CN101461963A - Multiplex composite bone tissue engineering bracket material capable of degrading gradiently and preparation method thereof - Google Patents

Abstract

The invention discloses a multiple composite and gradiently degradable bone tissue engineering scaffold material and a preparation method thereof. The composite scaffold material is composed of calcium phosphate bone cement, biocompatible and degradable synthetic polymers and biocompatible and degradable natural polymers. The multi-composite scaffold material has good mechanical properties and gradient degradation characteristics. At the same time, it can also induce the differentiation of stem cells into osteoblasts in the body by loading bone growth factors, so as to achieve the purpose of regeneration and repair of bone tissue defects, and can significantly improve the initial strength of the scaffold material. And toughness, to ensure that the scaffold material has sufficient strength and toughness during operation and implantation; due to the good flexibility of the scaffold after the composite polymer material, certain cutting and other mechanical processing can be performed.

Description

Multiple composite gradient degradable bone tissue engineering scaffold material and preparation method thereof

technical field

The invention belongs to the tissue engineering material technology in the field of biomedical engineering, and in particular relates to a multiple composite gradient degradable bone tissue engineering scaffold material and a preparation method thereof.

Background technique

There are many cases of bone defects caused by industrial accidents, traffic accidents, orthopedic inflammation, bone tumors, and age-related orthopedic diseases, which have seriously affected people's health and quality of life. The current clinical treatment methods for bone defects mainly include: autologous bone graft, allogeneic bone graft and artificial material repair. Although autologous bone is an ideal material for bone grafting, the sources of bone donors are limited. The second operation will bring pain to the patient and cause new damage to the donor. It is rich, can be stored in advance, and is convenient to use, but it has immune rejection, and the omissions in the treatment of allogeneic bone may also lead to recipient infection, and the possibility of causing infectious diseases and tumor formation, which also limits the development of allogeneic bone. It is widely used; while using artificial materials for direct replacement repair of bone defects, the repair effect is not ideal because it is difficult to achieve bone tissue regeneration at the defect site. Therefore, people have been looking for a more ideal method of biological reconstruction and repair of bone defects.

The development of tissue engineering provides an ideal and feasible way for the treatment and repair of bone defects. The core of tissue engineering is to establish a three-dimensional complex of cells and biomaterials, that is, living tissue with vitality, which is used to reconstruct the shape, structure and function of diseased tissues and achieve permanent replacement. Tissue engineering includes three key factors: signaling molecules (growth factors, inducing factors), scaffold materials and target cells. Scaffold materials play a pivotal role in the construction of tissue-engineered tissues or organs. They not only provide structural support for specific cells, but are also conducive to cell adhesion, nutrient exchange, cell proliferation and differentiation, and migration to the inside of the scaffold, providing support for cell growth. Appropriate external environment, but also can act as a template to guide tissue regeneration and control tissue structure. An ideal tissue engineering scaffold material should have a three-dimensional interconnected porous structure, good biocompatibility, a degradation rate that matches tissue regeneration, a chemical surface that can promote tissue regeneration, and a certain mechanical strength.

Currently commonly used scaffold materials for tissue engineering include biodegradable bioactive inorganic materials, natural biopolymers, degradable synthetic polymer materials and their composites. Natural biopolymer materials are represented by collagen, gelatin, and chitosan. These materials have good biocompatibility and cell affinity and come from a wide range of sources. However, the properties of these materials are unstable, and most of them have poor mechanical properties. The molding process is limited, the molecular structure is complex, in addition, the degradation and absorption are too fast, and the controllable degradation is not good; the degradable synthetic polymer materials are polylactic acid (PLA), polyglycolic acid (PGA) and their copolymers (PLGA) As a representative, most of these materials have good mechanical properties, and can be prepared into three-dimensional porous materials by a variety of processing methods. The degradation rate and active group types can be designed and regulated by molecular design, but these materials The hydrophobicity is strong, the cell affinity is not ideal, and the degradation products are generally acidic, which is easy to cause aseptic inflammation and cause adverse effects on tissues; inorganic materials are represented by calcium phosphate ceramics and bioactive glass. The composition of the material is close to that of hard tissue, the biocompatibility and bioactivity are good, and the mechanical properties of the dense body are good. It has a better application prospect as a bone implant material instead of hard tissue. There are certain difficulties in the porosity of the three-dimensional interconnected scaffolds, and the strength of the high-porosity scaffolds is usually very low. In addition, due to the lack of reactive active groups similar to polymer materials, there are certain restrictions on the combination of bioactive molecules. , and its controllable degradability is not ideal. Although there have been many studies on tissue engineering scaffold materials in recent years, an ideal bone tissue engineering scaffold material has not been developed yet. How to prepare high-porosity scaffolds with biocompatibility, cell affinity, good mechanical properties, and material degradation that basically matches the speed of cell growth or osteogenesis is an urgent problem to be solved in tissue engineering research. One of the feasible ways to solve the problem is to combine two or more biodegradable materials with complementary characteristics, and conduct bionic design, simulation and experiment to prepare composite tissue engineering scaffold materials with excellent performance.

Calcium phosphate cement (CPC) is a new type of self-fixing bioactive material, which has good biocompatibility, osteoconductivity, degradability, plasticity, and less heat generated by the reaction. It is an ideal bone replacement and repair materials. Since self-solidifying calcium phosphate can be hydrated into weakly crystalline hydroxyapatite at room temperature, has good degradable absorption, and is easy to shape and form pores, it is an ideal material for preparing calcium phosphate porous tissue engineering scaffolds. Xu et al conducted a systematic study on the bone cement tissue engineering scaffold of tetracalcium phosphate-anhydrous calcium hydrogen phosphate system, and the in vitro cell culture of the scaffold showed that the calcium phosphate cement material has good biocompatibility [H.H.K.Xu, et al . Biomaterials 2005, 26: 1337-1348.]. However, during years of research and application, the problems of brittleness and low strength of calcium phosphate materials have not been well resolved, which limits its wide application. This directly results in the low strength of porous calcium phosphate tissue engineering scaffolds with high porosity. In order to solve this problem, people have carried out research from different angles, including: adopting different calcium phosphate formula systems, adding additives that can play a reinforcing role, etc.; choosing citric acid (sodium), malic acid, sodium phosphate, etc. Organic and inorganic solutions are used as blending liquids; in addition, traditional reinforcement methods are used for reference to compound with whiskers, organic polymer materials, short fibers, etc. Through these methods, the mechanical properties of calcium phosphate bone cement have been improved to varying degrees, but the effect is still unsatisfactory, especially for calcium phosphate tissue engineering scaffold materials with high porosity, poor mechanical properties are still the key to hindering its practical application . At present, the method of using composite polymer materials is more researched and the effect is better. Xu et al. respectively studied the addition of absorbable suture fibers directly into the bone cement matrix [H.H.K.Xu et al.J.Biomed.Mater.Res.Part A.2005, 75:966-975.], adding PLGA network reinforcement [H.H.K.Xu et al.J.Biomed.Mater.Res.Part A.2004, 69:267-278.], adding chitosan [ H.H.K Xu et al.Biomaterials 2005, 6:1337-1348.], to improve the mechanical properties of bone cement, so that the mechanical properties of porous bone cement scaffolds have been significantly improved. However, since the added polymer material degrades faster than calcium phosphate bone cement, once the reinforcement randomly mixed with bone cement degrades, the strength of the scaffold will decrease significantly, which may make the material unable to bear the original load and collapse. On the other hand, these enhancers do not improve the biocompatibility and cell affinity of the scaffold, and some (such as PLGA) even decrease the biocompatibility and cell affinity of the scaffold.

The research on self-curing calcium phosphate tissue engineering scaffold materials generally does not consider the regulation of the degradation rate of scaffold materials.

Contents of the invention

In order to overcome the shortcomings of the above-mentioned prior art, the object of the present invention is to provide a multi-composite gradient degradable bone tissue engineering scaffold material and its preparation method, the composite scaffold material is composed of calcium phosphate bone cement, biocompatible and degradable Composed of polymers and biocompatible degradable natural polymers, after being implanted in the body, the rapidly degraded natural polymers first degrade and form connected large pores in situ in the composite scaffold for the growth of new bone tissue, which acts as a scaffold Function: Synthetic polymers that degrade relatively slowly are attached to the pore wall of the porous scaffold to strengthen the calcium phosphate porous scaffold matrix, so that the scaffold can maintain good strength for a period of time. With the gradual degradation of the synthetic polymer and calcium phosphate bone cement porous matrix, cells and tissues continue to grow in, and eventually the entire composite scaffold is completely degraded, and the implanted site is replaced by new bone tissue. The multiple composite scaffold material has good mechanical properties and gradient degradation properties. At the same time, it can also induce the differentiation of stem cells into osteoblasts in the body by loading bone growth factors, so as to achieve the purpose of regeneration and repair of bone tissue defects, and can meet the requirements of clinical use.

The purpose of the present invention is achieved through the following technical solutions: a preparation method of multiple composite gradient degradable bone tissue engineering scaffold materials, comprising the following steps:

(1) dissolving the biocompatible and degradable synthetic polymer material in a solvent, preparing a solution with a mass percentage concentration of 1-20%, then stirring or standing for 1-48 hours, and filtering to obtain a synthetic polymer solution A; During the dissolution process, heating (40-80°C) can be used to promote the dissolution of synthetic polymer materials as needed;

dissolving the biocompatible and degradable natural polymer material in a solvent, preparing a solution with a mass percentage concentration of 5-25%, then stirring or standing for 1-48 hours, and filtering to obtain a natural polymer solution B; during the dissolution process In the process, heating (30-100°C) can be used as needed to promote the dissolution of natural polymer materials;

(2) Dry the calcium phosphate bone cement porous scaffold with a total porosity of 70 to 95% at 30 to 100° C. for 1 to 48 hours; soak the calcium phosphate bone cement porous scaffold into the synthetic polymer prepared in step (1) In solution A, vacuum-dry for 0.5 to 5 hours, and the synthetic polymer solution is poured into the pores of the calcium phosphate bone cement porous scaffold under the action of pressure and capillary force to obtain a calcium phosphate bone cement porous scaffold filled with synthetic polymer materials;

The calcium phosphate bone cement porous scaffold can be prepared with reference to the following method: method 1 (H.H.K.Xu, S.Takagi, J.B.Quinn, et al.J.Biomed.Mater.Res., 2004, 68A:725-734.); method 2 (A.Almirall, G.Larrecq, J.A.Delgado, et al.Biomaterials, 2004, 25:3671-3680); method 3 (X.Miao, Y.Hu, J.Liu, et al.Materials Letters, 2004, 58:397-402); method 4 (X.P.Qi, J.D.Ye, Y.J.Wang.J Biomed Mater Res Part A, DOI: 10.1002/jbm.a.32054, in press).

(3) Dry the surface of the calcium phosphate bone cement porous scaffold perfused with synthetic polymer materials, and pre-freeze at -60 to -4°C for 1 to 48 hours; freeze-dry the obtained frozen composite material for 2 to 48 hours, and dry Finally, the polymer forms a film layer on the porous calcium phosphate pore wall, and the porous tissue engineering scaffold material composited with the synthesized polymer and calcium phosphate is obtained. The thickness of the film can be changed by changing the concentration of the polymer solution as needed.

(5) Soak the porous tissue engineering scaffold material composited by the synthetic polymer and calcium phosphate obtained in step (3) into the natural polymer solution B of step (1), and vacuum-dry for 0.5 to 5 hours. Infused into the pores of the calcium phosphate bone cement porous scaffold under the action of capillary force, the calcium phosphate bone cement porous scaffold perfused with the natural polymer solution is obtained;

(6) Dry the surface of the calcium phosphate bone cement porous scaffold perfused with natural polymer solution, and pre-freeze at -60 to -4°C for 1 to 48 hours; then freeze-dry the obtained frozen material for 2 to 48 hours, and dry The post-natural polymer is filled in the pores of the porous tissue engineering scaffold material composited by the synthetic polymer and calcium phosphate obtained in step (3) with a porous or network structure, that is, the multi-composite gradient degradable bone tissue engineering scaffold material.

In order to better realize the present invention, the biocompatible and degradable synthetic polymer materials include polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid/polyglycolic acid copolymer (PLGA), polylactic acid/starch Any one or two or more of blends, polycaprolactone (PCL) or polycaprolactone (PCL)/starch blends, etc.

The biocompatible and degradable natural polymer material includes any one or two or more materials among collagen, gelatin, chitosan, silk fibroin, sodium alginate or cellulose derivatives.

The solvent in which the biocompatible and degradable synthetic polymer material is dissolved includes 1,4-dioxane, acetone, methylene chloride, chloroform, dimethyl sulfoxide, or trichloroacetic acid.

The solvent in which the biocompatible and degradable natural polymer material is dissolved is deionized water, distilled water, acetic acid aqueous solution, phosphoric acid aqueous solution, absolute ethanol and its aqueous solution, calcium chloride solution, or acetone.

The calcium phosphate bone cement includes amorphous calcium phosphate (ACP) + calcium hydrogen phosphate dihydrate (DCPD) system bone cement, tricalcium phosphate (TCP) + calcium hydrogen phosphate dihydrate (DCPD) system bone cement, α-phosphoric acid Tricalcium (α-TCP) + calcium hydrogen phosphate dihydrate (DCPD) + calcium carbonate (CaCO ₃ ) + hydroxyapatite (HA) system bone cement, α-tricalcium phosphate (α-TCP) + hydrogen phosphate dihydrate Calcium (DCPD) + calcium carbonate (CaCO ₃ ) + hydroxyapatite (HA) + magnesium phosphate (Mg ₃ (PO ₄ ) ₂ ) + sodium biphosphite (NaHSO ₃ ) system bone cement, tetracalcium phosphate (TTCP) + Anhydrous calcium hydrogen phosphate (DCPA) + calcium carbonate (CaCO ₃ ) system bone cement, β-tricalcium phosphate (β-TCP) + sodium pyrophosphate (Na ₄ P ₂ O ₇ ) system bone cement or partially crystalline calcium phosphate (PCCP) + anhydrous calcium hydrogen phosphate (DCPA) system bone cement.

A multiple composite gradient degradable calcium phosphate bone tissue engineering scaffold is prepared by the above preparation method.

Compared with the prior art, the tissue engineering scaffold material used for the regeneration and repair of bone tissue defects according to the present invention has the following characteristics and advantages:

1. The multi-composite gradient degradable bone tissue engineering scaffold material prepared by the present invention is composed of calcium phosphate bone cement, synthetic polymer, and natural polymer triple compound, which can significantly improve the initial strength and toughness of the scaffold material, and ensure that it is stable during operation and implantation. The stent material has sufficient strength and toughness during the insertion process; moreover, due to the good flexibility of the stent after the composite polymer material, certain mechanical processing such as cutting can be performed.

2. After the multi-composite gradient degradable bone tissue engineering scaffold material prepared by the present invention is implanted in the body, the degradation rate of the degradable natural polymer material filled in the pores of the calcium phosphate bone cement porous scaffold is the fastest, and the porous calcium phosphate pores The synthetic polymer film on the wall degrades relatively slowly, while the bone cement degrades the slowest, so the degradation of the multiple composite scaffold material is a gradient process in which the three constituent materials gradually degrade. Due to the gradient degradation of natural polymers and synthetic polymer materials, although the strength of the scaffold material will decrease, it will not suddenly drop and collapse significantly, and with the gradual growth of new bone, the overall strength of the implanted site can be maintained. The degradation of the scaffold material and the ingrowth of new bone have a good match.

3. After the multi-composite gradient degradable bone tissue engineering scaffold material prepared by the present invention is implanted in the body, the natural polymer material degrades to form a connected porous structure in the scaffold material, providing space for the growth of cells and tissues. Moreover, since the natural polymer therein has a porous or network structure and degrades quickly, cells can be directly cultured on the scaffold.

4. After the multi-composite gradient degradable bone tissue engineering scaffold material prepared by the present invention is implanted in the body, the natural polymer in it can significantly improve the cell affinity of the scaffold before it is completely degraded. Moreover, growth factors can be introduced into natural polymers as needed to induce differentiation of stem cells cultured in vitro or in vivo, endow the scaffold with osteoinductive function, and promote the growth of new bone.

5. The preparation method of the multi-composite, gradiently degradable bone tissue engineering scaffold material for bone defect regeneration and repair of the present invention has a wide range of applications and a simple process.

Detailed ways

The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

Embodiment one

1, polylactic acid/polyglycolic acid copolymer (PLGA) is dissolved in methylene chloride, is made into the solution of 10% mass percent concentration, stirs with magnetic stirrer 6 hours, obtains polylactic acid/polyglycolic acid copolymer solution, Put in a container for later use.

2. Select a calcium phosphate bone cement porous scaffold with a total porosity of about 80% and a pore size of 200-400 μm, and put the calcium phosphate bone cement porous scaffold into the In a vacuum drying oven, vacuum dry at 50°C for 6 hours, take it out, soak it in the polylactic acid/polyglycolic acid copolymer (PLGA) solution prepared in step 1, put it into a vacuum desiccator, and vacuum it for 2 hours, and the polylactic acid/polyglycolic acid copolymer (PLGA) solution The polyglycolic acid copolymer (PLGA) solution was perfused into the pores of the calcium phosphate bone cement porous scaffold under the action of pressure and capillary force.

3. Take out the calcium phosphate bone cement porous scaffold infused with PLGA in the pores, dry the surface with filter paper, put it in a -18°C refrigerator for 24 hours, and then freeze-dry it at -40°C for 24 hours to obtain a composite phosphoric acid The calcium porous scaffold is a porous tissue engineering scaffold material composed of synthetic polymers and calcium phosphate.

4, chitosan is dissolved in the dilute acetic acid aqueous solution of 2% by volume, is mixed with the chitosan solution that mass percentage concentration is 15%, stirs 2 hours with magnetic stirrer, filters, obtains chitosan solution, waits use.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the chitosan solution prepared in step 4, put it in a vacuum desiccator, and evacuate it for 0.5 hours. It is poured into the pores of the primary composite calcium phosphate porous scaffold to obtain a calcium phosphate bone cement porous scaffold filled with the chitosan solution.

6. Take out the calcium phosphate bone cement porous scaffold that has been multiplexed with PLGA and chitosan, that is, the porous scaffold prepared in step 5, dry the surface with filter paper, and put it in -18°C for 12 hours; freeze-dry the obtained material After 48 hours, the multi-layer compound gradient degradable bone tissue engineering scaffold material is ready. The final porosity of the prepared multi-composite gradiently degradable bone tissue engineering scaffold material is 63%, and the compressive strength is 4.7MPa. Chitosan is porous, with a pore size between 20 and 80 μm. Although the pore size of the chitosan filler is relatively small, as the chitosan degrades rapidly, the scaffold can form large pores of more than 100 μm, and the porosity increases accordingly. After the chitosan is completely degraded, the pore size can reach more than 200 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment two

1. Dissolve polylactic acid (PLA) in 1,4-dioxane, prepare a polylactic acid solution with a concentration of 5% by mass, heat to 40°C, and stir for 12 hours until the polylactic acid is fully dissolved to obtain polylactic acid solution, ready to use.

2. Select a calcium phosphate bone cement porous scaffold with a total porosity of about 95% and a pore size of 100-300 μm, and put the porous scaffold into In an ordinary electric drying oven, dry at 100°C for 1 hour, take it out, soak it in the polylactic acid solution prepared in step 1, put it in a vacuum desiccator, and vacuum it for 4 hours. The polylactic acid solution is perfused under pressure and capillary force. Calcium phosphate cement in the pores of porous scaffolds.

3. Take out the scaffold material filled with PLA in the above-mentioned pores, dry the surface with filter paper, put it in a -40°C refrigerator for 6 hours, and then freeze-dry it at -40°C for 36 hours to obtain a composite calcium phosphate matrix. Tissue engineering scaffold materials are porous tissue engineering scaffold materials composed of synthetic polymers and calcium phosphate.

4. Dissolving the collagen in an acetic acid solution with a volume percentage of 1%, and preparing a collagen solution with a mass percentage concentration of 10%, stirring with a magnetic stirrer for 2 hours to obtain a collagen solution for use.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the collagen solution prepared in step 4, put it in a vacuum desiccator, and vacuumize for 2 hours. The collagen solution is perfused into the calcium phosphate under the action of pressure and capillary force. In the pores of the bone cement porous support, a calcium phosphate bone cement porous support perfused with collagen solution is obtained.

6. Take out the calcium phosphate bone cement porous scaffold that has been multiplexed with PLA and collagen, that is, the porous scaffold prepared in step 5, dry the surface with filter paper, and put it in -60°C for 1 hour; freeze-dry the obtained material for 24 hours, That is to say, it is multi-layer composite and gradiently degradable scaffold material for bone tissue engineering. The final porosity of the prepared multi-composite gradient-degradable bone tissue engineering scaffold material is 75%, and the compressive strength is 2.5MPa. Collagen is a network porous structure with a pore size between 50 and 120 μm. Although the pore size of the collagen filler is relatively small, as the collagen degrades rapidly, the scaffold can form large pores of more than 100 μm, and the porosity increases accordingly. After the collagen is completely degraded, the pore size can reach 100-300 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment three

1, polycaprolactone (PCL)/starch blend is dissolved in dimethyl sulfoxide, is mixed with the solution of 20% mass percentage concentration, is heated to 80 ℃, stands for 48 hours, waits for polycaprolactone ( PCL)/starch blend is fully dissolved to obtain polycaprolactone (PCL)/starch blend solution, which is ready for use.

2. Select a calcium phosphate bone cement porous scaffold with a _total porosity of about 80% and _a pore size of _200-400 μm. Put the bracket into a vacuum drying oven, dry it in vacuum at 60°C for 4 hours, take it out, soak it in the polycaprolactone (PCL)/starch blend solution prepared in step 1, put it in a vacuum dryer, and vacuum it for 3 hours , the polycaprolactone (PCL)/starch solution was perfused into the pores of the calcium phosphate bone cement porous scaffold under the action of pressure and capillary force.

3. Take out the scaffold material filled with the polycaprolactone (PCL)/starch blend in the pores prepared in step 2, wipe the surface dry with filter paper, put it in a -40°C refrigerator for 2 hours, and then store it at -50°C. Freeze-drying at ℃ for 30 hours to obtain a primary composite calcium phosphate-based tissue engineering scaffold material.

4. Put silk in 0.05% Na ₂ CO ₃ solution by mass, treat it at 100°C for 30 minutes, repeat 3 times, remove sericin from silk, and obtain refined silk after drying. Refined silk was dissolved in CaCl ₂ -CH ₃ CH ₂ OH-H ₂ O ternary solvent (molar ratio 1:2:8) at 70°C with stirring and dissolving, after dialysis and filtration, silk fibroin aqueous solution was obtained, and deionized water was used to prepare quality A silk fibroin solution with a percentage concentration of 5%; a sodium alginate solution with a mass percentage concentration of 8% was prepared with deionized water. Stand still for 24 hours, filter respectively, and then mix the two solutions according to the volume ratio of 1:1 to obtain silk fibroin/sodium alginate mixed solutions with the mass content of silk fibroin and sodium alginate being 2.5% and 4% respectively, stand-by.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the mixed solution of silk fibroin/sodium alginate prepared in step 4, put it in a vacuum desiccator, and vacuum for 5 hours, the silk fibroin/alginate The sodium mixed solution is perfused into the pores of the calcium phosphate bone cement porous scaffold under the action of pressure and capillary force to obtain the calcium phosphate bone cement porous scaffold perfused with the silk fibroin/sodium alginate mixed solution.

6. Take out the calcium phosphate bone cement porous scaffold compounded by polycaprolactone (PCL)/starch and silk fibroin/sodium alginate, dry the surface with filter paper, and put it into -40℃ for 24 hours to pre-freeze; the obtained The material was freeze-dried for 48 hours, that is, the multiple-composite gradient-degradable bone tissue engineering scaffold material. The final porosity of the prepared multi-composite gradient degradable bone tissue engineering scaffold material is 60%, and the compressive strength is 3.7MPa. The inner colloid fibroin/sodium alginate has a porous structure with a pore size between 30 and 70 μm. Although the pore size of the fibroin/sodium alginate filler is relatively small, with the rapid degradation of the fibroin/sodium alginate, the scaffold can form macropores of more than 100 μm, and the porosity increases accordingly. After the fibroin/sodium alginate is completely degraded, the pore size can reach more than 200 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment four

1. Dissolve polylactic acid/polyglycolic acid copolymer (PLGA) in acetone, prepare a PLGA solution with a concentration of 1% by mass, heat it to 60°C, and let it stand for 36 hours until PLGA is fully dissolved and ready for use.

2. Select the amorphous calcium phosphate (ACP) + anhydrous calcium hydrogen phosphate (DCPA) system calcium phosphate bone cement porous scaffold with a total porosity of about 85% and a pore size of 300-400 μm, and put the porous scaffold into a vacuum drying oven , vacuum-dried at 50°C for 12 hours, took it out, soaked it in the PLGA solution prepared in step 1, put it into a vacuum bottle, and vacuumed it for 0.5 hours. The PLGA solution was perfused into the calcium phosphate bone cement porous scaffold under the action of pressure and capillary force. in the pores.

3. Take out the scaffold material infused with PLGA in the pores, dry the surface with filter paper, put it in a -4°C refrigerator for 48 hours, and then freeze-dry it at -60°C for 36 hours to obtain a composite calcium phosphate-based tissue. Engineering support materials.

4. Prepare a sodium alginate solution with a mass percent concentration of 25% with distilled water, let it stand for 24 hours, and filter to obtain a sodium alginate solution for use.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the sodium alginate solution prepared in step 4, put it in a vacuum desiccator, and vacuumize it for 5 hours. perfusing into the pores of the calcium phosphate bone cement porous scaffold to obtain the calcium phosphate bone cement porous scaffold perfused with the sodium alginate solution.

6. Take out the calcium phosphate bone cement porous scaffold that has been compounded by PLGA and sodium alginate, dry the surface with filter paper, and put it in -60°C for 1 hour; freeze-dry the obtained material at -40°C for 12 hours , that is, a multi-layer compound gradient degradable scaffold material for bone tissue engineering. The final porosity of the prepared multi-composite gradient degradable bone tissue engineering scaffold material is 65%, and the compressive strength is 4.0MPa. Sodium alginate has a porous structure with a pore size between 30 and 60 μm. Although the pore size of the sodium alginate filler is relatively small, with the rapid degradation of sodium alginate, the scaffold can form macropores of more than 100 μm, and the porosity increases accordingly. After the sodium alginate is completely degraded, the pore size can reach more than 300 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment five

1. Dissolve polyglycolic acid (PGA) in trichloroacetic acid, prepare a polyglycolic acid solution with a concentration of 20% by mass, stir for 1 hour until the polyglycolic acid is fully dissolved, and set aside.

2. Select α-tricalcium phosphate (α-TCP) + calcium hydrogen phosphate dihydrate (DCPD) + calcium carbonate (CaCO ₃ ) + hydroxyapatite (HA) with a total porosity of about 80% and a pore size of 100-500 μm )+magnesium phosphate (Mg ₃ (PO ₄ ) ₂ )+sodium hydrogen phosphite (NaHSO ₃ ) system bone cement porous scaffold, put the above porous scaffold in a vacuum drying oven, vacuum dry at 80°C for 3 hours, take it out, Soak in the polyglycolic acid solution prepared in step 1, put it into a vacuum desiccator, and vacuumize for 5 hours, and pour the polyglycolic acid solution into the pores of the porous scaffold under the action of pressure and capillary force.

3. Take out the scaffold material infused with PGA in the pores, dry the surface with filter paper, put it in a -60°C refrigerator for 1 hour, and then freeze-dry it at -30°C for 2 hours to obtain a composite calcium phosphate-based tissue. Engineering support materials.

4. Prepare a gelatin solution with a mass percent concentration of 10% with distilled water, stir at 60° C. for 2 hours with a constant temperature magnetic stirrer, and filter to obtain a gelatin solution for use.

5. Soak the primary composite calcium phosphate-based porous scaffold into the gelatin solution prepared in step (4), put it in a vacuum desiccator, and vacuum for 4 hours. The gelatin solution is perfused into the calcium phosphate bone cement under the action of pressure and capillary force In the pores of the porous scaffold, a calcium phosphate bone cement porous scaffold perfused with a gelatin solution is obtained.

6. Take out the calcium phosphate bone cement porous scaffold that has been compounded with PGA and gelatin, dry the surface with filter paper, and put it in -30°C for 20 hours; then freeze-dry it at -50°C for 30 hours, that is, multiple compounding Gradient degradable scaffold material for bone tissue engineering. The final porosity of the prepared multi-composite gradient-degradable bone tissue engineering scaffold material is 55%, and the compressive strength is 3.3MPa. Gelatin has a porous structure with a pore size between 50 and 80 μm. Although the pore size of the gelatin filler is relatively small, as the gelatin degrades rapidly, the scaffold can gradually form large pores of more than 100 μm, and the porosity increases accordingly. After the gelatin is completely degraded, the pore size can reach 100-500 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment six

1, polylactic acid/polyglycolic acid copolymer (PLGA) is dissolved in chloroform, is made into the solution of 15% mass percentage concentration, stirs with magnetic stirrer 6 hours, obtains polylactic acid/polyglycolic acid copolymer solution, Put in a container for later use.

2. Select a porous bone cement scaffold with a total porosity of about 75% and a pore size of 200-500 μm, and put the porous scaffold in a vacuum drying oven for 37 Vacuum-dry at ℃ for 36 hours, take it out, soak it in the PLGA solution prepared in step 1, put it into a vacuum desiccator, and evacuate it for 4 hours, and the PLGA solution is poured into the pores of the porous scaffold under the action of pressure and capillary force.

3. Take out the scaffold material infused with PLGA in the pores, dry the surface with filter paper, put it in a -20°C refrigerator for 12 hours, and then freeze-dry it at -40°C for 12 hours to obtain a composite calcium phosphate-based tissue Engineering support materials.

4. Prepare carboxymethyl cellulose solution and sodium alginate solution with a concentration of 6% and 9% in distilled water; prepare a chitosan solution with a concentration of 6% in dilute acetic acid solution with a concentration of 2% in volume. Leave to stand for 12 hours, filter respectively, and then mix the three solutions according to the volume ratio of 1:1:1 to obtain carboxymethyl cellulose, sodium alginate and chitosan with a mass content of 2%, 3% and 2% respectively Carboxymethylcellulose/sodium alginate/chitosan mixed solution, set aside.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the mixed carboxymethyl cellulose/sodium alginate/chitosan solution prepared in step 4, put it in a vacuum desiccator, and vacuumize for 2 hours. The mixed solution of carboxymethyl cellulose/sodium alginate/chitosan was perfused into the pores of calcium phosphate bone cement porous scaffold under the action of pressure and capillary force, and the perfused carboxymethyl cellulose/sodium alginate/chitosan Mixing solutions of calcium phosphate cement porous scaffolds.

6. Take out the calcium phosphate bone cement porous scaffold that has been compounded by PLGA and carboxymethyl cellulose/sodium alginate/chitosan, dry the surface with filter paper, and put it in -30°C for 20 hours; Freeze-drying under the condition of ℃ for 48 hours, that is, the multi-layer compound gradient degradable bone tissue engineering scaffold material. The final porosity of the prepared multi-composite gradiently degradable bone tissue engineering scaffold material is 52%, and the compressive strength is 4.2MPa. Carboxymethyl cellulose/sodium alginate/chitosan has a porous structure with a pore size between 30 and 70 μm. Although the pore size of the carboxymethyl cellulose/sodium alginate/chitosan filler is relatively small, with the rapid degradation of the carboxymethyl cellulose/sodium alginate/chitosan, the scaffold can gradually form a pore size larger than 100 μm. pores, the porosity increases accordingly. After the carboxymethyl cellulose/sodium alginate/chitosan is completely degraded, the pore size can reach more than 200 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

Embodiment seven

1. Dissolve the polylactic acid (PLA)/starch blend in chloroform, prepare a solution with a concentration of 15% by mass, stir to make it fully dissolved, and set aside.

2. Select the β-tricalcium phosphate (β-TCP) + sodium pyrophosphate (Na ₄ P ₂ O ₇ ) system bone cement porous scaffold with a total porosity of about 70% and a pore size of 200-400 μm. Put it in a vacuum drying oven, dry it under vacuum for 48 hours at 30°C, take it out, soak it in the PLA/starch solution prepared in step 1, put it in a vacuum dryer, and vacuum it for 4 hours. perfused into the pores of the porous scaffold.

3. Take out the scaffold material filled with PLA/starch solution in the pores, dry the surface with filter paper, put it in a -30°C refrigerator for 6 hours, and then freeze-dry it at -40°C for 16 hours to obtain a composite phosphoric acid Calcium-based tissue engineering scaffold materials.

4. Prepare a collagen solution with a mass percentage concentration of 5% with a dilute acetic acid solution with a volume percentage of 1%, and stir and mix with a magnetic stirrer for 12 hours to obtain a collagen solution for use.

5. Soak the primary composite calcium phosphate-based porous scaffold prepared in step 3 into the solution prepared in step 4, put it in a vacuum desiccator, and vacuumize for 2 hours. The collagen solution is perfused into the calcium phosphate bone under the action of pressure and capillary force. In the pores of the cement porous scaffold; the calcium phosphate bone cement porous scaffold perfused with the collagen solution is obtained.

6. Take out the calcium phosphate bone cement porous scaffold that has undergone multiple compounding of PLA/starch and collagen, dry the surface with filter paper, put it in -30°C for 20 hours, and then freeze-dry it at -40°C for 48 hours to obtain Multi-composite gradient degradable scaffold material for bone tissue engineering. The final porosity of the prepared multi-composite gradient-degradable bone tissue engineering scaffold material is 47%, and the compressive strength is 5.5MPa. The PLA/starch forms a film and adheres to the pore wall of the calcium phosphate bone cement porous scaffold, filling the scaffold The inner collagen is a network porous structure with a pore size between 50 and 120 μm. Although the pore size of the collagen filler is relatively small, with the rapid degradation of the collagen, the scaffold can gradually form large pores of more than 100 μm, and the porosity increases accordingly. After the collagen is completely degraded, the pore size can reach more than 200 μm. The composition, structure and performance of the material are suitable for use as a scaffold for bone tissue engineering. Moreover, growth factors can be introduced into the natural polymer as needed to endow the scaffold with osteoinductive function and promote the growth of new bone.

The specific implementation described above is a preferred embodiment of the present invention, and cannot limit the claims of the present invention. Any other changes or other equivalent replacement methods that do not deviate from the technical solution of the present invention are included in the present invention. within the scope of protection.

Claims (7)

1, a kind of preparation method of multiplex composite bone tissue engineering bracket material capable of degrading gradiently is characterized in that comprising the steps:

(1) the bio-compatible degradable synthesized polymer material is dissolved in the solvent, is mixed with mass percent concentration and is 1～20% solution, stirred then or leave standstill 1～48 hour, filter, obtain synthetic high polymer solution;

Bio-compatible degradable natural macromolecular material is dissolved in the solvent, is mixed with mass percent concentration and is 5～25% solution, stirred then or leave standstill 1～48 hour, filter, obtain natural polymer solution;

(2) be 70～95% calcium phosphate bone cement porous support with overall porosity 30～100 ℃ dry 1～48 hour down; The calcium phosphate bone cement porous support is dipped in the synthetic high polymer solution that step (1) prepared, and vacuum drying 0.5～5 hour has obtained pouring into the calcium phosphate bone cement porous support of synthesized polymer material;

(3) the calcium phosphate bone cement porous support that will pour into synthesized polymer material was wiped away and is done the surface ,-60～-4 ℃ of following pre-freezes 1～48 hour; Freeze the composite lyophilization 2～48 hours with what obtain, promptly get the compound multi-porous tissue engineering supporting material of synthetic high polymer and calcium phosphate;

(5) will be dipped in the natural polymer solution of step (1) by resultant synthetic high polymer of step (3) and the compound multi-porous tissue engineering supporting material of calcium phosphate, vacuum drying 0.5～5 hour has obtained pouring into the calcium phosphate bone cement porous support of natural polymer solution;

(6) the calcium phosphate bone cement porous support that will pour into natural polymer solution was wiped away and is done the surface ,-60～-4 ℃ of following pre-freezes 1～48 hour; With the frozen material lyophilization that obtains 2～48 hours, promptly get multiplex composite bone tissue engineering bracket material capable of degrading gradiently then.

2, the preparation method of a kind of multiplex composite bone tissue engineering bracket material capable of degrading gradiently according to claim 1 is characterized in that: described bio-compatible degradable synthesized polymer material comprises in polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymer, polylactic acid and starch blend, polycaprolactone or the polycaprolactone/starch blend any one or two or more material.

3, the preparation method of a kind of multiplex composite bone tissue engineering bracket material capable of degrading gradiently according to claim 1 is characterized in that: described bio-compatible degradable natural macromolecular material comprises in collagen, gelatin, chitosan, fibroin albumen, sodium alginate or the cellulose derivative any one or two or more material.

4, the preparation method of a kind of multiplex composite bone tissue engineering bracket material capable of degrading gradiently according to claim 1, it is characterized in that: the solvent that described bio-compatible degradable synthesized polymer material is dissolved in the solvent is 1,4-dioxane, acetone, dichloromethane, chloroform, dimethyl sulfoxide or trichloroacetic acid.

5, the preparation method of a kind of multiplex composite bone tissue engineering bracket material capable of degrading gradiently according to claim 1 is characterized in that: the solvent that described bio-compatible degradable natural macromolecular material is dissolved in the solvent is deionized water, distilled water, aqueous acetic acid, phosphate aqueous solution, dehydrated alcohol and aqueous solution thereof, calcium chloride solution or acetone.

6, the preparation method of a kind of multiplex composite bone tissue engineering bracket material capable of degrading gradiently according to claim 1 is characterized in that: described calcium phosphate bone cement comprises unformed calcium phosphate+dicalcium phosphate dehydrate system bone cement, tricalcium phosphate+dicalcium phosphate dehydrate system bone cement, type alpha tricalcium phosphate+dicalcium phosphate dehydrate+calcium carbonate+hydroxyapatite system bone cement, type alpha tricalcium phosphate+dicalcium phosphate dehydrate+calcium carbonate+hydroxyapatite+magnesium phosphate+hydrogen phosphite sodium system bone cement, tetracalcium phosphate+calcium phosphate dibasic anhydrous+calcium carbonate system bone cement, bata-tricalcium phosphate+tetrasodium pyrophosphate system bone cement or partially crystallized calcium phosphate+calcium phosphate dibasic anhydrous system bone cement.

7, but a kind of MULTIPLE COMPOSITE gradient degraded calcium phosphate bone tissue engineering rack is prepared from by each described preparation method of claim 1～6 exactly.