CN109675118B - Composite tissue engineering scaffold material with enhanced mechanics and preparation method thereof - Google Patents

Composite tissue engineering scaffold material with enhanced mechanics and preparation method thereof Download PDF

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CN109675118B
CN109675118B CN201910119602.1A CN201910119602A CN109675118B CN 109675118 B CN109675118 B CN 109675118B CN 201910119602 A CN201910119602 A CN 201910119602A CN 109675118 B CN109675118 B CN 109675118B
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scaffold material
tissue engineering
engineering scaffold
porous microspheres
water
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CN109675118A (en
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周志敏
李晓凯
李敏
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Institute of Biomedical Engineering of CAMS and PUMC
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Abstract

The invention discloses a composite tissue engineering scaffold material with enhanced mechanics and a preparation method thereof. The composite material contains silk screen, gelatin, silk fibroin and porous microspheres. And blending the porous microspheres with silk fibroin aqueous solution and gelatin aqueous solution, injecting the mixture into a mold containing silk screen, freeze-drying to obtain silk screen/gelatin/porous microspheres/silk fibroin composite scaffold, crosslinking with 1% carbodiimide (EDC) for 8h, rinsing, and drying to obtain a mechanical enhancement final product. The scaffold material has good biocompatibility, degradability and mechanical adjustability, and is suitable for repairing and reconstructing cartilage and other relevant clinical defects. The preparation method disclosed by the invention is simple in preparation process, easy to operate, safe, green and environment-friendly in preparation process, low in cost and suitable for batch production.

Description

Composite tissue engineering scaffold material with enhanced mechanics and preparation method thereof
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a composite tissue engineering scaffold material with enhanced mechanics and a preparation method thereof.
Background
Tissue, organ defects or dysfunction due to various causes are one of the main causes of human health hazards [ Langer R. tissue engineering: a new field and its patents [ J ]. Pharmaceutical Research,1997,14(7):840-841 ], and tissue, organ defect repair and functional reconstruction are also significant challenges in the medical field. For tissue, organ defects or dysfunctions which are common in clinic, traditional treatment methods comprise tissue and organ transplantation, surgical reconstruction and the like, but the problem of donors is involved in the organ transplantation and the surgical reconstruction. The problems of immunological rejection and donor deficiency when the foreign body tissues or organs are used for treating the diseases cannot meet the existing clinical requirements. Autologous tissue transplantation is a repair mode of "treating wounds with wounds" at the expense of healthy tissues [ Zhou G, Jiang H, Yin Z, et al.
The rise of tissue engineering technology opens up a new way for the restoration and reconstruction of tissues and organs. The goal of tissue engineering is to develop tissue and organ substitutes to maintain, restore, or enhance the relevant functions of a patient's tissue and organs [ Khademhosseini A Langer R.A decapde of progression in tissue engineering [ J ]. Nature Protocols,2016,11(10):1775 ]. In recent years, tissue engineering technology has been rapidly developed as an important component of regenerative medicine, and the therapeutic concept of tissue and organ defects is gradually changed from tissue and organ transplantation to tissue and organ regeneration. Tissue engineering has been proposed as a novel therapeutic approach and is gradually becoming a very promising medical approach for tissue regeneration and organ reconstruction.
In the tissue engineering technology, the tissue engineering scaffold material is used as the 'soil' of seed cells and plays an important role in the research process of the tissue engineering technology. At present, the scaffold materials for tissue engineering are mainly divided into natural polymer materials, artificially synthesized polymer materials and composite scaffold materials. The natural polymer materials mainly comprise collagen, gelatin, chitosan, hyaluronic acid, alginic acid and the like, and the materials have good biocompatibility and are beneficial to the proliferation of cells, but have the defects of over-quick degradation and insufficient mechanical properties, and the new tissues can not be molded well; the artificially synthesized high polymer material mainly comprises polylactic acid (PLA), polyglycolic acid (PGA) or a copolymer (PLGA) of the PLA and the PGA and other polyester materials, and the materials have good plasticity, strong mechanical property and relatively long degradation period, can provide a stent function for a longer time, and can adjust the molecular weight according to the needs, but have the defects that the materials have certain hydrophobicity and are not beneficial to seed cell adhesion, and besides, the degradation products can cause aseptic inflammatory reaction; the mismatch between the degradation rate of the material and the tissue growth rate is a bottleneck problem which restricts the tissue repair material.
Disclosure of Invention
The invention aims to provide a composite tissue engineering scaffold material with enhanced mechanics and a preparation method thereof.
The composite tissue engineering scaffold material provided by the invention comprises a framework and fillers filled between the framework materials;
the material for forming the framework is a silk screen;
the filler is made of at least one material selected from gelatin, water-soluble silk fibroin, collagen and hyaluronic acid.
In the composite tissue engineering scaffold material, the silk screen is a silk screen or a polyester polymer material fiber screen;
the silk screen is a mulberry silk screen, a tussah silk screen or a wild silk screen;
the polyester polymer material fiber net is specifically a PLA fiber net, a PGA fiber net or a PCL fiber net;
the water-soluble silk fibroin is mulberry silk fibroin, tussah silk fibroin or tussah silk fibroin.
The water-soluble silk fibroin can be directly obtained by a commercial way, or the commercially obtained water-insoluble silk fibroin can be purified according to various conventional methods to ensure that the water-soluble silk fibroin has water solubility; the water-solubility can be specifically carried out according to the following method:
putting 2g of silk fibroin powder into 10mL of 9.3M LiBr solution, heating and stirring in an oil bath kettle at 60 ℃ for 4 hours to completely dissolve the silk fibroin powder, transferring the obtained solution into a dialysis bag (3500D), dialyzing with deionized water for 3-4 days, and replacing the deionized water once every 4 hours; centrifuging the obtained solution at 10000rpm for 10 min, repeating for 3 times, collecting supernatant, filtering with microporous membrane to obtain silk fibroin aqueous solution, storing at 4 deg.C, and freeze drying to determine silk fibroin concentration.
The material for forming the filler can be specifically composed of gelatin and water-soluble silk fibroin; the mass ratio of the gelatin to the water-soluble silk fibroin is 30-90 mg: 3-15 mg.
The composite tissue engineering scaffold material is of a three-dimensional porous structure, the porosity is 78.91 +/-0.89-94.10 +/-1.45%, and the mean value of the expansion rate is 115.65 +/-4.65%.
The composite tissue engineering scaffold material also comprises porous microspheres;
the porous microspheres are dispersed in the filler;
specifically, the porous microspheres are polylactic acid (PLA) porous microspheres or polylactic-co-glycolic acid (PLGA) porous microspheres; the polylactic acid (PLA) porous microspheres are specifically levorotatory polylactic acid (PLLA) porous microspheres;
the particle size of the porous microsphere is 100-300 mu m; specifically 245 +/-35 μm; the aperture is 20-40 μm; specifically, it may be 27. + -. 4.8. mu.m.
Specifically, the composite tissue engineering scaffold material consists of silk screens, gelatin filled among the silk screens, water-soluble silk fibroin and PLLA porous microspheres;
the mass ratio of the silk screen, the gelatin, the water-soluble silk fibroin and the PLLA porous microspheres is specifically 2-3 mg: 30-90 mg: 3-15 mg: 0-15 mg; more specifically, it may be 2 mg: 30 mg: 3 mg: 0. 3 mg: 90 mg: 15 mg: 15mg, 2.67 mg: 70 mg: 10 mg: 10.67mg, 2.67 mg: 90 mg: 3 mg: 0 or 2.67 mg: 90 mg: 3 mg: 13.3 mg.
The method for preparing the composite tissue engineering scaffold material provided by the invention is a physical blending method, and comprises the following steps:
1) spreading a material for forming the framework in a mould;
mixing the materials constituting the filler with water to obtain a suspension;
2) and (2) injecting the suspension into the mold obtained in the step 1), cooling and molding, and then immersing into a cross-linking agent for cross-linking to obtain the composite tissue engineering scaffold material.
In the suspension prepared by the method, the mass percentage concentration of the gelatin is 3-9%; specifically, it can be 7%; the concentration of the water-soluble silk fibroin is 3-15 mg/mL; specifically, the concentration may be 10 mg/mL.
In the step 1), the step of spreading the material for forming the framework in the mold may specifically be: uniformly spreading a silk net into a net structure, cutting the net structure into a silk net with the length of 7cm, the width of 4cm and the thickness of 0.5cm, wherein the mass of the silk net is 30-45mg, and spreading the silk net into a mould with the length of 7cm multiplied by 4cm multiplied by 1 cm;
the method further comprises the following steps: adding the porous microspheres when preparing the suspension in the step 1);
the mass percentage concentration of the porous microspheres in the suspension is 0-15mg/mL and is not 0; specifically, the concentration of the surfactant may be 10.67 mg/mL.
In the step 2), the cross-linking agent is an aqueous solution of carbodiimide (EDC);
the mass percentage concentration of the cross-linking agent is 0.5-2%; in particular to 1 percent;
in the crosslinking step, the temperature is room temperature; the time is 4-12 h; in particular 8 h.
The method may further comprise: before step 1), the material constituting the skeleton, in particular a silk net, is degummed. The degumming process may be any of various commonly known methods. The silk net can be degummed, for example, as follows: placing the silkworm cocoon shell in 0.6% NaHCO3Boiling in solution for 20min, rinsing in deionized water for 2-3 times, repeating the above operation for 3 times, and oven drying at 60 deg.C; placing the shell of tussah cocoon in 0.5% Na2CO3Boiling in the solution for 20min, rinsing in deionized water for 2-3 times, repeating the above operation for 3 times, and oven drying at 60 deg.C for use.
The method may further comprise: after the step 2) cooling and forming step and before the crosslinking step, freeze-drying a product obtained by cooling and forming; in the freeze-drying step, the freeze-drying method is various conventional methods; the freeze-drying temperature can be-55 ℃.
In addition, the application of the composite tissue engineering scaffold material provided by the invention in preparing tissue repair products also belongs to the protection scope of the invention. Wherein, the tissue repair product can be a cartilage defect tissue repair product.
The invention selects silk, gelatin and polylactic acid as raw materials to prepare the composite tissue engineering scaffold material. The silk is used as a natural polymer material, has very excellent mechanical property, and the RGD sequence naturally exists on the tussah silk, so that the adhesion of cells is facilitated; although gelatin is a product of collagen denaturation or hydrolysis, gelatin retains the arginine-glycine-aspartic acid (RGD) sequence in the collagen structure, facilitates cell attachment, and has lower immunogenicity than collagen; in addition, the gelatin also has a certain buffering effect and a certain neutralization effect on an acid product generated by degrading the polylactic acid; polylactic acid (PLA) material is an artificially synthesized high molecular material, has excellent mechanical property, biocompatibility and biodegradability, and can be clinically used after FDA approval; the polylactic acid is prepared into porous microspheres, so that the mechanical property of the composite material can be enhanced on the basis of reducing the dosage of the polylactic acid, and the aseptic inflammatory reaction caused by acidic degradation products of the polylactic acid can be reduced. The porous scaffold material composite porous microspheres realize a hierarchical porous structure, so that the problem of poor adhesion of cells on the surface of the polylactic acid porous spheres is solved, and the hierarchical porous structure is favorable for the cells to migrate to the inside of the material. Through blending of multiple materials, the mechanical property of the stent material can be enhanced, and the sterile inflammatory reaction caused by the acidic degradation product of the polylactic acid can be reduced. The mechanical property of the scaffold material is enhanced again by regulating the material dosage to reach the mechanical index of organism tissues, and the degradation time is prolonged, so that the material degradation and tissue growth rate are regulated, and the scaffold material has important application value in the field of tissue repair scaffold materials.
Drawings
FIG. 1 is an electron micrograph of porous microspheres used in the examples.
FIG. 2 shows non-degummed (a) and degummed (b) mulberry silk and non-degummed (c) and degummed (d) tussah silk.
FIG. 3 shows a composite tissue engineering scaffold material (a) prepared according to example 4 and (b) prepared according to example 5.
Fig. 4 is a graph showing mechanical properties of the composite tissue engineering scaffold material (a) prepared according to example 4 and (b) prepared according to example 5.
FIG. 5 shows (a) confocal laser scanning images and (b) SEM images of a composite scaffold prepared according to example 5, inoculated with cartilage cells of rabbit ears, and cultured in vitro for two weeks.
FIG. 6 shows (a) confocal laser scanning images and (b) SEM images of a composite scaffold prepared in example 6, inoculated with cartilage cells of rabbit ears, and cultured in vitro for two weeks.
FIG. 7 shows the result of cytotoxicity test of composite tissue engineering scaffold material.
FIG. 8 is the results of in vitro degradation tests of composite tissue engineering scaffold materials prepared according to example 5.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
The porous microspheres used in the following examples are all L-polylactic acid (PLLA) porous microspheres, which can be prepared as follows:
fully dissolving 200mg of L-polylactic acid in 8mL of dichloromethane to prepare an oil phase; adding 1% NH dropwise under stirring4HCO3The solution forms a primary emulsion; transferring the primary emulsion into a 0.1% PVA aqueous solution to form multiple emulsion, and stirring for 3 h; collecting polylactic acid porous microspheres, washing with deionized water for 3 times, washing with 0.1M NaOH for 20min, washing with deionized water for 3 times, subpackaging, and freeze-drying to obtain porous microsphere solid powder.
The porous polylactic acid microsphere has the pore diameter of 27 +/-4.8 microns and the particle size of 245 +/-35 microns.
The mulberry silk used in the following examples was degummed before step 1) as follows:
placing silkworm cocoon shell in 06% NaHCO3Boiling in solution for 20min, rinsing in deionized water for 2-3 times, repeating the above operation for 3 times, and oven drying at 60 deg.C; placing the shell of tussah cocoon in 0.5% Na2CO3Boiling in the solution for 20min, rinsing in deionized water for 2-3 times, repeating the above operation for 3 times, and oven drying at 60 deg.C for use.
The silk fibroin is purified according to the following method:
putting 2g of silk fibroin powder into 10mL of 9.3M LiBr solution, heating and stirring in an oil bath kettle at 60 ℃ for 4 hours to completely dissolve the silk fibroin powder, transferring the obtained solution into a dialysis bag (3500D), dialyzing with deionized water for 3-4 days, and replacing the deionized water once every 4 hours; centrifuging the obtained solution at 10000rpm for 10 min, repeating for 3 times, collecting supernatant, filtering with microporous membrane to obtain silk fibroin aqueous solution, storing at 4 deg.C, and freeze drying to determine silk fibroin concentration.
Example 1 preparation of composite scaffold Material
(1) Uniformly spreading degummed mulberry silk into a net structure, cutting into silk nets with the length of 7cm, the width of 4cm and the thickness of 0.5cm and the mass of 30mg, and spreading the silk nets into a mould with the length of 7cm multiplied by 4cm multiplied by 1 cm.
(2) 9g of gelatin was dissolved in deionized water so that the gelatin solution concentration was 30%. 0mg of polylactic acid porous microspheres, 2.25mL of 20mg/mL mulberry silk fibroin solution and 1.5mL of 30% gelatin solution are blended, the volume of the obtained suspension is 15mL, so that the concentration of the porous microspheres is 0mg/mL, the concentration of the mulberry silk fibroin is 3mg/mL, the concentration of the gelatin is 3 wt%, and the mass of the gelatin in the suspension is 450 mg.
(3) And (3) injecting the suspension in the step (2) into the mould in the step (1), cooling and forming at the temperature of minus 20 ℃, and primarily obtaining the composite scaffold material after vacuum freeze drying (the freeze-drying temperature is minus 55 ℃).
(4) And (4) immersing the composite scaffold material in the step (3) into a 1 wt% carbodiimide (EDC) aqueous solution for crosslinking for 8h at room temperature, washing with water, and freeze-drying to finally obtain the composite tissue engineering scaffold material provided by the invention.
Example 2 preparation of composite scaffold Material
(1) Uniformly spreading degummed mulberry silk into a net structure, cutting into silk nets with the length of 7cm, the width of 4cm and the thickness of 0.5cm and the mass of 45mg, and spreading the silk nets into a mould with the length of 7cm multiplied by 4cm multiplied by 1 cm.
(2) 9g of gelatin was dissolved in deionized water so that the gelatin solution concentration was 30%. 225mg of polylactic acid porous microspheres, 9mL of a mulberry silk fibroin solution with the concentration of 25mg/mL and 4.5mL of 30% gelatin solution are blended to obtain 15mL of suspension, so that the concentration of the porous microspheres is 15mg/mL, the concentration of the mulberry silk fibroin is 15mg/mL, the concentration of the gelatin is 9 wt%, and the mass of the gelatin in the suspension is 1350 mg.
(3) And (3) injecting the suspension in the step (2) into the mould in the step (1), cooling and forming at the temperature of minus 20 ℃, and primarily obtaining the composite scaffold material after vacuum freeze drying (the freeze-drying temperature is minus 55 ℃).
(4) And (4) immersing the composite scaffold material in the step (3) into a 0.5 wt% carbodiimide (EDC) aqueous solution for crosslinking for 12h at room temperature, washing with water, and freeze-drying to finally obtain the composite tissue engineering scaffold material.
Example 3 preparation of composite scaffold Material
(1) Uniformly spreading degummed tussah silk into a net structure, cutting into silk net with length of 7cm, width of 4cm and thickness of 0.5cm and mass of 40mg, and spreading the silk net into a mould with length of 7cm × 4cm × 1 cm.
(2) 9g of gelatin was dissolved in deionized water so that the gelatin solution concentration was 30%. 160mg of polylactic acid porous microspheres, 7.5mL of 20mg/mL mulberry silk fibroin solution and 3.5mL of 30% gelatin solution are blended to obtain 15mL of suspension, so that the concentration of the porous microspheres is 10.67mg/mL, the concentration of the mulberry silk fibroin is 10mg/mL, the concentration of the gelatin is 7 wt%, and the mass of the gelatin in the suspension is 1050 mg.
(3) And (3) injecting the suspension in the step (2) into the mould in the step (1), cooling and forming at the temperature of minus 20 ℃, and primarily obtaining the composite scaffold material after vacuum freeze drying (the freeze-drying temperature is minus 55 ℃).
(4) And (4) immersing the composite scaffold material in the step (3) into a 2 wt% carbodiimide (EDC) aqueous solution for crosslinking for 4h at room temperature, washing with water, and freeze-drying to finally obtain the composite tissue engineering scaffold material.
Example 4 preparation of composite scaffold Material
The preparation method of the composite stent material is the same as that in example 1, only the dosage of the silk screen is 40mg, the dosage of the 30% gelatin solution is 4.5mL, and the gelatin quality is correspondingly replaced according to the dosage.
Example 5 preparation of composite scaffold Material
The preparation method of the composite scaffold material is the same as that in example 1, only the dosage of the silk screen is 40mg, the dosage of the 30% gelatin solution is 4.5mL, the gelatin quality is correspondingly replaced according to the dosage, and the dosage of the polylactic acid porous microsphere is 200 mg.
Example 6 preparation of composite scaffold Material
The preparation method of the composite scaffold material is the same as that in example 1, only the dosage of the tussah silk screen is 40mg, the dosage of the 30% gelatin solution is 4.5mL, the gelatin mass is correspondingly replaced according to the dosage, and the dosage of the polylactic acid porous microsphere is 200 mg.
FIG. 3 shows a composite tissue engineering scaffold material (a) prepared according to example 4 and (b) prepared according to example 5. As can be seen from the figure, the composite tissue engineering scaffold material is in a three-dimensional porous structure, and the silk is uniformly distributed in the composite material. The porosity is 78.91 + -0.89% -94.10 + -1.45%, and the average expansion rate is 115.65 + -4.65%.
Fig. 4 is a graph showing mechanical properties of the composite tissue engineering scaffold material (a) prepared according to example 4 and (b) prepared according to example 5. As can be seen from the figure, the mechanical property of the composite scaffold material can be enhanced by adding the porous microspheres.
FIG. 5 shows (a) confocal laser scanning images and (b) scanning electron microscope images of a scaffold material for complex tissue engineering prepared in example 5, inoculated with rabbit ear osteocytes, cultured in vitro (37 ℃ C. in a constant temperature incubator containing 5% carbon dioxide, and the medium was changed every two days) for two weeks. As can be seen from the figure, the proliferation state of the rabbit ear cartilage cells on the composite scaffold material is better.
FIG. 6 shows (a) confocal laser scanning images and (b) scanning electron microscope images of a scaffold material for complex tissue engineering prepared in example 6, inoculated with rabbit ear cartilage cells, cultured in vitro (37 ℃ in a constant temperature incubator containing 5% carbon dioxide, and the medium replaced every two days) for two weeks. As can be seen from the figure, the proliferation state of the rabbit ear cartilage cells on the composite scaffold material is better.
FIG. 7 shows cytotoxicity test of composite tissue engineering scaffold material.
The specific test method is as follows:
taking rabbit ear cartilage cells growing to 80% fusion, digesting, centrifuging, resuspending, counting by a cell counter, and diluting the cell concentration to 1 × 104And each cell/mL is inoculated into a 96-well plate by 1mL per well, 10mg of the composite scaffold material prepared according to the embodiment 4, the embodiment 5 and the embodiment 6 is respectively added into each group after 24h of cell adherence, 4 groups are provided, each group is provided with 6 multiple wells, and the group without material is provided with a control group. Incubating the composite scaffold material and cells for 24h, removing the supernatant and the composite scaffold material, adding a CCK-8 detection system, and measuring absorbance at a wavelength of 450nm by using an enzyme-labeling instrument after 4hThe value is obtained.
The blank was not provided with any material, (a) was the composite tissue scaffold material prepared according to example 4, (b) was the composite tissue scaffold material prepared according to example 5, and (c) was the composite tissue scaffold material prepared according to example 6. As can be seen from the figure, the composite scaffold material is nontoxic to the ear osteocyte of the rabbit and has good in-vitro safety.
FIG. 8 is the results of in vitro degradation tests of composite tissue engineering scaffold materials prepared according to example 5.
The stent material degradation test method is specifically as follows:
the stent material was cut into rectangular blocks of approximately 6mm by 7mm by 3mm and weighed as m0Placing the materials in a 24-pore plate, carrying out ultraviolet irradiation for 3h, adding 2mL PBS into each pore, immersing the materials in the PBS, placing the materials in a constant-temperature incubator at 37 ℃ for incubation, replacing fresh PBS once every two days, taking out the materials according to a set time point, washing the materials with water for three times, freeze-drying and weighing the materials as mt
Figure BDA0001971397700000071
As can be seen, the scaffold material gradually degraded with increasing days of degradation.

Claims (10)

1. A composite tissue engineering scaffold material comprises a framework and fillers filled between the framework materials;
the material for forming the framework is a silk screen; the silk screen is a silk screen or a polyester polymer material fiber screen;
the material for forming the filler consists of gelatin and water-soluble silk fibroin;
the composite tissue engineering scaffold material also comprises porous microspheres; the porous microspheres are dispersed in the filler;
the porous microspheres are polylactic acid porous microspheres or polylactic acid-glycolic acid copolymer porous microspheres;
the aperture of the porous microsphere is 20-40 μm;
the method for preparing the composite tissue engineering scaffold material comprises the following steps:
1) spreading a material for forming the framework in a mould;
mixing the materials constituting the filler with water to obtain a suspension;
the method further comprises the following steps: adding the porous microspheres when preparing the suspension in the step 1);
the mass percentage concentration of the porous microspheres in the suspension is 0-15mg/mL and is not 0;
2) and (2) injecting the suspension into the mold obtained in the step 1), cooling and molding, and then immersing into a cross-linking agent for cross-linking to obtain the composite tissue engineering scaffold material.
2. The composite tissue engineering scaffold material of claim 1, wherein: the silk screen is a mulberry silk screen, a tussah silk screen or a wild silk screen;
the polyester polymer material fiber net is a PLA fiber net, a PGA fiber net or a PCL fiber net;
the water-soluble silk fibroin is water-soluble mulberry silk fibroin, water-soluble tussah silk fibroin or water-soluble tussah silk fibroin;
the composite tissue engineering scaffold material is of a three-dimensional porous structure, the porosity is 78.91 +/-0.89-94.10 +/-1.45%, and the mean value of the expansion rate is 115.65 +/-4.65%.
3. The composite tissue engineering scaffold material of claim 1 or 2, wherein: the particle size of the porous microsphere is 100-300 mu m.
4. The composite tissue engineering scaffold material of claim 3, wherein: the composite tissue engineering scaffold material consists of silk screens, gelatin filled among the silk screens, water-soluble silk fibroin and PLLA porous microspheres;
the mass ratio of the silk screen, the gelatin, the water-soluble silk fibroin and the PLLA porous microspheres is 2-3 mg: 30-90 mg: 3-15 mg: 0-15 mg.
5. A method of preparing the composite tissue engineering scaffold material of any one of claims 1-4, comprising:
1) spreading a material for forming the framework in a mould;
mixing the materials constituting the filler with water to obtain a suspension;
the method further comprises the following steps: adding the porous microspheres when preparing the suspension in the step 1);
the mass percentage concentration of the porous microspheres in the suspension is 0-15mg/mL and is not 0;
2) and (2) injecting the suspension into the mold obtained in the step 1), cooling and molding, and then immersing into a cross-linking agent for cross-linking to obtain the composite tissue engineering scaffold material.
6. The method of claim 5, wherein: in the suspension, the mass percentage concentration of gelatin is 3-9%; the concentration of the water-soluble silk fibroin is 3-15 mg/mL.
7. The method according to claim 5 or 6, characterized in that: in the step 2), the cross-linking agent is a carbodiimide water solution;
the mass percentage concentration of the cross-linking agent is 0.5-2%;
in the crosslinking step, the temperature is room temperature; the time is 4-12 h.
8. The method of claim 7, wherein: the mass percentage concentration of the cross-linking agent is 1 percent;
in the crosslinking step, the time is 8 hours.
9. Use of the composite tissue engineering scaffold material of any one of claims 1 to 4 in the preparation of a tissue repair product.
10. Use according to claim 9, characterized in that: the tissue repair product is a cartilage defect tissue repair product.
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