural coral was calcined at 500 ℃ for 5 hours, and XRD showed a calcium carbonate pattern (. + -.) with good crystallinity. b. Natural coral was boiled in 4M diammonium hydrogen phosphate solution in a common autoclave for 80 hours, and XRD showed a mixed pattern of calcium hydrogen phosphate (#) and HAP (+) (ratio 65: 35). c. Natural coral was boiled in 3M disodium hydrogen phosphate solution in a conventional autoclave for 24 hours, and XRD showed a mixed pattern of calcium hydrogen phosphate (#) and HAP (+) (ratio 65: 35). d. The natural coral was reacted in 4M ammonium dihydrogen phosphate solution at normal temperature and pressure for 12 hours, and XRD showed a mixed pattern (ratio 55: 45) of calcium carbonate (. + -.) and calcium hydrogen phosphate (#). e. The natural coral is reacted in 4M ammonium dihydrogen phosphate solution at normal temperature and pressure for 72 hr, and XRD shows the pattern (#) of calcium hydrogen phosphate. f. Soaking natural coral in 4M ammonium hydrogen phosphate solution, oven drying, calcining at 1000 deg.C for 4 hr, and steaming (without CO)₂) Post XRD showed calcium hydroxide ($) and tricalcium phosphate ($)&) Mixed mode (ratio 52: 48).

FIG. 3 shows that the calcium hydrogen phosphate/HAP Biphasic coral has good porosity and communication pores (40X) observed by scanning electron microscope. (B) The material is formed by stacking long spindle or needle-shaped fine particles, wherein cellular micropores with the diameter of about 2-3 mu m (2000 x) are formed among the fine particles.

FIG. 4 is a scanning electron microscope observation: various gels and BMSCs mixtures were loaded on BCB for 4 days. (a) In the calcium alginate group, many spherical cell contours (→) were visible on the surface of the calcium alginate gel. (b) In the agarose group, the contours of many cells were visible on the surface of the agarose gel, and some cells (→) grew out of the gel and attached to the surface of the gel and BCB. (c) In the collagen gel group, many cells (→) grow in the network of collagen or on the surface of BCB. (d) In the collagen surface coating group, BMSCs (→) were fully confluent at the BCB surface (a and b:. times.500; c and d:. times.200).

Figure 5. nude mice intramuscular induction osteogenesis assay: the bone marrow stromal cells transfected with adenovirus-mediated human BMP-2 gene and the gel mixture were injected 5 weeks after the nude mice were intramuscularly. In the collagen group (a), the hyaluronate group (b), and the calcium alginate group (c), a large amount of new bones were formed in the muscle of nude mice; in agarose group (d) there was a small amount of bone formation. In the calcium alginate group (c), and especially the agarose group (d), there are many growth plate-like chondrogenesis (HE staining, x 40).

Figure 6. nude mice subcutaneous induced osteogenesis experiment: adenovirus-mediated bone marrow stromal cells transfected with the human BMP-2 gene were loaded in calcium alginate (a), agarose (b), collagen (c) gel and Biphasic Calcined Bone (BCB) and implanted subcutaneously in nude mice, after 5 weeks, large amounts of new bone (red) were formed in BCB (HE staining,. times.40).

FIG. 7 shows that 5 weeks after adenovirus-mediated complex artificial bone of BMSCs transfected by human BMP-2 gene, collagen gel and coral is implanted into the defect of rabbit radius shaft bone, a large amount of callus is formed in the pores of the coral (HE 40X).

Detailed Description

The invention uses porous material and gel to compound as the carrier of bone and cartilage tissue engineering. The composite material can be used as a carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is particularly suitable for cells transfected by Bone Morphogenetic Protein (BMP) genes, seed cells and a carrier of BMP, transforming growth factors, Vascular Endothelial Growth Factors (VEGF) or fibroblast growth factors (bFGF) mixtures. Can be used for constructing tissue engineering artificial bone, repairing bone and cartilage injury, bone and cartilage defect, bone nonunion, periodontal defect, spinal fusion, enhancing stability of artificial prosthesis or enhancing tendon or ligament attachment on bone surface.

1. The porous material can be calcined bone and related materials, coral and related materials after conversion, porous organic polymers (such as PLA, PGA and the like), artificially synthesized porous ceramic materials, porous HAP/collagen composite materials and other porous materials.

2. The porous material alpha-tricalcium phosphate (alpha-TCP, Ca) synthesized by the method of the invention₃(PO₄)₂) Hydroxyapatite (HAP, Ca)₁₀(PO₄)₆(OH)₂) Biphasic calcined bone and coral, beta-TCP/HAP biphasic calcined bone and coral, calcium hydrogen phosphate (CaHPO)₄) (HAP) biphasic coral, calcium carbonate/TCP biphasic coral, calcium carbonate (CaCO)₃) Calcium hydrogen phosphate biphase coral, calcium hydrogen phosphate coral, HAP coral, beta-TCP or alpha-TCP calcined bone and coral, artificially synthesized porous ceramic material, porous HAP/collagen composite material, etc. These materials can be used alone as carriers of cells or growth factors of bone tissue engineering, and can also be used alone to repair bone defect and bone nonunion.

3. The method for preparing the TCP/HAP biphasic calcined bone comprises the following steps: HAP in the calcined bone is partially or completely converted into beta-TCP or alpha-TCP by impregnating ammonium phosphate solutions of different concentrations (0.05M-2M), calcining and applying different calcination temperatures (800-900 ℃ or 1200-1600 ℃ combined quenching). The specific method comprises the following steps:

sources of cancellous bone: taking the centrum or the femoral head of the cattle and the pig and the lower end of the femur, and drilling the cancellous bone at the center of the centrum or the lower end of the femur by using a hollow drill.

Pretreatment: the aim is to remove as much as possible impurities, soft tissues, fat and bone marrow. Rinsing with running water, rinsing with distilled water, boiling with deionized water for 5-12 hr (preferably 10 hr), rinsing with high-pressure running water for 2 times, and oven drying at 40-100 deg.C for 4 days.

Primary calcination: calcining in a high-temperature furnace at 400-900 ℃ for 3 hours at the heating rate of 2-10 ℃/min. This step may be omitted to simplify the steps.

Dipping: soaking the calcined bone blocks in 0.05-2M ammonium phosphate solution (diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, etc., or orthophosphoric acid can be used for replacing) for 24 hours, removing redundant liquid, and drying at 40-100 deg.C for 4 days. The concentrations of ammonium phosphate solution at which four multi-purpose calcined bones were prepared (TCP/HAP ratios 4/6, 6/4, 8/2, and 10/0, respectively) were about 0.3M, 0.6M, 1.0M, and 1.5M, respectively.

And (3) calcining again: naturally cooling in a high temperature furnace at 800-1300 deg.c (for example, 800-900 deg.c with diammonium hydrogen phosphate) for 1-10 hr at 2-10 deg.c/min to convert HAP in the calcined bone partially or completely into beta-TCP. Or quenching in a high temperature furnace at 1300-1600 deg.c for 1-8 hr and at 2-10 deg.c/min to convert HAP in the calcined bone into alpha-TCP partially or completely. Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature.

4. The adopted natural coral is of order of Halorales in Laurencia serrulata, and commonly used species are Porites (Porites), Orthosiphon (Goniopora) and Alveolopora, and can be collected from the south China sea, southeast Asia or the vicinity of Pacific island. Cutting natural coral into small blocks of proper size, calcining at 400-600 deg.c for several hr to eliminate organic components from coral, raise the crystallinity of calcium carbonate, raise the biological mechanical strength and slow down the biological degradability of calcium carbonate. Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature.

5. The method for preparing the calcium hydrophosphate/HAP double-phase coral or coral hydroxyapatite comprises the following steps: cutting natural coral into small blocks of appropriate size, washing with flowing water, rinsing with 5% sodium hypochlorite for 24 hr, rinsing with flowing water for 1 hr, ultrasonically soaking with distilled water, rinsing with distilled water, and oven drying at 50-100 deg.C. By impregnating various concentrations (0.05M to 7M) of phosphate solutions (disodium hydrogen phosphate (potassium), diammonium hydrogen phosphate, triammonium phosphate, trisodium phosphate (potassium), calcium hydrogen phosphate, monocalcium phosphate, etc.) formulated with deionized or triple distilled water, placing in a high temperature resistant glass bottle (e.g. 250ml, Schott Duran, Germany), unscrewing the bottle cap, and carrying out hydrothermal exchange reaction (1 to 500 hours) at low temperature (< 200 ℃) and low pressure (< 100 bar) in an open ordinary autoclave filled with water to two thirds of the height. Boiling with slow fire, and changing or adding liquid once in 6-12 hr. By controlling the time and the concentration of ammonium phosphate, calcium carbonate in the coral is partially or completely converted into calcium hydrogen phosphate/HAP double-phase coral or hydroxyapatite (coral hydroxyapatite). Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature.

6. The method for preparing the calcium carbonate/calcium hydrophosphate double-phase coral comprises the following steps: rinsing natural coral with 5% sodium hypochlorite, ultrasonic washing, soaking in phosphate solution (ammonium dihydrogen phosphate, or orthophosphoric acid, sodium (potassium) dihydrogen phosphate, calcium dihydrogen phosphate, etc.) of different concentrations (0.05-4M), exchange reaction at low temperature or normal temperature and pressure, and controlling time (2-200 hr), temperature (0-50 deg.C) and phosphate concentration to convert calcium carbonate in coral into calcium hydrogen phosphate. Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature.

7. The method for preparing the TCP/HAP biphasic coral comprises the following steps: soaking coral hydroxyapatite in ammonium phosphate solution (diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, etc. or orthophosphoric acid) of different concentrations (0.05-2M) for 24 hr, removing excessive liquid, and oven drying at 40-100 deg.C for 4 days. Naturally cooling at 800-1000 deg.c for 1-10 hr and 2-10 deg.c/min to convert HAP in coral into beta-TCP. Or quenching at 1300-1600 deg.C for 1-8 hr in a high-temperature furnace at a temperature rise rate of 2-10 deg.C/min to convert HAP in coral into alpha-TCP. Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature. The concentrations of ammonium phosphate solution were about 0.3M, 0.6M, 1.0M and 1.5M for the preparation of four multi-purpose corals (TCP/HAP ratios 4/6, 6/4, 8/2 and 10/0, respectively).

8. The method for preparing the calcium carbonate/TCP double-phase coral comprises the following steps: rinsing natural coral with 5% sodium hypochlorite for 24 hr, rinsing with flowing water for 1 hr, ultrasonic soaking in distilled water, rinsing with distilled water, and drying at 40-100 deg.c for several days. By dipping phosphate solutions (diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, etc., and can also be replaced by orthophosphoric acid) with different concentrations (1M-7M) for several hours to several days, the solution can be aided by heating. Calcining and quenching at different calcining temperatures (800-1300 deg.C or 1200-1600 deg.C) to obtain calcium oxide (CaO)/beta-TCP or CaO/alpha-TCP diphase coral. The heating rate is 2-10 deg.C/min. Placing CaO/TCP coral in a saturated humidity carbon dioxide incubator or a container with steam and high concentration carbon dioxide gas for several days to several weeks until calcium carbonate/beta-TCP or calcium carbonate/alpha-TCP biphasic coral is formed. Flushing with running water, removing

9. Rinsing with ionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature for use.

10. The preparation method of the porous ceramic (beta-TCP/HAP, alpha-TCP/HAP, beta-TCP or alpha-TCP ceramic) comprises the following steps: mixing beta-TCP/HAP or beta-TCP powder with plastic wires with diameter of 100 microns and length of 1-10mm, which can be nylon, polycarbonate, acrylic polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate, polyethylene and the like, according to different proportions (such as volume ratio of 1-3: 1), adding 1-15% of gelatin solution for bonding and pressurizing (100MPa-2000MPa), and sintering at 900-1300 ℃ for 1-8 hours to form a beta-TCP/HAP or beta-TCP material; or sintering at 1200-1600 deg.c for 1-8 hr, and quenching to form alpha-TCP/HAP or alpha-TCP material. The heating rate is 2-10 deg.C/min. Washing with running water, rinsing with deionized water, drying at 40-100 deg.C for several days, and sterilizing at high temperature.

11. The preparation method of the artificially synthesized porous HAP/collagen composite material comprises the following steps: dissolving the dialyzed soluble collagen in 60mM-1.08M orthophosphoric acid solution to prepare 0.2% -5% collagen phosphoric acid solution. The calcium hydroxide powder or the thick suspension is mixed with naphthalene particles or plastic wires (the length is 1-10mm, and the length can be polycarbonate, acrylic acid polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate, polyethylene, nylon and the like) with the diameter of 100-1000 microns, and the volume ratio of the calcium hydroxide powder to the naphthalene particles or the plastic wires is 1: 0.3-2. Then mixing with collagen phosphoric acid solution according to the calcium-phosphorus ratio of 5: 3, quickly stirring, and regulating pH to 6.5-9. Placing in 35-41 deg.C water bath box for 4 hr, performing ultrafiltration under heavy pressure for more than 24 hr, and air drying. Cut into small pieces and placed in a suitable organic solvent such as acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate, hexafluoroisopropanol (hexafluoroisopropanol) and the like until the naphthalene particles or plastic are completely dissolved. And (5) drying in the air to remove the organic solvent. Collagen accounts for 10-40% of the dry weight. Sterilizing with gamma ray for later use.

12. The ceramic material can be compounded with PGA and/or PLA to improve the biomechanical strength of the ceramic material. PGA and/or PLA are first dissolved in an organic solvent such as acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate, hexafluoroisopropanol (hexafluoroisopropanol) and the like to prepare a high-concentration solution, the porous material is put into the solution after appropriate heating, vacuum-pumping is carried out for several times, and the solution is taken out and dried. The steps can be repeated for a plurality of times, and the soaking time is less than 10 minutes when the steps are repeated. Sterilizing with ethylene oxide or gamma ray.

13. Coating the calcined bone and porous material with cellulose (FN) surface, soaking sterile porous material in FN (dissolved in PBS) 10-500 microgram/mL, incubating at 37 deg.C for several hours, and air drying.

14. The preparation method of the collagen gel solution comprises the following steps: removing epidermis, muscle and fat from dermis and tendon of pig, cattle, mouse, human, etc., mincing, adding 3% acetic acid or citric acid (pH 3), mixing with pepsin (100-. Adjusting pH to neutral (pH 6.5-7.5) by adding 4N NaOH solution by low salt neutralization method, centrifuging at 12000rpm for 30min, collecting precipitate, and cooling at 4 deg.C. Adding NaOH solution with pH of 10-11.5 to dissolve collagen precipitate, inactivating pepsin, centrifuging at 4000rpm for 30min, collecting supernatant, and cooling at 4 deg.C. Adding into diluted HCl solution, adjusting pH to 3. Acid salting-out (adding NaCl to concentration of 1.0-3.0M, pH 3) to purify the collagen solution, centrifuging at 12000rpm for 30min, collecting the precipitate, and cooling to 4 deg.C. Freeze-drying, and refrigerating at 4-6 deg.C. Dilute deionized water of hydrochloric acid at pH 1-4 was dialyzed under sterile conditions to sterilize, remove salts and organic acids, and facilitate gel formation. Dissolving collagen with dilute hydrochloric acid solution with pH of 1-4 to obtain collagen solution, and refrigerating at 4-6 deg.C. Putting soluble collagen solution into dialysis bag, placing in 4-6 deg.C cold storage chamber, dialyzing with dilute hydrochloric acid solution with pH of 1 prepared with deionized water for 2 days or more for sterilization, dialyzing with dilute hydrochloric acid solution with pH of 2-4 for 4 days or more, magnetically stirring, and changing solution every 100ml collagen solution for 1.5-2L dialysate every two days for 2-4 times.

15. Collagen surface coating: adjusting the concentration of dialyzed collagen to 0.1-2mg/ml with dilute hydrochloric acid deionized water solution with pH of 2-4, ultrafiltering with needle filter, sterilizing, and refrigerating at 4-6 deg.C. Taking out from refrigerator before use, mixing with 10 times of NaCl-free PBS (1/10 volume), immediately adding high-temperature sterilized calcined bone into the solution, or dripping the mixture onto the calcined bone, vacuumizing, repeating for three times, incubating at 37 deg.C for 0.5-2 hr to make collagen form into gel, placing in sterile culture dish, opening cover, air drying or freeze vacuum drying in ultra-clean bench, and packaging in sterile container without ethylene oxide sterilization. Coating with undialyzed and unsterilized collagen solution, air drying, washing with deionized water, and sterilizing with ethylene oxide or gamma ray.

16. The gel solution compounded with the porous material comprises collagen, alginate, sodium hyaluronate, agarose, fibrinogen, Pluronic, chitosan, polyvinylpyrrolidone, polypropylene fumarate gel and the like, is mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof (without xenogeneic serum), is compounded with the porous material in vitro, forms gel through treatment, and is implanted to repair bone and cartilage defects, or can be mixed in vitro in open surgery or in vivo in defects. Such complexes are cell-free and can be stored or shipped lyophilized. The cell and carrier complex can be stored and transported at 1-8 deg.C, and culture medium without blood type matching alloserum or autologous serum can be added with collagen, alginate, agarose, fibrinogen gel.

17. The compounding method of calcium alginate and porous materials comprises the following steps: sodium alginate is available in various viscosities from Sigma (st. louis, MO) and the like. Mixing 0.3-3% sodium alginate PBS with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or suspension of mixture thereof, sucking into the porous material, and dripping 0.5-0.2M calcium chloride, calcium gluconate or calcium sulfate solution to form gel. Excess solution was removed and xeno-free α -MEM or DMEM was added.

18. Compounding collagen solution with porous material: the soluble collagen solution can be derived from Patnogen S of Gattefosse, France, Vitrogen brand collagen of Cohesion, Roche and Sigma, or extracted from dermis and tendon of pig, cattle, mouse, and human by the method of this patent, and is refrigerated at 4-6 deg.C, pH2-4, and concentration is 2-8 mg/ml. Dilute deionized water of hydrochloric acid at pH 1-4 was dialyzed under sterile conditions to sterilize, remove salts and organic acids, and facilitate gel formation. Mixing the collagen gel solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixture solution or suspension thereof (in PBS or culture medium) to make pH approximately equal to neutral, dropping into the porous material, or putting the porous material into the suspension, vacuumizing, one atmosphere at 10-20 seconds each time, 5-15 seconds apart, repeating three times, rapidly sucking into the porous material, incubating at 37 deg.C, and forming gel for 30 min. The ratio of collagen to suspension was 1: 1.

19. The method for compounding the low-melting-point agarose solution with the porous material comprises the following steps: low-melting agarose (26-29 ℃ C.) was used, available from Sigma (A9414) and the like. Firstly, 0.3-10% agarose solution (39 ℃) is mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixture suspension (37 ℃) thereof, and is quickly absorbed into the porous material, and the porous material is cooled at 0-25 ℃ to form gel.

20. Compounding fibrinogen solution with porous material: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or their mixture, sucking into porous material, dripping 10-3000IU/ml thrombin (0.01-0.2M calcium chloride), and incubating at 37 deg.C for 5 min to form gel.

21. The collagen gel solution can be independently used as an injectable carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is applied to bone and cartilage tissue engineering. The collagen gel solution (concentration 1-15mg/ml) is mixed with seed cells, gene-transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or a mixture solution or suspension thereof (in PBS or culture medium) to make pH about neutral, and is injected into the bone and cartilage defect site immediately thereafter, or may be injected into the bone or cartilage defect site in open surgery for repairing bone or cartilage damage. The ratio of collagen to suspension was 1: 1.

22. Compounding the sodium hyaluronate solution with the porous material: the sodium hyaluronate gel solution can be mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, sucked into the porous material and implanted into the defect part of bone and cartilage, and can also be mixed with the porous material in the defect part of bone or cartilage in vivo in open surgery for repairing bone or cartilage injury.

23. The alginate solution can be used alone as an injectable carrier with seed cells, genetically transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof, for use in bone and cartilage tissue engineering. The alginate injection method: mixing 0.3-3% sodium alginate with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, and injecting sodium alginate mixed solution and 0.5-0.2M calcium chloride, calcium gluconate or calcium sulfate solution into the same part of bone or cartilage defect in vivo by using double-cavity injection needle, bone puncture needle or lumbar puncture needle to form gel in vivo. For example, one volume of 2.4% sodium alginate in Phosphate Buffered Saline (PBS) is mixed with two volumes of BMSCs cell suspension and one volume of 0.1M CaCl₂A double lumen injection needle is injected into the bone defect.

24. The low melting point agarose solution can be independently used as an injectable carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is applied to bone and cartilage tissue engineering. The agarose solution injection method: 0.3% -3% low melting point agarose solution (39 ℃) is mixed with suspension (37 ℃) of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixture thereof, and injected into the bone or cartilage defect in vivo by a large-size needle to form gel in vivo. The gel formation can be promoted by topical cold application of 4 deg.C cold water and ice cubes.

25. The fibrinogen gel solution can be independently used as an injectable carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is applied to bone and cartilage tissue engineering. Fibrinogen gel solution injection method: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, and injecting fibrinogen mixed solution and 10-3000IU/ml thrombin (0.01-0.2M calcium chloride) into the same part of the bone or cartilage defect in vivo by using a double-cavity injection needle, a bone puncture needle or a waist puncture needle to form gel in vivo.

26. The sodium hyaluronate gel solution can be independently used as an injectable carrier of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, and is applied to bone and cartilage tissue engineering.

27. The seed cells can be autologous or allogeneic bone marrow-derived cells (including bone marrow stromal cells), myogenic cells, osteoblasts, bone precursor cells, periosteal cells, fibroblasts, mesenchymal cells, stem cells (including embryonic stem cells), chondrocytes and the like.

Wherein the growth factor can be bone morphogenetic proteins, transforming growth factors, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, vascular endothelial growth factors, etc. The hormone may be calcitonin, parathyroid hormone, vitamin D, growth hormone, etc.

The transfected gene may be a bone morphogenetic protein, transforming growth factor, insulin-like growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, transcription factor such as core binding factor (CBFA-1), calcitonin, parathyroid hormone or growth hormone, etc. The gene transfection method can be adenovirus, adeno-associated virus, retrovirus, herpes virus, tobacco virus, liposome, plasmid or electroporation.

The immunosuppressant can be CTLA-4Ig, FK506, or Anti-CD154, Anti-CD80, Anti-CD86 monoclonal antibody, which is combined with gene transfected cells or allogeneic cells and loaded in the above vector to reduce the immune response of the body to the gene transfected cells or allogeneic cells, and is especially suitable for adenovirus-mediated gene therapy or allogeneic cell transplantation.

A method for preparing a TCP/HAP biphasic calcined bone comprises the following steps:

sources of cancellous bone: taking a bovine vertebral body or a femoral head and the lower end of a femur, and drilling spongy bone at the center of the vertebral body or the lower end of the femur by using a hollow electric drill with the inner diameter of 16-18 mm. Pretreatment: rinsing with running water, rinsing with distilled water, boiling with deionized water for 10 hours, rinsing with high-pressure running water, rinsing with deionized water for 2 times, drying at 70 ℃ and standing for 4 days. Primary calcination: calcining in a high-temperature furnace at 800 ℃ for 3 hours at the heating rate of 5-10 ℃/min. This step may be omitted to simplify the steps. Dipping: soaking the calcined bone blocks in 0.3-1.5M ammonium hydrogen phosphate or ammonium dihydrogen phosphate solution for 24 hr, removing excessive liquid, and oven drying at 70 deg.C for 4 days. The concentrations of ammonium phosphate solution were 0.3M, 0.6M, 1.0M and 1.5M for the preparation of four multi-purpose calcined bones (TCP/HAP ratios 4/6, 6/4, 8/2 and 10/0, respectively). And (3) calcining again: and (3) naturally cooling in a high-temperature furnace at 880 ℃ (diammonium hydrogen phosphate) or 1000 ℃ (ammonium dihydrogen phosphate) for 5 hours at the heating rate of 5 ℃/min to form the beta-TCP/HAP biphase calcined bone. Or quenching and quickly cooling in a high temperature furnace at 1200-1600 ℃ for 3 hours to form the alpha-TCP/HAP biphase calcined bone. Washing with running water, rinsing with deionized water for 2 times, drying at 50-100 deg.C for 4 days, and sterilizing at high temperature.

The adopted natural coral is of order of Halorales in Laurencia serrulata, and commonly used species are Porites (Porites), Orthosiphon (Goniopora) and Alveolopora, and can be collected from the south China sea, southeast Asia or the vicinity of Pacific island. Cutting natural coral into small blocks with proper size, calcining at 500 deg.C for 4 hr, removing organic components from coral, increasing crystallinity of calcium carbonate, improving biomechanical strength, and slowing down biodegradability in vivo. Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Secondly, the method for preparing the calcium hydrophosphate/HAP double-phase coral or coral hydroxyapatite comprises the following steps: cutting natural coral into small blocks of appropriate size, washing with flowing water, rinsing with 5% sodium hypochlorite for 24 hr, rinsing with flowing water for 1 hr, ultrasonically soaking in distilled water for half an hour, rinsing with distilled water for several days, and oven drying at 70 deg.C. Soaking 3M disodium hydrogen phosphate (potassium) or 4M diammonium hydrogen phosphate solution (prepared with deionized water or triple distilled water), placing in a high temperature resistant glass bottle (such as 250ml, Schott Duran, Germany), unscrewing the bottle cap, performing hydrothermal exchange reaction in an open type common pressure cooker with water added to two thirds of the height at low temperature and low pressure for 8-300 hours, and boiling with slow fire. The pressure is about 80kPa, and liquid is added or changed once every 8 to 12 hours. By controlling the time, calcium carbonate in the coral is partially or completely converted into calcium hydrophosphate/HAP double-phase coral or hydroxyapatite (coral hydroxyapatite). Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Thirdly, the method for preparing calcium carbonate/calcium hydrophosphate biphase coral:

cutting natural coral into small blocks (thickness of 6mm) with proper size, washing with flowing water, rinsing with 5% sodium hypochlorite for 24 hr, rinsing with flowing water for 1 hr, ultrasonically soaking in distilled water for half an hour, rinsing with distilled water for several days, and oven drying at 70 deg.C. The exchange reaction is carried out at low temperature (4 ℃) and normal pressure by dipping 1M ammonium dihydrogen phosphate solution, and calcium carbonate in the coral is partially or completely converted into calcium hydrophosphate by controlling the time (2-120 hours). Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Fourthly, the method for preparing the TCP/HAP biphasic coral comprises the following steps:

soaking coral hydroxyapatite in ammonium hydrogen phosphate or ammonium dihydrogen phosphate solution of different concentration (0.3-1.5M) for 24 hr, taking out, removing excessive liquid, and oven drying at 70 deg.C for 4 days. Naturally cooling at 950 deg.c for 5 hr in a high temperature furnace at the rate of 5 deg.c/min to convert HAP in coral into beta-TCP partially or completely. Or quenching at 1400 deg.C for 3 hr in a high temperature furnace at a temperature rise rate of 5 deg.C/min to partially or completely convert HAP in coral into alpha-TCP. Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Fifthly, a method for preparing calcium carbonate/TCP (1: 1) biphase coral: rinsing natural coral with 5% sodium hypochlorite for 24 hr, rinsing with running water for 1 hr, ultrasonically soaking and washing with distilled water, rinsing with distilled water, and oven drying at 70 deg.C for 3 days. Soaking in heated and dissolved 4M diammonium hydrogen phosphate solution for 48 hours, taking out, and drying at 70 ℃ for 3 days. Calcining at 1000 deg.C or 1400 deg.C combined with quenching) to form calcium oxide (CaO)/beta-TCP or CaO/alpha-TCP diphasic coral. The heating rate is 2-10 deg.C/min. Placing CaO/TCP coral in a saturated humidity high concentration carbon dioxide incubator for 3 weeks until calcium carbonate/beta-TCP or calcium carbonate/alpha-TCP diphasic coral is formed. Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Sixthly, a porous ceramic (beta-TCP/HAP, alpha-TCP/HAP, beta-TCP or alpha-TCP ceramic) preparation method comprises the following steps: mixing beta-TCP/HAP or beta-TCP powder with nylon wire with diameter of 300-5 microns and length of 1-5mm according to the volume ratio of 1: 0.6, adding 4% gelatin solution for bonding and pressurizing, sintering at 1000 deg.C (forming beta-TCP/HAP or beta-TCP material) or sintering at 1400 deg.C in combination with quenching (forming alpha-TCP/HAP or alpha-TCP material). Washing with running water, rinsing with deionized water, drying at 70 deg.C for several days, and sterilizing at high temperature.

Seventhly, the preparation method of the artificially synthesized porous HAP/collagen composite material comprises the following steps: the dried soluble collagen after dialysis was dissolved in 100ml of 360mM orthophosphoric acid solution to prepare a 2% collagen phosphoric acid solution. The calcium hydroxide powder or the thick suspension is mixed with naphthalene particles or plastic wires (the length is 1-5mm, and the length can be polycarbonate, acrylic acid polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate, polyethylene, nylon and the like) with the diameter of 100-1000 microns, and the volume ratio of the calcium hydroxide powder to the naphthalene particles or the plastic wires is 1: 0.6. Then mixing with collagen phosphate solution at a ratio of 5: 3, stirring rapidly for 1 min, and adjusting pH to 7. Placing in a 37 ℃ water bath cabinet for 4 hours, gradually adding heavy pressure ultrafiltration for 48 hours under 500MPa, and drying in the air. Cutting into small pieces, and placing in chloroform and the like until the naphthalene particles or the plastic are completely dissolved. And (5) drying in the air to remove the organic solvent. Collagen makes up 25% of the dry weight. Sterilizing with gamma ray for later use.

Eighthly, a ceramic material and PGA/PLA (for example, the ratio is 50: 50) compounding method is adopted, so that the biomechanical strength of the ceramic material is improved. Dissolving PGA/PLA in organic solvent (such as chloroform) to obtain 5.0-10% PGA/PLA solution, heating, adding the porous material into the solution, repeatedly vacuumizing for 4 times, taking out, and air drying. This step can be repeated 3 times, with the soaking time being less than 5 minutes. Sterilizing with ethylene oxide or gamma ray.

Ninthly, adopting a surface coating of cellulose (FN) to the porous material, soaking the porous material in 100 microgram/mL FN (dissolved in PBS), incubating at 37 ℃ for 2 hours, and drying in an ultra-clean bench.

Tenthly, the preparation method of the collagen gel solution comprises the following steps: it is prepared from dermis and tendon of cattle or pig by removing epidermis, muscle and fat, mincing, adding 3% acetic acid (pH 3), mixing with pepsin (250mg/kg), digesting, extracting at 11 deg.C for 10 days, centrifuging at 4000rpm, and collecting supernatant, 30min, 4 deg.C. The pH was adjusted to neutral (pH 7.0) by neutralization with low salt with 4N NaOH solution, centrifuged at 12000rpm for 30min, and the precipitate was collected at 4 ℃. Adding NaOH solution with pH of 11 to dissolve collagen precipitate, adjusting pH of 11, inactivating pepsin, centrifuging at 4000rpm for 30min, collecting supernatant, and cooling at 4 deg.C. Adding into diluted HCl solution, adjusting pH to 3. The collagen solution was purified by acid salting out (NaCl to a concentration of 2.0M and pH 3), centrifuged at 12000rpm for 30min, and the precipitate was collected at 4 ℃. Freeze-drying, and refrigerating at 4-6 deg.C. Dissolving collagen with dilute hydrochloric acid solution with pH of 3 to obtain collagen solution, and refrigerating at 4-6 deg.C. Putting soluble collagen solution into a dialysis bag, placing in a refrigerating chamber at 4-6 deg.C, dialyzing with dilute hydrochloric acid solution with pH of 1 prepared with deionized water for 4 days or more for sterilization, dialyzing with dilute hydrochloric acid solution with pH of 3 under aseptic condition for 4 days or more, magnetically stirring, and changing the solution every 100ml of collagen solution for 2L of dialysate every two days for 3-4 times. Transferring into sterile bottle, and refrigerating at 4-6 deg.C.

Eleven, collagen surface coating: adjusting the concentration of the dialyzed collagen to 1mg/ml with dilute hydrochloric acid deionized water solution with pH of 3.0, ultrafiltering with needle filter, sterilizing, and refrigerating at 4-6 deg.C. Taking out from refrigerator before use, mixing with 10 times of NaCl-free PBS (1/10 volume), immediately adding high-temperature sterilized calcined bone into the solution, vacuumizing, repeating for three times, or dripping the mixture onto calcined bone, incubating at 37 deg.C for 1 hr to make collagen form into gel, placing in sterile culture dish, opening cover, drying by ultraviolet irradiation in ultra-clean bench, and packaging in sterile container without ethylene oxide sterilization. Coating with collagen solution without dialysis and ultrafiltration, air drying, washing with deionized water, and sterilizing with ethylene oxide or gamma ray.

Twelve, compounding calcium alginate with porous materials: medium viscosity sodium alginate (a2033, etc.) is available from Sigma (st. Mixing 1.6% sodium alginate PBS solution with bone marrow stromal cell suspension (optionally containing 1-5mg CTLA4Ig) transfected by adenovirus-mediated human BMP (Adv-hBMP) gene, or mixing 1.6% sodium alginate PBS solution with bone marrow stromal cells and 5-10 mg BMP suspension (optionally containing 1-100 μ g Vascular Endothelial Growth Factor (VEGF) or basic fibroblast growth factor (bFGF)), mixing with porous material, sucking into porous material, vacuumizing, repeating for three times, and adding 0.1M calcium chloride solution dropwise to form gel. The excess calcium chloride solution is removed and alpha-MEM or DMEM containing blood group matched allogenic serum or autologous serum can be added. The cell and carrier compound can be placed in a culture solution without xenogeneic serum (can contain blood type matched allogenic serum or autologous serum), and stored and transported at 1-8 ℃. Can be mixed with porous material in bone defect in vivo, 0.1M calcium chloride solution is dripped to form gel, and excessive calcium chloride solution is removed.

Thirteen, calcium alginate is compounded with porous materials as a carrier of growth factors: mixing 2% sodium alginate PBS solution and 10-5 mg BMP suspension (containing 1-100 μ g VEGF or bFGF) in the same amount, mixing with porous material, sucking into the porous material, vacuumizing, repeating for three times, and adding 0.1M calcium chloride solution dropwise to form gel. Removing excessive calcium chloride solution, lyophilizing, and refrigerating.

Fourteen, collagen solution and porous material compounding method: the soluble collagen solution can be derived from Patnogen S of Gattefosse, France, and Vitrogen collagen of Cohesion, or extracted from dermis and tendon of pig, cattle, and human by the method of this patent, and has pH of 3 and concentration of 3-6mg/ml (optimally 5 mg/ml). Dialyzing with dilute hydrochloric acid solution of pH 3 under aseptic condition for 8 days at 4-6 deg.C. Collagen gel solution and 1X 10⁷Mixing the A dv-hBMP gene transfected marrow stromal cell suspension (in PBS or culture medium) with equal amount, making pH approximately equal to neutral, dripping into calcined bone, or putting calcined bone into suspension, vacuumizing, one atmosphere, 10-20 seconds each time, 5-15 seconds at intervals, repeating for three times, quickly sucking into porous material, incubating at 37 deg.C, and forming gel for 30 min.

Fifteen, compounding the collagen solution and the porous material as a carrier of the growth factor:

the concentration of the soluble collagen solution is 3-6mg/ml (optimally 5mg/ml), and the pH is 3. Dialyzing with dilute hydrochloric acid solution of pH 3 under aseptic condition for 8 days at 4-6 deg.C. Mixing collagen gel solution with 10 μ g-5mg BMP suspension (optionally containing 1 μ g-100 μ g VEGF or bFGF) at equal amount, making pH about neutral, dripping into calcined bone, or putting calcined bone into suspension, vacuumizing, repeating for three times, rapidly sucking into porous material, incubating at 37 deg.C for 30min to form gel, lyophilizing, and refrigerating.

Sixthly, compounding the low-melting-point agarose solution with the porous material:

low-melting agarose (26-29 ℃) was obtained from Sigma (A9414). A1.6% agarose solution (39 ℃) was first incubated with 1X 10⁷The bone marrow stromal cell suspension (in PBS or culture medium, 37.5 ℃) (optionally containing 1-5mg CTLA4Ig) transfected by Adv-hBMP gene is mixed in equal amount, or mixed with bone marrow stromal cell and suspension (37.5 ℃) of 5-10 mg BMP (optionally containing 1-100 ug VEGF or bFGF) in equal amount, and then the mixture is quickly sucked into the porous material, vacuumized, repeated three times, and cooled at 4 ℃ to form gel.

Seventhly, compounding the low-melting-point agarose solution and a porous material to serve as a carrier of the growth factor:

low-melting agarose (26-29 ℃) was obtained from Sigma (A9414). Mixing 2% agarose solution (39 deg.C) and 10 μ g-5mg BMP suspension (37.5 deg.C, optionally containing 1 μ g-100 μ g VEGF or bFGF) in equal amount, quickly sucking into porous material, vacuumizing, repeating for three times, cooling at 4 deg.C to form gel, lyophilizing, and refrigerating.

Eighteen, compounding the sodium hyaluronate solution with the porous material:

1% sodium hyaluronate (Sofast) with a molecular weight of 160-250kD from Furrida, Dafurida, Shandong can be used. 1% sodium hyaluronate gel solution with 1/2 volumes of 2X 10⁷The/ml bone marrow stromal cell suspension (in PBS or culture medium) transfected by the Adv-hBMP-2 gene is mixed and then absorbed into the porous material to be implanted into the defect part of the bone and the cartilage, and can also be mixed with the porous material in the defect part of the bone or the cartilage in vivo in the open surgery to repair the bone or the cartilage injury.

Nineteen, a method for compounding a fibrinogen solution with a porous material:

human fibrinogen is available from RAAS corporation (FIBRORAAS). Human fibrinogen was dissolved in a solution at 37 deg.C, and a 1% fibrinogen solution was mixed with a suspension of Adv-hBMP-2 gene-transfected bone marrow stromal cells (in PBS or culture medium) (optionally containing 1-5mg CTLA4Ig), aspirated into the porous material, evacuated, repeated three times, added 1 volume of 100IU/ml thrombin (0.05M calcium chloride in PBS), and incubated at 37 deg.C for 5 minutes to form a gel.

Twenty, collagen gel solution as injectable carrier method:

the collagen gel solution (concentration 5mg/ml) was dialyzed against a dilute hydrochloric acid solution having a pH of 3, and then mixed with 1X 10⁷The bone marrow stromal cell suspension (in PBS or culture medium) transfected by Adv-hBMP-2 gene is mixed in equal amount to make pH about neutral, and then injected into bone and cartilage defect site immediately or in open surgery to form gel in vivo for repairing bone or cartilage wound, such as cartilage defect, bone defect and fracture disunion.

Twenty-one, sodium hyaluronate solution as injectable carrier method:

1% sodium hyaluronate (Sofast) with a molecular weight of 160-250kD from Furrida, Dafurida, Shandong can be used. The 1% sodium hyaluronate gel solution can be mixed with 1/2 volume bone marrow stromal cell suspension and 5 microgram-10 mg human BMP-2 and then injected into bone and cartilage defect sites, and can also be injected into bone or cartilage defect sites in open surgery for repairing bone or cartilage wounds, such as cartilage defects, bone defects and nonunion.

Twenty three, alginate solution as an injectable carrier method:

the alginate injection method: firstly, 1.6% sodium alginate PBS solution and 1 × 10⁷The bone marrow stromal cell suspension (which may contain 1-5mg CTLA4Ig) transfected by Adv-hBMP-2 gene is mixed in equal amount or mixed with bone marrow stromal cell suspension (which may contain 5 ug-10 mg human BMP-2) in equal amount, and then the mixture of sodium alginate cells and 1/2 volume of 0.1M calcium chloride solution are injected into the same site of the bone or cartilage defect to form gel in vivo.

Twenty-four, low melting point agarose solution as injectable carrier method:

the agarose solution injection method: a1.6% agarose solution (39 ℃) is mixed with a bone marrow stromal cell suspension (37.5 ℃ C., in PBS or culture medium) transfected by Adv-hBMP-2 gene (which may contain 1-5mg of CTLA4Ig), or mixed with the same amount of bone marrow stromal cell suspension (37.5 ℃ C., which may contain 5. mu.g-10 mg of human BMP-2), and the mixture is injected into a bone or cartilage defect in vivo by using a large-size needle to form a gel in vivo. The gel formation can be promoted by topical cold application of 4 deg.C cold water and ice cubes.

Twenty five, a method of using fibrinogen gel solution as an injectable carrier:

human fibrinogen is available from RAAS corporation (FIBRORAAS). Human fibrinogen is dissolved in a solution at 37 ℃, 1% fibrinogen solution is mixed with a bone marrow stromal cell suspension (in PBS or culture medium) transfected by Adv-hBMP-2 gene (which can contain 1-5mg of CTLA4Ig), and fibrinogen mixed solution and 200IU/ml thrombin (in 0.05M calcium chloride PBS) are respectively injected into the same part of the bone or cartilage defect in vivo by using a double-cavity injection needle, a bone puncture needle or a lumbar puncture needle to form gel in vivo.

Claims

1. The material is formed by compounding a porous material and collagen gel, wherein the porous material is one or a combination of alpha-tricalcium phosphate/hydroxyapatite two-phase calcined bone and coral, beta-tricalcium phosphate/hydroxyapatite two-phase calcined bone and coral, calcium hydrophosphate/hydroxyapatite, calcium carbonate/tricalcium phosphate, calcium carbonate/calcium hydrophosphate two-phase coral, calcium hydrophosphate coral, hydroxyapatite coral, alpha-tricalcium phosphate or beta-tricalcium phosphate calcined bone and coral, natural coral, allogeneic or xenogeneic cancellous bone, an artificially synthesized porous ceramic material and a porous hydroxyapatite/collagen composite material; the gel is an injectable gel, and is one of collagen, alginate, sodium hyaluronate, agarose, chitosan, Pluronic, polyvinylpyrrolidone, polypropylene fumarate gel, cellulose and fibrinogen gel.

2. The material of claim 1, wherein the tricalcium phosphate/hydroxyapatite biphasic calcined bone is prepared by: ammonium dihydrogen phosphate, triammonium phosphate or orthophosphoric acid with the concentration of 0.05M/L-2M/L is impregnated, and the HAP in the calcined bone is partially or completely converted into beta-tricalcium phosphate or alpha-tricalcium phosphate by calcination and calcination at the temperature of 800-1300 ℃ or 1200-1600 ℃ in combination with quenching.

3. The material of claim 2, wherein the tricalcium phosphate/hydroxyapatite biphasic calcined bone is prepared by: the hydroxyapatite in the calcined bone is partially or completely converted into beta-tricalcium phosphate or alpha-tricalcium phosphate by soaking diammonium hydrogen phosphate solution with the concentration of 0.05-2M/L, calcining at the temperature of 800-900 ℃ or 1200-1600 ℃ and quenching.

4. The porous material and gel composite material of claim 1, wherein said coral is a modified coral obtained by calcining natural coral at 400 ℃ to 600 ℃ to remove organic components and increase the crystallinity of calcium carbonate.

5. The material compounded of porous material and gel as claimed in claim 1, wherein the calcium hydrogen phosphate/hydroxyapatite biphasic coral is prepared by the following steps: the natural coral is immersed in 0.05-7M/L phosphate solution, hydrothermal exchange reaction is carried out in an autoclave at open low temperature of less than 200 ℃ and low pressure of less than 100 bar, calcium carbonate in the coral is partially or completely converted into calcium hydrogen phosphate/hydroxyapatite biphase coral or hydroxyapatite by controlling the time for 1-500 hours and the concentration of phosphate, wherein the phosphate is one or combination of dipotassium hydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, triammonium phosphate, tripotassium phosphate, trisodium phosphate, calcium hydrogen phosphate and calcium dihydrogen phosphate.

6. The material compounded of porous material and gel as claimed in claim 1, wherein the calcium carbonate/calcium hydrogen phosphate biphasic coral is prepared by: the natural coral is subjected to exchange reaction at low temperature or normal temperature and normal pressure by dipping in a phosphate solution with the concentration of 0.05-4M/L, wherein the time is controlled to be 2-200 hours, the low temperature or normal temperature is 0-50 ℃, calcium carbonate in the coral is partially or completely converted into calcium hydrophosphate, and the phosphate is at least one of ammonium dihydrogen phosphate, orthophosphoric acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate and calcium dihydrogen phosphate.

7. The material of claim 1, wherein the tricalcium phosphate/hydroxyapatite biphasic coral is prepared by: the coral hydroxyapatite is prepared through soaking orthophosphoric acid or its ammonium phosphate solution in the concentration of 0.05-2M/L, calcining at 800-1000 deg.c or 1200-1600 deg.c, and quenching to convert HAP in coral into beta-tricalcium phosphate or alpha-tricalcium phosphate, with the ammonium phosphate being at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate and triammonium phosphate.

8. The material of claim 1, wherein the calcium carbonate/tricalcium phosphate biphasic coral is prepared by: the natural coral is produced through soaking in 1-7M/L phosphate solution, calcining at 800-1300 deg.c or 1200-1600 deg.c, quenching to form biphase calcium oxide/beta-tricalcium phosphate or CaO/alpha-tricalcium phosphate coral, setting the CaO/tricalcium phosphate coral in saturated humidity carbon dioxide incubator or steam and high concentration carbon dioxide gas container for at least two days to form biphase calcium carbonate/beta-tricalcium phosphate or calcium carbonate/alpha-tricalcium phosphate coral, and replacing phosphate with at least one of diammonium hydrogen phosphate, ammonium dihydrogen phosphate, triammonium phosphate, calcium hydrogen phosphate and calcium dihydrogen phosphate.

9. The material of claim 1, wherein the porous bioceramic is one of α -tricalcium phosphate/hydroxyapatite ceramic, β -tricalcium phosphate ceramic, or α -tricalcium phosphate ceramic, and the preparation method comprises: mixing beta-tricalcium phosphate/hydroxyapatite and beta-tricalcium phosphate calcium phosphorus powder with plastic wires with the diameter of 100 microns and the length of 1-10mm, adding 1-15% of gelatin solution for bonding, pressurizing, drying in the air, and sintering at 900-1600 ℃, wherein the plastic wires are one or the combination of nylon, polycarbonate, acrylic acid polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate and polyethylene.

10. The material compounded with porous material and gel according to claim 9, characterized in that the porous ceramic material is compounded with polyglycolic acid (PGA) and/or polylactic acid (PLA) by the following method: dissolving PGA and/or PLA in organic solvent, heating, adding the porous material into the solution, vacuumizing, taking out, and air drying, wherein the organic solvent is acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate, and hexafluoroisopropanol.

11. The material compounded with porous material and gel as claimed in claim 1, characterized in that the preparation method of the artificially synthesized porous HAP/collagen composite material comprises: dissolving dialyzed soluble collagen in 60mM/L-1.08M/L orthophosphoric acid solution to prepare 0.2% -5% collagen phosphoric acid solution, mixing calcium hydroxide powder or suspension (more than 100mM/L) with naphthalene granules with the diameter of 100 microns or plastic wires with the length of 1-10mM, wherein the volume ratio of the calcium hydroxide powder to the naphthalene granules or the plastic wires is 1: 0.3-3, then mixing the calcium hydroxide powder and the naphthalene granules or the plastic wires with the collagen phosphoric acid solution according to the calcium-phosphorus ratio of 5: 3, rapidly stirring, adjusting the pH value to be 6.5-9, placing the mixture in a 35-41 ℃ water bath box for 4 hours, carrying out heavy pressure ultrafiltration for not less than 24 hours, drying, cutting into small blocks, placing the small blocks in an organic solvent until the naphthalene granules or the plastic are completely dissolved, removing the organic solvent by drying, wherein the collagen accounts for 10-40% of the dry weight, wherein the plastic wires are polycarbonate and acrylic acid polystyrene, one or a combination of polystyrene, acrylonitrile-butadiene-styrene, polymethyl methacrylate, polyethylene or nylon, wherein the organic solvent is at least one of acetone, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate and hexafluoroisopropanol.

12. The material compounded of porous material and gel as claimed in claim 1, wherein the collagen gel solution is prepared by the following steps: digesting dermis or tendon of mammal with pepsin, extracting with 3% acetic acid or citric acid at 4-11 deg.C and pH2-4 at pH of above three days, adjusting pH to alkaline, adjusting pH to 10-11.5, inactivating pepsin, and refrigerating at 0-6 deg.C; finally, a low-salt neutralization method is adopted, namely the pH value is 6.5-7.5; acid salting-out method, namely NaCl concentration is 1-3M/L, and pH is 2-4; and a dialysis method for extracting and purifying the collagen solution by combining the dialysis method, wherein the dialysis method comprises the step of performing dialysis on dilute hydrochloric acid deionized water with the pH value of 1-4 under the aseptic condition to sterilize and remove salt and organic acid.

13. The material of claim 12, wherein the calcined bone and the porous material are coated with collagen gel, 0.1-2mg/ml collagen solution is mixed with PBS with various concentrations, incubated at 37 ℃ to form gel, and dried.

14. The material of claim 1, wherein the porous material is coated with a surface layer of cellulose, and the porous material is soaked in FN at a concentration of 10-500 μ g/mL, incubated at 37 deg.C, and air-dried.

15. The material compounded by the porous material and the gel as claimed in claim 1, wherein the gel is compounded with the porous material by mixing a gel solution with the compound and compounding the gel solution with the porous material in vitro to form a gel layer, wherein the compound is one of seed cells, gene transfected cells, growth factors, hormones, immunosuppressants and antibiotics or a mixture thereof.

16. The material compounded with porous material and gel according to claim 15, characterized in that the seed cells can be autologous or allogeneic bone marrow-derived cells including bone marrow stromal cells, myogenic cells, osteoblasts, bone precursor cells, periosteal cells, fibroblasts, mesenchymal cells, stem cells including embryonic stem cells and chondrocytes.

17. Use of a material composited with a porous material and a gel according to claim 15, characterized in that said cells transfected with genes can be bone morphogenic proteins, transforming growth factors, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, vascular endothelial growth factors, transcription factors such as core binding factors, calcitonin, parathyroid hormone or growth hormones; the method of gene transfection may be adenovirus, adeno-associated virus, retrovirus, herpes virus, tobacco virus, liposome, plasmid or electroporation.

18. Use of a porous material and gel composite according to claim 15, characterized in that said growth factors can be bone morphogenetic proteins including BMPs, transforming growth factors, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, vascular endothelial growth factors.

19. Use of a porous material and a gel composite according to claim 15, characterized in that said hormone can be calcitonin, parathyroid hormone, vitamin D, growth hormone.

20. The use of a porous material complexed with a gel as claimed in claim 15, wherein the immunosuppressive agent is CTLA-4Ig, FK506, or Anti-CD154, Anti-CD80, Anti-CD86 monoclonal antibody, conjugated to genetically transfected cells or allogeneic cells and loaded into the vector.

21. The method for storing and transporting a material compounded of a porous material and a gel as claimed in claim 15, wherein the compound is stored and transported in a freeze-dried state when the compound is free of cells.

22. The method for storing and transporting a material compounded of a porous material and a gel as claimed in claim 15, wherein the compound is a compound containing cells and a carrier, and is stored and transported at 1-8 ℃.

23. The material of claim 1, wherein said alginate is calcium alginate, and is compounded with the porous material by the method comprising: mixing 0.3-3% sodium alginate with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, sucking into porous material, and dripping 0.05-0.2M/L calcium chloride, calcium gluconate or calcium sulfate solution to form calcium alginate gel.

24. The material compounded with a porous material and a gel according to claim 1, characterized in that the collagen and porous material compounding method: mixing the collagen gel solution with concentration of 1-15mg/ml after dialysis with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or their mixture and phosphate buffer or culture solution, allowing pH to be neutral, rapidly absorbing into the porous material, and incubating at 37 deg.C to form gel.

25. The material of claim 1, wherein the agarose is a low-melting agarose, and the low-melting agarose is complexed with the porous material by: firstly, 0.3% -6% of agarose solution with 37-39 ℃ is mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixture suspension thereof at 37 ℃, and then is quickly absorbed into the porous material, and the porous material is cooled at 0-25 ℃ to form gel.

26. The material of claim 1, wherein the fibrinogen is complexed with the porous material by a method comprising: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, sucking into the porous material, and dripping 0.01-0.2M calcium chloride containing 100-3000IU/ml thrombin to form gel.

27. The material of claim 1, wherein the sodium hyaluronate is compounded with the porous material by the following steps: the sodium hyaluronate gel solution can be mixed with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or a mixture thereof, then compounded with a porous material, implanted into bone and cartilage defect sites, or mixed with the porous material in vivo bone or cartilage defects in open surgeries.

28. Use of the porous material and gel composite according to claim 1 as a carrier for bone and cartilage tissue engineering.

29. Use of a porous material and gel composite according to claim 1, characterized in that said composite is used as a carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof, in particular for cells transfected with Bone Morphogenetic Protein (BMP) genes, seed cells and BMP, transforming growth factors, Vascular Endothelial Growth Factor (VEGF), or Fibroblast Growth Factor (FGF) mixtures.

30. The use of the porous material and gel composite material according to claim 28 as a carrier for cells or drugs for the construction of tissue engineered artificial bones, repair of bone and cartilage injuries, bone and cartilage defects, bone fractures, nonunions, periodontal defects, spinal fusion, enhanced stability of prostheses, enhanced tendon or ligament attachment to the bone surface.

31. Use of a porous material and gel composite according to claim 28, characterized in that the collagen gel is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixtures thereof.

32. Use of a porous material and gel composite according to claim 31, characterized in that a 1-15mg/ml concentration of collagen gel solution after dialysis, and growth factors, hormones solution or/and cell suspension are mixed in PBS or culture medium at a volume ratio of 1: 1 to make pH neutral and injected into bone and cartilage defect sites to form gel immediately or into bone or cartilage defect sites in open surgery to form gel.

33. Use of a porous material and gel composite according to claim 28, characterized in that the alginate solution is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or mixtures thereof.

34. Use of a material compounded of a porous material and a gel according to claim 31, characterized in that it is used in bone and cartilage tissue engineering, wherein the calcium alginate injection method: mixing 0.3-3% sodium alginate with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture, and injecting sodium alginate mixed solution and 0.05-0.2M calcium chloride, calcium gluconate or calcium sulfate solution into the same part of the defect of bone or cartilage to form gel at the defect position of bone or cartilage.

35. Use of a porous material and gel composite according to claim 28, characterized in that a low melting point agarose solution is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof.

36. Use of a porous material and gel composite according to claim 35, characterized in that said agarose injection method: mixing 0.3% -3% agarose solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics or their mixture at 39 deg.C, and injecting into bone or cartilage defect with large-size needle to form gel.

37. Use of a porous material and gel composite material according to claim 28, characterized in that the fibrinogen gel solution is used alone as an injectable carrier for seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or mixtures thereof.

38. Use of a porous material and gel composite according to claim 37, characterized in that the injection fibrinogen gel method: mixing 0.1-3% fibrinogen solution with seed cells, gene transfected cells, growth factors, hormones, immunosuppressants, antibiotics, or their mixture, and injecting fibrinogen mixture and 10-3000IU/ml thrombin in 0.01-0.2M calcium chloride into the same part of bone or cartilage defect to form gel.

39. The use of the porous material and gel composite according to claim 28, wherein the sodium hyaluronate gel solution is used alone as an injectable carrier of seed cells, growth factors, hormones, immunosuppressants, or a mixture thereof, for bone and cartilage tissue engineering, and can be injected into a bone or cartilage defect site for repairing bone or cartilage damage in open surgery, and the seed cells do not include bone marrow stromal cells, gene transfected cells, and a carrier of bone marrow stromal cells transfected with BMP-2 gene.