Composite silk fibroin modified biomimetic bone material and application thereof

Abstract

The application discloses a kind of composite silk fibroin modified biomimetic bone material and its application, belong to bone tissue engineering material technical field.The preparation method of the disclosed biomimetic bone material includes: gradient stirring, standing forming, freeze-drying are carried out to the mixed solution of silk fibroin solution and calcium phosphate oligomer aqueous solution, and the biomimetic bone material is obtained.In the material preparation process, the mineralization reaction condition and gradient shear force are regulated, so that the material is integrated to form outer dense plate layer structure and inner porous network structure, wherein the outer layer simulates cortical bone to provide higher mechanical strength and elastic modulus, and the inner layer simulates cancellous bone to facilitate cell ingrowth, tissue regeneration and angiogenesis.The biomimetic bone material has good mechanical support performance, biocompatibility, osteogenic induction ability and proangiogenic potential, and can be used for load-bearing or non-load-bearing bone defect repair, and has good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and bone tissue engineering technology, specifically relating to a composite silk fibroin modified biomimetic bone material, its preparation method, and its application. Background Technology

[0002] Silk fibroin is widely used in bone tissue engineering due to its good biocompatibility, biodegradability, and processability. However, existing silk fibroin-based bone repair materials often suffer from insufficient mechanical properties, simple structure, limited mineralization, and difficulty in simulating the natural bone gradient structure, which limits their application in load-bearing and complex bone defect repair. Summary of the Invention

[0003] In view of the defects or deficiencies of the existing technology, the present invention provides a composite silk fibroin modified biomimetic bone material.

[0004] Therefore, the preparation method of the biomimetic bone material provided by the present invention includes: gradient stirring of a mixture of silk fibroin solution and calcium phosphate oligomer aqueous solution, static molding, and freeze-drying to obtain the biomimetic bone material; the preparation method of the calcium phosphate oligomer includes: reacting H3PO4 ethanol solution with an ethanol solution containing calcium salt and triethylamine, and washing the ethanol and triethylamine in the solid reactants to obtain calcium phosphate oligomers; the pH of the mixture is 7.0-7.6; the gradient stirring includes: stirring at 400-800 rpm for 1-4 h at 2-8℃, followed by stirring at 100-300 rpm for 0.5-3 h at 15-30℃.

[0005] Furthermore, the biomimetic bone material package contains an internal porous body and an external dense plate layer that wraps around the internal porous body circumferentially, and the internal porous body and the external dense plate layer are an integral structure; the elastic modulus and hardness of the external dense plate layer are both higher than those of the internal porous body.

[0006] An alternative is that the elastic modulus of the internal porous body is 0.2–0.5 GPa and the hardness is 20–30 MPa, while the elastic modulus of the external dense plate layer is 9.0–9.5 GPa and the hardness is 195–202 MPa.

[0007] Alternatively, the biomimetic bone material may have an elastic modulus of 9.0–9.7 GPa and a hardness of 205–230 MPa. For example, it may have an elastic modulus of 9.45 GPa and a hardness of 227 MPa.

[0008] An alternative approach is to use calcium phosphate oligomers accounting for 10% to 20% of the total mass of the dried silk fibroin solution and the calcium phosphate oligomers.

[0009] Alternatively, the calcium salt may be selected from calcium chloride or calcium phosphate.

[0010] An alternative approach is to place the static molding process at -20℃ to 0℃ for 5 to 6 hours.

[0011] An alternative approach is to use a stepped temperature freeze-drying method, including freeze-drying at -20℃ for 12–36 h, freeze-drying at -10℃ for 12–36 h, freeze-drying at 0℃ for 6–24 h, and freeze-drying at 5℃ for 6–24 h.

[0012] This invention combines regenerated silk fibroin with calcium phosphate oligomers, utilizing the silk fibroin molecular chains, β-lamellae structure, and amorphous regions as mineralization nucleation sites to enable in-situ nucleation, growth, and deposition of calcium phosphate within the silk fibroin matrix. Simultaneously, during the preparation process, gradient shearing is applied to regulate the mineralization reaction kinetics and material forming process. Specifically, under low-temperature conditions and relatively high stirring speed, the calcium phosphate oligomers are rapidly mineralized within the silk fibroin matrix, inducing the formation of a highly solidified inner layer structure. In this stage, the higher shearing promotes the rearrangement of silk fibroin molecular chains and the nucleation and deposition of calcium phosphate oligomers within the silk fibroin, thereby forming a mineralized structure with porous network characteristics. Subsequently, stirring continues at room temperature with a relatively low stirring speed, allowing the partially solidified composite mineralization system to continue to mineralize slowly under lower shearing, gradually coating the inner layer structure. In this stage, the lower shearing facilitates the formation of a relatively dense layered stacked structure, thus obtaining a dense outer lamellar region.

[0013] This constructs an integrated biomimetic bone scaffold with an outer dense lamellar structure and an inner porous network structure. The outer dense lamellar region simulates the structure of natural cortical bone, while the inner porous structure simulates the structure of natural cancellous bone. The outer dense lamellar region and the porous structure are tightly integrated. The inner porous structure has interconnected pores that promote cell adhesion, cell migration, nutrient exchange, blood vessel ingrowth, and bone regeneration. This invention relates to the application of the silk fibroin-modified rigid material in the preparation of bone tissue repair materials. Attached Figure Description

[0014] Figure 1 The images show the external structure (a), electron microscope structure (b), and Fourier transform infrared spectrum (c) of the biomimetic gradient bone scaffold of this invention.

[0015] Figure 2 shows the appearance (a) and electron microscope image (b) of the material prepared in the comparative example.

[0016] Figure 3 shows the mechanical property test results of the materials prepared in the embodiments and comparative examples of the present invention; Figure 3(a) shows the elastic modulus test results of the internal and external structures of the material prepared in Example 1; Figure 3(b) shows the hardness test results of the internal and external structures of the material prepared in Example 1; Figure 3(c) shows the elastic modulus test results of the materials prepared in Example 1 and each comparative example; Figure 3(d) shows the hardness test results of the materials prepared in Example 1 and each comparative example.

[0017] Figure 4 The results of biocompatibility testing (a) and statistical analysis (b) of the materials prepared in the examples and comparative examples are shown, with green representing live cells.

[0018] Figure 5 This is a graph showing the in vitro osteogenic capacity test results of the biomimetic mineralized material prepared in the embodiments of the present invention; Figure 5 (a) and (b) show the ALP staining results and the same statistical analysis; Figure 5 (c) and (d) show the ARS staining results and the same statistical analysis. Detailed Implementation

[0019] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0020] The silk fibroin solution of the present invention is obtained by degumming, dissolving, and dialysis of natural mulberry silk. The preparation method of the silk fibroin solution used in the following examples is as follows:

[0021] (1) Weigh 20 g of shredded high-quality silkworm cocoons and place them in 1.5 L of 0.02 M sodium carbonate aqueous solution. After the sodium carbonate solution boils, add the silkworm cocoons and continue boiling for 30 min. Stir continuously during the boiling process to prevent the silkworm silk from tangling into bundles. (2) After boiling, remove the degummed silk and wash it thoroughly with enough deionized water 5 times to remove residual sodium carbonate and sericin. Place the washed degummed silk in a 37°C oven or fume hood to dry for later use. (3) Weigh 15 g of dried degummed silk and add it to 60 ml of 9.3 M lithium bromide solution to fully soak and dissolve it. Then heat the mixture in a 60°C water bath for 4 h to obtain silk fibroin solution. (4) Transfer the obtained silk fibroin solution to a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze it in distilled water for 3 days, changing the distilled water every 8 hours; (5) After dialysis, the silk fibroin solution in the dialysis bag is collected into a centrifuge tube and centrifuged at 4℃ and 8000 rpm for 10 min. The centrifugation is repeated twice, and the supernatant is collected to obtain the regenerated silk fibroin solution (the silk fibroin solution is dried and the solid is collected. The solid accounts for 7% of the mass volume of the corresponding silk fibroin solution, that is, the solid concentration of the silk fibroin solution is 70 mg / ml). The obtained regenerated silk fibroin solution is stored in a refrigerator at 4℃ for later use.

[0022] Example 1: Preparation of calcium phosphate oligomers: (1) At room temperature, weigh 5.88 g CaCl2·2H2O and add it to 800 mL of ethanol. Mix for 15 min under stirring to disperse it fully and obtain CaCl2 alcohol solution. (2) Then add 110.9 mL of triethylamine to the above CaCl2 alcohol solution and continue stirring at room temperature for 30 min to form a uniform calcium-containing solution; (3) Take another 2.09 mL of H3PO4, dissolve it in 40 mL of ethanol, and prepare a phosphoric acid solution; (4) Under stirring conditions, the phosphoric acid solution was slowly added to the calcium-containing solution to form calcium phosphate oligomers. The calcium phosphate oligomers were centrifuged at 4000 rpm for 5 min and the precipitate was collected. The precipitate was washed three times with ethanol to remove residual triethylamine. Then it was washed twice with deionized water to remove residual ethanol and obtain CPO gel (0.311 g of CPO can be collected after drying). Finally, it was resuspended in distilled water to obtain a calcium phosphate oligomer suspension with a concentration of 40 mg / mL.

[0023] Preparation of composite silk fibroin modified biomimetic bone material: Take 30 mL of regenerated silk fibroin solution and, under stirring, add 10 mL of calcium phosphate oligomer suspension. After mixing, adjust the pH of the system to 7.2–7.4 with dilute sodium hydroxide (NaOH) solution (0.1 mol / L) and dilute hydrochloric acid (HCl) solution (0.1 mol / L) to obtain a silk fibroin / calcium phosphate oligomer composite mineralization system. Shear mineralization: Gradient shearing was applied to the composite mineralization system: First, the system was stirred at 500 rpm for 2 hours at 4°C to rapidly mineralize the calcium phosphate oligomers within the silk fibroin matrix and induce the formation of a highly solidified inner layer structure. Subsequently, the stirring rate was reduced to 200 rpm, and stirring was continued for 1 hour at room temperature (26°C) to allow the partially solidified composite mineralization system to continue to slowly mineralize under lower shearing and gradually coat the inner layer structure.

[0024] Shaping and freeze-drying: After shearing and mineralization, the material is placed at -4℃ for 4 hours to allow it to set and then subjected to gradient freeze-drying in a freeze dryer. The preferred freeze-drying program is: freeze-drying at -20℃ for 24 h, freeze-drying at -10℃ for 24 h, freeze-drying at 0℃ for 12 h, and freeze-drying at 5℃ for 12 h. After freeze-drying, an integrated composite silk fibroin-modified biomimetic bone material with a dense outer lamellar structure and an internal porous network structure is obtained.

[0025] Comparative Example 1: The difference between this comparative example and Example 1 is that no calcium phosphate oligomer suspension was added, but the rest of the operation was the same as in Example 1, to obtain unmineralized silk fibroin scaffold material.

[0026] Comparative Example 2: The difference between this comparative example and Example 1 is that the calcium phosphate oligomers were replaced with hydroxyapatite powder (289396, Sigma). Specifically, the hydroxyapatite powder was resuspended in distilled water (40 mg / ml) and then added to the regenerated silk fibroin solution under stirring. The remaining preparation conditions were the same as in Example 1.

[0027] Comparative Example 3: The difference between this comparative example and Example 1 is that only a single stirring rate was used for the mineralization reaction during the shear mineralization process. Specifically, the stirring speed was 200 rpm and the duration was 3 hours. The rest of the operation was the same as in Example 1.

[0028] The biomimetic gradient bone scaffold structure was validated using the materials prepared in the above embodiments and comparative examples: The materials obtained in Example 1 and each comparative example were observed by macroscopic morphology and scanning electron microscopy; at the same time, the materials prepared in Example 1 were analyzed by Fourier transform infrared spectroscopy.

[0029] The specific method is as follows: Take the corresponding material, first use a digital camera to observe and record its overall macroscopic morphology; then after the material is sputtered with gold, use a scanning electron microscope to observe the cross-sectional morphology and the distribution of inner and outer layer structures.

[0030] Meanwhile, Fourier transform infrared spectroscopy was performed on the material sample prepared in Example 1 to analyze the characteristic structure of silk fibroin and calcium phosphate mineralization phase in the material.

[0031] The results are as follows Figure 1 As shown in Figure -2. Figure 1 a is a macroscopic morphology diagram of the material, which shows that the material is fully formed. Figure 1b is a scanning electron microscope image of the material as a whole and a local magnification, showing that the material has obvious inner and outer layer structures. Further observation can be made that the outer layer has a relatively dense lamellar structure, the inner layer has a porous network structure, and the inner and outer layers are tightly bonded as an integral structure. The dense lamellar region of the outer layer is denoted as the SF-C200 structure region, and the porous network region of the inner layer is denoted as the SF-C500 structure region.

[0032] Compared with the electron microscope image b of the material in Example 1, Comparative Examples 1, 2 and 3 did not form obvious inner and outer layer partitioning structures, while the material in Example 1 showed obvious layered structure. This indicates that the present invention uses in-situ mineralization of calcium phosphate oligomers combined with gradient shear regulation to achieve the integrated construction of an outer dense lamellar structure and an inner porous network structure, forming a gradient structure that mimics natural bone.

[0033] The above results show that the present invention achieves the integrated construction of an outer dense lamellar structure and an inner porous network structure through in-situ mineralization and gradient shear regulation. It can better simulate the highly ordered gradient structure characteristics formed by the high-density, high-strength cortical bone of the outer layer and the porous, reticular cancellous bone of the inner layer in natural bone.

[0034] Figure 1 c is the Fourier transform infrared spectrum of the material prepared in Example 1. The infrared spectroscopy results show that characteristic absorption peaks related to amide bonds of silk fibroin and phosphate-related absorption peaks can be detected in the material, indicating that the calcium phosphate mineralization phase has been successfully introduced into the silk fibroin matrix.

[0035] The above results indicate that the composite silk fibroin modified biomimetic bone material prepared in this invention has an organic / inorganic composite mineralization structure and can form an integrated biomimetic gradient bone scaffold with distinct internal and external partitions and a continuous structure.

[0036] The mechanical properties of the materials prepared in the above embodiments and comparative examples were tested. To verify the mechanical reinforcement effect of the material of the present invention, the mechanical properties of the scaffolds obtained in Example 1 (SF-C), Comparative Example 1 (SF), Comparative Example 2 (SF-HAP), and Comparative Example 3 ((SF-C) without gradient) were tested. The materials of each group were cut to the same size, and the hardness and elastic modulus of different regions of the material were tested using a nanoindenter.

[0037] Referring to Figures 3(c, d), the overall elastic modulus of the material obtained in Example 1 is 9.45 GPa and the hardness is 227 MPa, both superior to those of the comparative examples. Compared with Comparative Example 1, Example 1 significantly improved the mechanical properties of the silk fibroin scaffold through in-situ calcium phosphate mineralization; compared with Comparative Example 2, the calcium phosphate mineralized phase in Example 1 is more tightly bound to the silk fibroin matrix, which is superior to the physical blending and reinforcement effect of hydroxyapatite powder; compared with Comparative Example 3, Example 1 forms a gradient structure with a dense outer layer and a porous inner layer, further improving the mechanical support capacity of the material. The overall elastic modulus of the material obtained in Example 1 of this invention is 9.45 GPa and the hardness is 227 MPa, exhibiting good mechanical support performance. This result indicates that in-situ mineralization of calcium phosphate oligomers and gradient shear regulation synergistically improve the structural stability and mechanical properties of the silk fibroin scaffold.

[0038] Meanwhile, the material prepared in Example 1 possesses a dense lamellar structure on the outer layer and a porous network structure on the inner layer, which can respectively simulate natural cortical bone and cancellous bone in terms of structure and function. Further mechanical testing was performed on the inner and outer layers of the material prepared in the example. The composite material was placed in an operating table, and the outer and inner layers were slowly peeled off along the interface using a sterile, fine surgical blade to obtain the outer and inner layers. These were then cut to the same size, and the hardness and elastic modulus of different regions of the material were measured using a nanoindentation instrument. Figure 3a Results b show that the inner porous SF-C500 structural region has an elastic modulus of 0.3 GPa and a hardness of 25 MPa, while the outer dense plate SF-C200 structural region has a higher elastic modulus of 9.3 GPa and a hardness of 197 MPa.

[0039] Natural bone tissue exhibits typical structural and mechanical partitioning characteristics, comprising an outer cortical bone and an inner cancellous bone. Cortical bone is dense, possessing high elastic modulus and hardness; its elastic modulus is typically 10–30 GPa, and its hardness is typically 0.2–0.8 GPa (200–800 MPa), primarily serving as a support and load-bearing component. Cancellous bone, on the other hand, is porous and has relatively low mechanical strength; its apparent elastic modulus is typically 0.05–2 GPa, and its hardness is typically 0.05–0.3 GPa (5–300 MPa), facilitating cell ingrowth, nutrient exchange, angiogenesis, and new bone formation.

[0040] Therefore, the material of this invention is suitable for repairing bone defects that require both mechanical support and tissue regeneration, especially for repairing cortical-cancellous bone composite defects. The outer dense structure provides initial mechanical support to the defect area, while the inner porous structure provides space for cell adhesion, migration, and new bone ingrowth, thereby achieving structural reconstruction and functional repair of the bone defect site.

[0041] The biocompatibility of the materials prepared in the above embodiments and comparative examples was verified. Cell compatibility was tested on the scaffolds obtained in Example 1 (SF-C), Comparative Example 1 (SF), Comparative Example 2 (SF-HAP), and Comparative Example 3 (SF-C without gradient).

[0042] Cell viability was determined using a live / dead cell staining method: First, bone marrow mesenchymal stem cells (BMSCs, primary isolated from SD rat bone marrow, autonomously isolated and cultured) were cultured. Then, materials from each group were cut into identical sizes (20 mm diameter discs, 1 mm thickness), sterilized (autoclaved at 121 ℃ (0.1 MPa, 20 min)) and co-cultured with BMSCs in 6-well cell culture plates (1 × 10⁶ cells per well). 4 cells / cm 2 α-MEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin was added at a rate of 2 mL per well. On days 1 and 3 of co-culture, the medium was aspirated from the wells, and the cells were gently washed twice with PBS to remove serum and dead cell debris. The cell suspension was collected, centrifuged, and the supernatant was discarded. The cells were washed once with PBS and then resuspended, adjusting the concentration to approximately 1 × 10⁻⁶. 5 –1×10 6 Cells / mL, add 1 ml of Calcein-AM / PI staining working solution, incubate at 37°C in the dark for 30 minutes, and then take pictures and analyze using a fluorescence microscope.

[0043] Cell proliferation was assessed using the CCK-8 assay: First, bone marrow mesenchymal stem cells (BMSCs) were cultured. Then, materials from each group were cut into identical sizes (20 mm diameter discs, 1 mm thick), sterilized, and co-cultured with BMSCs in 48-well cell culture plates (5 × 10⁶ cells per well). 3 cells / cm 2 The culture medium was α-MEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, with 500 μL added to each well. On days 1, 3, and 5 of co-culture, 10 μL of CCK-8 (Dojindo, Japan) staining solution was added to each well of the cells cultured in the plate. The cells were incubated in a 37°C 5% CO2 cell culture incubator for 1 h. The absorbance was measured at 450 nm using a microplate reader. Intergroup comparisons were made and statistical graphs were plotted.

[0044] See Figure 4Results a and 4b showed that none of the materials exhibited significant cytotoxicity, and cells were able to adhere and grow on the material surface. Specifically, cells in both Example 1 and the comparative examples showed good survival and sufficient cell spread; furthermore, the proliferation in Example 1 was slightly better than in the comparative examples. Compared to Comparative Example 1, the calcium phosphate mineralization phase in Example 1 improved the biomimetic mineralization microenvironment on the material surface; compared to Comparative Example 2, Example 1 formed a more stable organic / inorganic composite structure through in-situ mineralization, reducing the local aggregation that might be caused by the physical mixing of hydroxyapatite particles; compared to Comparative Example 3, the gradient structure of Example 1 was more conducive to cell adhesion, migration, and growth. This indicates that the integrated biomimetic mineralization material of the present invention has good biocompatibility and can provide a suitable cell growth microenvironment for bone tissue repair.

[0045] The in vitro osteogenic capacity of the materials prepared in the above embodiments and comparative examples was verified. To verify the in vitro osteogenic induction capacity of the material of the present invention, scaffolds obtained from Example 1 (SF-C), Comparative Example 1 (SF), Comparative Example 2 (SF-HAP), and Comparative Example 3 ((SF-C) without gradient) were used for in vitro osteogenic capacity testing. The in vitro osteogenic capacity of the material was evaluated using alkaline phosphatase (ALP) activity assay (Beyotime, China) and Alizarin Red (ARS) staining (Sigma, USA). First, bone marrow mesenchymal stem cells (BMSCs) were cultured. Materials from each group were cut to the same size (20mm diameter circular slices, 1mm thickness), sterilized, and then co-cultured with BMSCs in 24-well cell culture plates (1×10⁶ cells per well). 4 cells / cm 2 The culture medium was α-MEM complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics, with 1 mL added to each well. After the cells adhered well, the original medium was discarded, and the cells were gently washed once with PBS. The medium was then replaced with an equal volume of osteogenic induction medium (the osteogenic induction medium was prepared by adding 100 nM dexamethasone, 10 mM sodium β-glycerophosphate, and 50 μg / mL ascorbic acid to α-MEM complete medium as a base). The osteogenic induction medium was replaced with fresh osteogenic induction medium every 2–3 days during the induction period.

[0046] On days 7 and 14 of induction culture, ALP staining and photographic analysis and quantitative detection of ALP activity were performed, respectively. ALP staining and photographic analysis: After washing the cells three times with PBS, fix them with 4% paraformaldehyde for 20 minutes, wash them three times with PBS, add 150 μL of ALP staining working solution to each well of a 24-well plate, and then incubate at 37 ℃ in the dark for 30 minutes. After removing the staining solution, wash the cells three times with PBS and take photographs for analysis using an inverted microscope.

[0047] ALP activity absorbance assay: After aspirating the culture medium, the cells were digested with trypsin, resuspended, and the culture medium was aspirated again. The cells were washed three times with PBS, and 100 μL of 0.1% Triton-X100 solution was added. The cells were shaken on a microplate for 30 minutes. 50 μL of cell lysate was added to a 96-well plate pre-cooled at 4°C, and 150 μL of ALP substrate reaction solution was added. The plate was incubated in a shaker for 30 minutes. The reaction was terminated with 0.1 mol / L NaOH, and the absorbance was measured at 405 nm using a microplate reader.

[0048] On days 14 and 21 of induction culture, ARS staining and photographic analysis and quantitative detection of ARS activity were performed, respectively. ARS staining and imaging analysis: After rinsing with PBS, cells were fixed with 4% paraformaldehyde for 20 minutes. The fixative was then removed, and the cells were washed twice with PBS. Alizarin Red S staining solution was added to cover the samples, and staining was performed for 5 minutes. After staining, the culture medium was removed, and the cells were washed twice with PBS. Calcium deposits were observed using an inverted microscope. After imaging, the PBS was discarded, and 500 μL of 10% cetylpyridine chloride solution was added to each well. The cells were incubated at room temperature with shaking for 30 minutes until the dye was completely dissolved. The cells were then gently mixed by pipetting, and 200 μL of eluent was transferred from each well to a 96-well plate. The absorbance at 562 nm was measured using a microplate reader.

[0049] See Figure 5 a, b, ALP staining results showed that the staining intensity of the Example 1 group was significantly stronger than that of the other pairs, indicating that the material obtained in Example 1 can more effectively promote early osteogenic differentiation.

[0050] See Figure 5c and d, ARS staining results showed that the Example 1 group had more mineralized nodules and more obvious calcium salt deposition, indicating that it had superior late-stage osteogenic mineralization capacity. Statistical analysis results showed that the ALP activity and ARS quantification results of the Example 1 group were higher than those of Comparative Examples 1, 2, and 3, and the differences were statistically significant. Compared with Comparative Example 1, the calcium phosphate mineralization phase in Example 1 provided a biomimetic mineralization microenvironment for osteogenic differentiation; compared with Comparative Example 2, Example 1 formed a more stable organic / inorganic composite structure through in-situ mineralization of calcium phosphate oligomers, which was superior to the physical mixing effect of hydroxyapatite powder; compared with Comparative Example 3, the outer dense and inner porous gradient structure formed in Example 1 was more conducive to cell ingrowth, nutrient exchange, and osteogenic differentiation.

[0051] The above results indicate that the integrated biomimetic mineralization material of the present invention can significantly promote osteogenic differentiation and mineralization deposition of osteogenic cells and has good in vitro osteogenic induction ability.

Claims

1. A composite silk fibroin modified biomimetic bone material, characterized in that, The preparation method of the biomimetic bone material includes: gradient stirring of a mixture of silk fibroin solution and calcium phosphate oligomer aqueous solution, static molding, and freeze-drying to obtain the biomimetic bone material; The method for preparing the calcium phosphate oligomer includes: reacting an H3PO4 ethanol solution with an ethanol solution containing calcium salt and triethylamine, and then washing the solid reactants to remove the ethanol and triethylamine to obtain the calcium phosphate oligomer; The pH of the mixture is 7.0–7.6; The gradient stirring includes: stirring at 400-800 rpm for 1-4 hours at 2-8°C, followed by stirring at 100-300 rpm for 0.5-3 hours at 15-30°C.

2. The biomimetic bone material according to claim 1, characterized in that, The biomimetic bone material package contains an internal porous body and an external dense plate layer that wraps around the internal porous body circumferentially, with the internal porous body and the external dense plate layer forming an integral structure; the elastic modulus and hardness of the external dense plate layer are both higher than those of the internal porous body.

3. The biomimetic bone material according to claim 1, characterized in that, The internal porous body has an elastic modulus of 0.2–0.5 GPa and a hardness of 20–30 MPa, while the external dense plate layer has an elastic modulus of 9.0–9.5 GPa and a hardness of 195–202 MPa.

4. The biomimetic bone material according to claim 1, characterized in that, The biomimetic bone material has an elastic modulus of 9.0–9.7 GPa and a hardness of 205–230 MPa.

5. The bone-like matrix composite silk fibroin modified rigid material according to claim 1, characterized in that, The mass of calcium phosphate oligomers accounts for 10% to 20% of the total mass of the dried silk fibroin solution and the total mass of calcium phosphate oligomers.

6. The bone-like matrix composite silk fibroin modified rigid material according to claim 1, characterized in that, The calcium salt is selected from calcium chloride or calcium phosphate.

7. The bone-like matrix composite silk fibroin modified rigid material according to claim 1, characterized in that, The static molding process involves placing the sample at -20℃ to 0℃ for 5 to 6 hours.

8. The bone-like matrix composite silk fibroin modified rigid material according to claim 1, characterized in that, The freeze-drying process is a stepped temperature freeze-drying method, including freeze-drying at -20℃ for 12–36 h, freeze-drying at -10℃ for 12–36 h, freeze-drying at 0℃ for 6–24 h, and freeze-drying at 5℃ for 6–24 h.

9. The application of the silk fibroin modified rigid material according to claim 1 in the preparation of bone tissue repair materials.

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