Preparation method of porous sponge scaffold loaded with vitamin C microspheres

Abstract

The present invention discloses a preparation method of a porous sponge scaffold loaded with vitamin C microspheres. The method first uses cortical bone to prepare bone matrix gel, then prepares vitamin C microspheres, adds vitamin C microspheres and BMP protein to the bone matrix gel, forms a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and making a film. Finally, by cross-linking, a porous sponge scaffold loaded with vitamin C microspheres is made. The porous sponge scaffold loaded with vitamin C microspheres prepared by the present invention can significantly promote cell proliferation, differentiation and angiogenesis under external ultrasonic stimulation, and is used in conjunction with surgical treatment. It is expected that the accessibility of clinical treatment can be improved and clinical treatment efficiency can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair, and particularly relates to a method for preparing an engineered decalcified bone matrix porous sponge scaffold loaded with vitamin C microspheres. Background Art

[0002] In clinical practice, the repair and treatment of bone defects caused by trauma, infection, and tumors has always been a challenging problem. Because it is accompanied by damaged blood vessels, poor blood supply seriously affects the efficiency of bone healing and reconstruction. Taking tibial shaft fractures as an example, more than 20% of patients have not achieved healing of the fracture site after more than 8 months after surgery. Traditional autologous bone and allogeneic bone graft materials are limited in source channels, resulting in a shortage of donors that cannot meet actual needs, and artificial bone materials have the problem of poor compatibility.

[0003] Demineralized bone matrix (DBM) is a xenogeneic bone material that has been stripped of inorganic minerals, leaving only the organic "collagen" matrix. It exhibits strong osteoinductive properties. Furthermore, DBM, the collagen scaffold left behind after natural bone is decalcified, exhibits piezoelectric properties. Previously, a bioactive bone matrix gel was developed, giving DBM a formable and injectable gel state (CN115645617A). Furthermore, the piezoelectric effect of engineered DBM has been explored by manipulating its porous structure (CN117618675A).

[0004] Bone morphogenetic proteins (BMPs) are multifunctional proteins belonging to the transforming growth factor-β (TGF-β) superfamily, possessing important biological functions and broad application prospects. BMP proteins consist of a signal peptide, a pro-domain, and a mature domain. The mature form is a dimer composed of two polypeptide chains linked by a disulfide bond. These polypeptide chains contain seven cysteine ​​residues, ensuring protein stability. The BMP family is divided into several subgroups based on gene homology and function, including BMP2, BMP4, BMP7, BMP12, and BMP13. As key signaling molecules inducing bone formation, BMPs bind tightly to collagen within the bone matrix, promoting the differentiation of mesenchymal cells into osteoblasts and ultimately forming bone tissue.

[0005] Vitamin C, also known as ascorbic acid, is an important redox agent that plays an important role in bone salt metabolism and bone formation. Ascorbic acid can increase the activity of osteoblasts, increase intracellular oxidation and reduction reactions, increase the deposition of calcium salts, and stimulate the formation of mineralized nodules in cultured cells. Ascorbic acid also plays a role in the development and differentiation of osteoblasts. Ascorbic acid is also a cofactor in the collagen synthesis process, participating in the hydroxylation of proline to synthesize collagen. The results of the research team's previous studies showed that the use of vitamin C to culture bone marrow mesenchymal stem cells significantly promoted the growth of new blood vessels. Since vitamin C is soluble in water, it is encapsulated by chitosan microspheres, which allows vitamin C to be continuously released while enhancing the piezoelectric properties. At present, there are no reports on the preparation of bone repair materials by combining BMP and vitamin C into DBM. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing a porous sponge scaffold loaded with vitamin C microspheres.

[0007] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0008] (1) Fresh cortical bone was taken, soft tissue was removed, and the cortical bone was cut into small pieces. The pieces were rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The pieces were ground into 80-120 mesh particles using a grinder, non-collagenous impurities were removed with 0.05-0.15 mol / L NaOH, and decalcified with 0.4-0.8 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.5-2.0, 0.5-1.5 mol / L acetic acid and 8-12% of the cortical bone mass of pepsin were added, and the pieces were gelled on a shaker at room temperature for 1-3 days. The acid- and enzymatic-hydrolyzed samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.0-6.5, and salting was carried out for 12-36 hours. The pieces were dialyzed against acetic acid and deionized water overnight to prepare bone matrix gel.

[0009] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%, add 5-20 mg / ml vitamin C solution to the chitosan solution and mix; add the mixed solution dropwise to 40-60 ml of liquid paraffin containing 2-3 ml of Tween-80 at 600-1000 rpm and 50-70 ° C and stir for 1-2 hours; add 0.5-1.5 ml of 25% glutaraldehyde dropwise to the above solution and stir for 8-12 minutes; collect by centrifugation; wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres;

[0010] (3) Adding vitamin C microspheres and BMP protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and forming a film;

[0011] (4) The film prepared in step (3) was immersed in a 0.03-0.07 mol / L MES solution for 20-40 min, EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare an 8-12 mmol / L crosslinker, crosslinked at room temperature for 10-14 h, and washed with PBS and deionized water 2-4 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0012] The cortical bone is bovine cortical bone, porcine cortical bone or the same kind of cortical bone.

[0013] The size of the small pieces in step (1) is 0.5 cm*0.5 cm*0.5 cm.

[0014] The amount of vitamin C solution added in step (2) is 3-8 ml.

[0015] The amount of vitamin C microspheres added in step (3) is 0.5-2% of the mass of the bone matrix gel.

[0016] The amount of BMP protein added in step (3) is 0.01-0.1% of the mass of the bone matrix gel.

[0017] The BMP protein is one or more of BMP2 protein, BMP4 protein, BMP7 protein and BMP13 protein.

[0018] The BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1.

[0019] The present invention has the following beneficial effects: The porous sponge scaffold loaded with vitamin C microspheres prepared by the present invention can significantly promote cell proliferation, differentiation, and angiogenesis under external ultrasonic stimulation. The application of engineered decalcified bone matrix and its endogenous bone morphogenetic proteins, BMP2 and BMP13, in combination with vitamin C microspheres in the treatment of fractures and bone defects simultaneously recruits in situ bone marrow mesenchymal stem cells and promotes their osteogenic differentiation. Combined with surgical treatment, this is expected to improve the accessibility and efficiency of clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 SEM image of the porous sponge scaffold loaded with vitamin C microspheres. DETAILED DESCRIPTION

[0021] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Example 1

[0022] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0023] (1) Fresh bovine cortical bone was taken, and soft tissue was removed. The cortical bone was cut into small pieces of 0.5 cm*0.5 cm*0.5 cm. The cortical bone was rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The bone was ground into 100 mesh particles using a grinder, and non-collagenous impurities were removed with 0.1 mol / L NaOH. The bone was decalcified with 0.6 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.8, and 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone. The bone was shaken at room temperature for 2 days for gelation. The acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and salting was carried out for 24 hours. The bone matrix gel was prepared.

[0024] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%. Add 10 mg / ml vitamin C solution to the chitosan solution and mix. The amount of vitamin C solution added is 5 ml. Add the mixed solution dropwise to 50 ml of liquid paraffin containing 2.5 ml of Tween-80 at 800 rpm and 60 °C and stir for 1.5 h. Add 1 ml of 25% glutaraldehyde dropwise to the above solution and stir for 10 min. Collect by centrifugation. Wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres.

[0025] (3) Adding vitamin C microspheres and BMP protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and preparing a film; the BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1; the amount of vitamin C microspheres added is 1% of the mass of the bone matrix gel; the amount of BMP protein added is 0.05% of the mass of the bone matrix gel;

[0026] (4) The film prepared in step (3) was immersed in a 0.05 mol / L MES solution for 30 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 10 mmol / L crosslinker. The film was cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres. Example 2

[0027] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0028] (1) Fresh porcine cortical bone was taken, and soft tissue was removed. The porcine cortical bone was cut into small pieces of 0.5cm*0.5cm*0.5cm. The bone was rinsed with a high-pressure water gun to remove blood and bone marrow residues. TritonX-100 and H2O2 were used for degreasing and decellularization in sequence. The bone was ground into 80-mesh particles using a grinder, and non-collagenous impurities were removed with 0.05mol / L NaOH. The bone was decalcified with 0.4mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.5, and 0.5mol / L acetic acid and pepsin were added according to 8% of the weight of the cortical bone. The bone was gelled on a shaker at room temperature for 3 days. The acid- and enzymatic-hydrolyzed samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.0, and salting was carried out for 20 hours. The bone matrix gel was prepared.

[0029] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%. Add 8 mg / ml of vitamin C solution to the chitosan solution and mix. The amount of vitamin C solution added is 8 ml. Add the mixed solution dropwise to 60 ml of liquid paraffin containing 3 ml of Tween-80 at 600 rpm and 50 °C and stir for 2 h. Add 0.5 ml of 25% glutaraldehyde dropwise to the above solution and stir for 8 min. Collect by centrifugation. Wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres.

[0030] (3) Adding vitamin C microspheres and BMP protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and preparing a film; the BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1; the amount of vitamin C microspheres added is 0.5% of the mass of the bone matrix gel; the amount of BMP protein added is 0.02% of the mass of the bone matrix gel;

[0031] (4) The film prepared in step (3) was immersed in a 0.03 mol / L MES solution for 20 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare an 8 mmol / L crosslinking agent. The film was cross-linked at room temperature for 14 h. The film was washed with PBS and deionized water twice / h to prepare a porous sponge scaffold loaded with vitamin C microspheres. Example 3

[0032] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0033] (1) Fresh allogeneic cortical bone was obtained, and the soft tissue was removed. The allogeneic cortical bone was cut into small pieces of 0.5 cm * 0.5 cm * 0.5 cm. The blood and bone marrow residues were washed with a high-pressure water gun, and Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The particles were ground into 120 mesh particles using a grinder, and non-collagenous impurities were removed with 0.15 mol / L NaOH. The particles were decalcified with 0.8 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 2.0, and 1.5 mol / L acetic acid and pepsin were added according to 12% of the weight of the cortical bone. The particles were gelled on a shaker at room temperature for 1 day. The acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.5, and salting was carried out for 12 hours. The samples were dialyzed against acetic acid and deionized water overnight to prepare bone matrix gel.

[0034] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%. Add 15 mg / ml vitamin C solution to the chitosan solution and mix. The amount of vitamin C solution added is 3 ml. Add the mixed solution dropwise to 40 ml of liquid paraffin containing 2 ml of Tween-80 at 1000 rpm and 70 °C and stir for 1 hour. Add 1.5 ml of 25% glutaraldehyde dropwise to the above solution and stir for 8 minutes. Collect by centrifugation. Wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres.

[0035] (3) Adding vitamin C microspheres and BMP protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and preparing a film; the BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1; the amount of vitamin C microspheres added is 2% of the mass of the bone matrix gel; the amount of BMP protein added is 0.08% of the mass of the bone matrix gel;

[0036] (4) The film prepared in step (3) was immersed in a 0.07 mol / L MES solution for 20 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 12 mmol / L crosslinker. The film was cross-linked at room temperature for 10 h. The film was washed with PBS and deionized water 4 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0037] Comparative Example 1

[0038] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0039] (1) Fresh bovine cortical bone was taken, and soft tissue was removed. The cortical bone was cut into small pieces of 0.5 cm*0.5 cm*0.5 cm. The cortical bone was rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The bone was ground into 100 mesh particles using a grinder, and non-collagenous impurities were removed with 0.1 mol / L NaOH. The bone was decalcified with 0.6 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.8, and 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone. The bone was shaken at room temperature for 2 days for gelation. The acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and salting was carried out for 24 hours. The bone matrix gel was prepared.

[0040] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%. Add 10 mg / ml vitamin C solution to the chitosan solution and mix. The amount of vitamin C solution added is 5 ml. Add the mixed solution dropwise to 50 ml of liquid paraffin containing 2.5 ml of Tween-80 at 800 rpm and 60 °C and stir for 1.5 h. Add 1 ml of 25% glutaraldehyde dropwise to the above solution and stir for 10 min. Collect by centrifugation. Wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres.

[0041] (3) Adding vitamin C microspheres and BMP2 protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and preparing a film; the amount of vitamin C microspheres added is 1% of the mass of the bone matrix gel; the amount of BMP2 protein added is 0.05% of the mass of the bone matrix gel;

[0042] (4) The film prepared in step (3) was immersed in a 0.05 mol / L MES solution for 30 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 10 mmol / L crosslinker. The film was cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0043] Comparative Example 2

[0044] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps:

[0045] (1) Fresh bovine cortical bone was taken, and soft tissue was removed. The cortical bone was cut into small pieces of 0.5 cm*0.5 cm*0.5 cm. The cortical bone was rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The bone was ground into 100 mesh particles using a grinder, and non-collagenous impurities were removed with 0.1 mol / L NaOH. The bone was decalcified with 0.6 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.8, and 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone. The bone was shaken at room temperature for 2 days for gelation. The acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and salting was carried out for 24 hours. The bone matrix gel was prepared.

[0046] (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%. Add 10 mg / ml vitamin C solution to the chitosan solution and mix. The amount of vitamin C solution added is 5 ml. Add the mixed solution dropwise to 50 ml of liquid paraffin containing 2.5 ml of Tween-80 at 800 rpm and 60 °C and stir for 1.5 h. Add 1 ml of 25% glutaraldehyde dropwise to the above solution and stir for 10 min. Collect by centrifugation. Wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres.

[0047] (3) Adding vitamin C microspheres and BMP13 protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and preparing a film; the amount of vitamin C microspheres added is 1% of the mass of the bone matrix gel; the amount of BMP13 protein added is 0.05% of the mass of the bone matrix gel;

[0048] (4) The film prepared in step (3) was immersed in a 0.05 mol / L MES solution for 30 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 10 mmol / L crosslinker. The film was cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0049] Comparative Example 3

[0050] A method for preparing a porous sponge scaffold is carried out according to the following steps:

[0051] (1) Fresh bovine cortical bone was taken, and soft tissue was removed. The cortical bone was cut into small pieces of 0.5 cm*0.5 cm*0.5 cm. The cortical bone was rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The bone was ground into 100 mesh particles using a grinder, and non-collagenous impurities were removed with 0.1 mol / L NaOH. The bone was decalcified with 0.6 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.8, and 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone. The bone was shaken at room temperature for 2 days for gelation. The acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and salting was carried out for 24 hours. The bone matrix gel was prepared.

[0052] (2) adding BMP protein to the bone matrix gel to form a hydrogel complex, freeze-drying, and preparing a film; the BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1; the amount of BMP protein added is 0.05% of the mass of the bone matrix gel;

[0053] (3) The film prepared in step (2) was immersed in a 0.05 mol / L MES solution for 30 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 10 mmol / L crosslinking agent. The film was crosslinked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold.

[0054] Comparative Example 4

[0055] A method for preparing a porous sponge scaffold is carried out according to the following steps:

[0056] (1) Fresh bovine cortical bone was taken, and soft tissue was removed. The cortical bone was cut into small pieces of 0.5cm*0.5cm*0.5cm, and washed with a high-pressure water gun to remove blood and bone marrow residues. TritonX-100 and H2O2 were used for degreasing and decellularization in sequence. The bone powder was ground into 100-mesh particles using a grinder, and non-collagenous impurities were removed with 0.1mol / L NaOH. The bone powder was decalcified with 0.6mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.8, and 1mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone. The bone powder was gelled on a shaker at room temperature for 2 days. The acid- and enzymatic-hydrolyzed samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and salting was carried out for 24 hours. The bone matrix gel was prepared by acetic acid and deionized water dialyzation overnight, and freeze-dried to prepare a film.

[0057] (2) The film prepared in step (1) was immersed in a 0.05 mol / L MES solution for 30 min. EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare a 10 mmol / L crosslinking agent. The film was crosslinked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold.

[0058] Experimental example:

[0059] 1. Micromorphology characterization: Scanning electron microscopy (SEM) was used to characterize the surface microstructure of the porous sponge scaffold loaded with vitamin C microspheres prepared in Example 1. Figure 1 As can be seen in the figure, the porous sponge scaffold exhibits a porous structure with pore sizes ranging from 50μm to 150μm. Furthermore, scanning electron microscopy images clearly show good physical adhesion between the vitamin C-loaded chitosan microspheres and the sponge scaffold. The distribution and aggregation of microspheres, with particle sizes ranging from 20μm to 80μm, were observed on the scaffold surface.

[0060] 2. Cell proliferation: BMSCs were cultured on the sponge scaffolds prepared in Examples 1-3 and Comparative Examples 1-4 for 3 days, and piezoelectric stimulation was performed daily. After 3 days, the effect of the scaffolds on BMSC cell proliferation was evaluated using CCK-8 reagent.

[0061] The formula for calculating cell proliferation rate (RGR) is: Cell proliferation rate = (OD value of experimental group - OD value of control group) / OD value of control group × 100%

[0062] The formula is used to calculate the degree of cell proliferation. The OD value (optical density) is measured by a microplate reader and reflects the metabolic activity and number of cells.

[0063] The experimental results were statistically analyzed using SPSS 24.0 software. The measurement data were expressed as `x ± (mean ± standard deviation). The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The measurement results are shown in Table 1:

[0064] Table 1

[0065]

[0066] 3. ALP activity detection: BMSCs cells were cultured on the sponge scaffolds prepared in Examples 1-3 and Comparative Examples 1-4 for 7 days and piezoelectrically stimulated every day. After 7 days, the cells were treated with lysis buffer and the supernatant was collected. ALP activity was determined using the pNPP liquid substrate system. At the same time, 10 μL of lysate was taken and the protein concentration was determined using the BCA protein method; finally, the alkaline phosphatase (ALP) activity in the sample was calculated according to the definition of enzyme activity unit (enzyme activity definition: 1 μmol PNPP is hydrolyzed per milligram of protein per minute at 37°C to produce PNP, which is defined as 1 enzyme activity unit). A standard curve was drawn based on the standard test value, and the standard curve equation was calculated: y=52.039x+0.0019; calculated according to the sample protein concentration: ALP (μmol / min / mg prot)=[(ΔA-0.0019)÷52.039]÷(Cpr×V1)÷T×D=0.128×(ΔA-0.0019)÷Cpr×D;

[0067] V1: added sample volume, 0.01 mL; T: reaction time, 15 min; D: dilution factor, undiluted is 1; Cpr: sample protein concentration (BCA result), mg / mL; ΔA = A measurement - A blank.

[0068] The experimental results were statistically analyzed using SPSS 24.0 software. The measurement data were expressed as `x ± (mean ± standard deviation). The Kolmogorov-Smirnov test was used to test the normality of the data. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. The difference was considered statistically significant when P < 0.05. The measurement results are shown in Table 2:

[0069] Table 2

[0070]

[0071] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a porous sponge scaffold loaded with vitamin C microspheres, characterized in that: Follow these steps: (1) Fresh cortical bone was taken, soft tissue was removed, and the cortical bone was cut into small pieces. The pieces were rinsed with a high-pressure water gun to remove blood and bone marrow residues. Triton X-100 and H2O2 were used for degreasing and decellularization in sequence. The pieces were ground into 80-120 mesh particles using a grinder, non-collagenous impurities were removed with 0.05-0.15 mol / L NaOH, and decalcified with 0.4-0.8 mol / L hydrochloric acid. The pH of the decalcified bone powder particles was adjusted to 1.5-2.0, 0.5-1.5 mol / L acetic acid and 8-12% of the cortical bone mass of pepsin were added, and the pieces were gelled on a shaker at room temperature for 1-3 days. The acid- and enzymatic-hydrolyzed samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.0-6.5, and salting was carried out for 12-36 hours. The pieces were dialyzed against acetic acid and deionized water overnight to prepare bone matrix gel. (2) Dissolve 0.05 g of chitosan in 5 ml of 3% glacial acetic acid to prepare a solution with a chitosan content of 1%, add 5-20 mg / ml vitamin C solution to the chitosan solution and mix; add the mixed solution dropwise to 40-60 ml of liquid paraffin containing 2-3 ml of Tween-80 at 600-1000 rpm and 50-70 ° C and stir for 1-2 hours; add 0.5-1.5 ml of 25% glutaraldehyde dropwise to the above solution and stir for 8-12 minutes; collect by centrifugation; wash the microspheres with petroleum ether and ethanol, and then freeze-dry to prepare vitamin C microspheres; (3) Adding vitamin C microspheres and BMP protein to the bone matrix gel to form a hydrogel complex loaded with vitamin C microspheres, freeze-drying, and forming a film; The BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1; (4) The film prepared in step (3) was immersed in a 0.03-0.07 mol / L MES solution for 20-40 min, EDC and NHS were added to the MES solution at a molar ratio of 4:1 to prepare an 8-12 mmol / L crosslinker, crosslinked at room temperature for 10-14 h, and washed with PBS and deionized water 2-4 times / h to prepare a porous sponge scaffold loaded with vitamin C microspheres.

2. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The cortical bone is bovine cortical bone, porcine cortical bone or the same kind of cortical bone.

3. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The size of the small pieces in step (1) is 0.5 cm*0.5 cm*0.5 cm.

4. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The amount of vitamin C solution added in step (2) is 3-8 ml.

5. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The amount of vitamin C microspheres added in step (3) is 0.5-2% of the mass of the bone matrix gel.

6. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The amount of BMP protein added in step (3) is 0.01-0.1% of the mass of the bone matrix gel.

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