Preparation method of porous sponge scaffold loaded with vitamin C microspheres

Abstract

The invention discloses a preparation method of a porous sponge scaffold loaded with vitamin C microspheres. The method comprises the following steps: firstly, preparing bone matrix gel from cortical bones, then preparing vitamin C microspheres, adding the vitamin C microspheres and BMP protein into the bone matrix gel to form a hydrogel complex loaded with the vitamin C microspheres, freeze-drying, preparing a film, and finally preparing the porous sponge scaffold loaded with the vitamin C microspheres through crosslinking. The porous sponge stent loaded with the vitamin C microspheres, prepared by the invention, can obviously promote proliferation and differentiation of cells and formation of blood vessels under external ultrasonic stimulation, and is expected to be capable of improving accessibility of clinical treatment and improving clinical treatment efficiency when being used in cooperation with operative treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair, and in particular 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 have limited source channels, resulting in a number of donors that are difficult to meet actual needs, and artificial bone materials have the problem of poor compatibility.

[0003] Decalcified bone matrix (DBM) is a xenogeneic bone material that has been removed of inorganic minerals and only retains the organic "collagen" matrix. It has strong osteoinduction ability. At the same time, DBM is the collagen scaffold left after the natural bone is decalcified and has piezoelectric properties. In the early stage, a bioactive bone matrix gel has been developed, which gives DBM a shapeable and injectable gel state (CN115645617A). In addition, the piezoelectric effect of engineered DBM is explored by regulating the porous structure (CN117618675A).

[0004] BMP proteins (Bone Morphogenetic Proteins) are a class of multifunctional proteins belonging to the transforming growth factor-β (TGF-β) superfamily, with important biological functions and broad application prospects. BMP proteins are composed of signal peptides, precursor regions and mature regions. Their mature form is a dimer formed by two polypeptide chains connected by a disulfide bond. These polypeptide chains contain 7 cysteine ​​residues, which ensure the stability of the protein. Based on gene homology and function, the BMP family is divided into multiple subgroups, including BMP2, BMP4, BMP7, BMP12, BMP13, etc. As a key signaling molecule that induces bone formation, BMP is closely bound to collagen in the bone matrix, which can promote the differentiation of mesenchymal cells into osteoblasts and ultimately form 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 process of collagen synthesis, participating in the lightening of proline to synthesize collagen. The results of the previous research of the research group 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, chitosan microspheres are used to encapsulate it, so that vitamin C can be continuously released while the piezoelectric properties are also enhanced. At present, there are no reports on the preparation of bone repair materials by combining BMP and vitamin C with 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: (1) Take fresh cortical bone, remove soft tissue, cut the cortical bone into small pieces, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2 Degreasing and decellularizing in sequence; grinding into particles with a particle size of 80-120 using a grinder, removing non-collagen impurities with 0.05-0.15 mol / L NaOH, and decalcifying with 0.4-0.8 mol / L hydrochloric acid; adjusting the pH of the decalcified bone powder particles to 1.5-2.0, adding 0.5-1.5 mol / L acetic acid and pepsin according to 8-12% of the cortical bone mass, and gelling treatment on a shaker at room temperature for 1-3 days; then centrifuging the acid and enzymatic hydrolyzed samples, taking the precipitate, adjusting the pH to 6.0-6.5, and salting out for 12-36 hours, dialysis with acetic acid and deionized water overnight to prepare a 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 into 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 into 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, and freeze-drying to form a film; (4) Soak the film prepared in step (3) in a 0.03-0.07 mol / L MES solution for 20-40 min, add EDC and NHS to the MES solution at a molar ratio of 4:1 to prepare an 8-12 mmol / L cross-linking agent, cross-link at room temperature for 10-14 h, and wash with PBS and deionized water 2-4 times / h, respectively, to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0008] The cortical bone is bovine cortical bone, porcine cortical bone or the same kind of cortical bone.

[0009] The size of the small pieces in step (1) is 0.5 cm*0.5 cm*0.5 cm.

[0010] The amount of vitamin C solution added in step (2) is 3-8 ml.

[0011] The amount of vitamin C microspheres added in step (3) is 0.5-2% of the mass of the bone matrix gel.

[0012] The amount of BMP protein added in step (3) is 0.01-0.1% of the mass of bone matrix gel.

[0013] The BMP protein is one or more of BMP2 protein, BMP4 protein, BMP7 protein and BMP13 protein.

[0014] The BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1.

[0015] Beneficial effects of the present invention: 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 combined with vitamin C microspheres in the treatment of fractures and bone defects can simultaneously recruit in situ bone marrow mesenchymal stem cells and promote the osteogenic differentiation of bone marrow mesenchymal stem cells. When used in conjunction with surgical treatment, it is expected to improve the accessibility of clinical treatment and improve the efficiency of clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 SEM image of the porous sponge scaffold loaded with vitamin C microspheres. DETAILED DESCRIPTION

[0017] In order to facilitate the 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

[0018] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps: (1) Take fresh bovine cortical bone, remove soft tissue, cut the cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2 Degreasing and decellularization were performed in sequence; grinding was performed into particles with a particle size of 100 using a grinder, non-collagen impurities were removed with 0.1 mol / L NaOH, and decalcification was performed with 0.6 mol / L hydrochloric acid; the pH of the decalcified bone powder particles was adjusted to 1.8, 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone, and gelation was performed on a shaker at room temperature for 2 days; the acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and then salting was performed for 24 hours, and acetic acid and deionized water were dialyzed overnight to prepare bone matrix gel; (2) Dissolve 0.05 g chitosan in 5 ml 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 into 50 ml liquid paraffin containing 2.5 ml Tween-80 at 800 rpm and 60°C and stir for 1.5 h; add 1 ml 25% glutaraldehyde dropwise into the above solution and stir for another 10 min; 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 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; (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 cross-linking agent, and cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h, respectively, to prepare a porous sponge scaffold loaded with vitamin C microspheres. Example 2

[0019] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps: (1) Take fresh pig cortical bones, remove soft tissue, cut the pig cortical bones into small pieces of 0.5cm*0.5cm*0.5cm, wash with a high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2O 2 Degreasing and decellularizing in sequence; grinding into 80-mesh particles using a grinder, removing non-collagen impurities with 0.05 mol / L NaOH, and decalcifying with 0.4 mol / L hydrochloric acid; adjusting the pH of the decalcified bone powder particles to 1.5, adding 0.5 mol / L acetic acid and pepsin according to 8% of the cortical bone mass, and gelling treatment on a shaker at room temperature for 3 days; then centrifuging the acid and enzymatic hydrolyzed samples, taking the precipitate, adjusting the pH to 6.0, and salting out for 20 hours, dialysis with acetic acid and deionized water overnight to prepare bone matrix gel; (2) Dissolve 0.05 g chitosan in 5 ml 3% glacial acetic acid to prepare a solution with a chitosan content of 1%, add 8 mg / ml vitamin C solution to the chitosan solution and mix, the amount of vitamin C solution added is 8 ml; add the mixed solution dropwise into 60 ml liquid paraffin containing 3 ml Tween-80 at 600 rpm and 50°C and stir for 2 h; add 0.5 ml 25% glutaraldehyde dropwise into the above solution and stir for another 8 min; 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 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; (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 cross-linking agent, and 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

[0020] A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps: (1) Take fresh cortical bone of the same type, remove soft tissue, cut the same cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with a high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2Degreasing and decellularizing in sequence; grinding into 120 mesh particles with a grinder, removing non-collagen impurities with 0.15 mol / L NaOH, and decalcifying with 0.8 mol / L hydrochloric acid; adjusting the pH of the decalcified bone powder particles to 2.0, adding 1.5 mol / L acetic acid and pepsin according to 12% of the cortical bone mass, and gelling treatment on a shaker at room temperature for 1 day; then centrifuging the acid and enzymatic hydrolyzed samples, taking the precipitate, adjusting the pH to 6.5, and salting out for 12 hours, and dialyzing with acetic acid and deionized water overnight to prepare bone matrix gel; (2) Dissolve 0.05 g chitosan in 5 ml 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, and the amount of vitamin C solution added is 3 ml; add the mixed solution dropwise into 40 ml liquid paraffin containing 2 ml Tween-80 at 1000 rpm and 70°C and stir for 1 hour; add 1.5 ml 25% glutaraldehyde dropwise into the above solution and stir for another 8 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 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; (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 cross-linking agent, and cross-linked at room temperature for 10 h. The film was washed with PBS and deionized water 4 times / h, respectively, to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0021] Comparative Example 1 A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps: (1) Take fresh bovine cortical bone, remove soft tissue, cut the cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2Degreasing and decellularization were performed in sequence; grinding was performed into particles with a particle size of 100 using a grinder, non-collagen impurities were removed with 0.1 mol / L NaOH, and decalcification was performed with 0.6 mol / L hydrochloric acid; the pH of the decalcified bone powder particles was adjusted to 1.8, 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone, and gelation was performed on a shaker at room temperature for 2 days; the acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and then salting was performed for 24 hours, and acetic acid and deionized water were dialyzed overnight to prepare bone matrix gel; (2) Dissolve 0.05 g chitosan in 5 ml 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 into 50 ml liquid paraffin containing 2.5 ml Tween-80 at 800 rpm and 60°C and stir for 1.5 h; add 1 ml 25% glutaraldehyde dropwise into the above solution and stir for another 10 min; 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 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; (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 cross-linking agent, and cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h, respectively, to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0022] Comparative Example 2 A method for preparing a porous sponge scaffold loaded with vitamin C microspheres is carried out according to the following steps: (1) Take fresh bovine cortical bone, remove soft tissue, cut the cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2 Degreasing and decellularizing were performed in sequence; grinding was performed into particles with a particle size of 100 using a grinder, non-collagen impurities were removed with 0.1 mol / L NaOH, and decalcification was performed with 0.6 mol / L hydrochloric acid; the pH of the decalcified bone powder particles was adjusted to 1.8, 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone, and gelation was performed on a shaker at room temperature for 2 days; the acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and then salting was performed for 24 hours, and acetic acid and deionized water were dialyzed overnight to prepare bone matrix gel; (2) Dissolve 0.05 g chitosan in 5 ml 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 into 50 ml liquid paraffin containing 2.5 ml Tween-80 at 800 rpm and 60°C and stir for 1.5 h; add 1 ml 25% glutaraldehyde dropwise into the above solution and stir for another 10 min; 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 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; (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 cross-linking agent, and cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h, respectively, to prepare a porous sponge scaffold loaded with vitamin C microspheres.

[0023] Comparative Example 3 A method for preparing a porous sponge scaffold is carried out according to the following steps: (1) Take fresh bovine cortical bone, remove soft tissue, cut the cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2 Degreasing and decellularization were performed in sequence; grinding was performed into particles with a particle size of 100 using a grinder, non-collagen impurities were removed with 0.1 mol / L NaOH, and decalcification was performed with 0.6 mol / L hydrochloric acid; the pH of the decalcified bone powder particles was adjusted to 1.8, 1 mol / L acetic acid and pepsin were added according to 10% of the weight of the cortical bone, and gelation was performed on a shaker at room temperature for 2 days; the acid and enzymatic hydrolysis samples were then centrifuged, the precipitate was taken, the pH was adjusted to 6.2, and then salting was performed for 24 hours, and acetic acid and deionized water were dialyzed overnight to prepare bone matrix gel; (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; (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 cross-linking agent, and cross-linked at room temperature for 12 h. The film was washed with PBS and deionized water 3 times / h respectively to prepare a porous sponge scaffold.

[0024] Comparative Example 4 A method for preparing a porous sponge scaffold is carried out according to the following steps: (1) Take fresh bovine cortical bone, remove soft tissue, cut the cortical bone into small pieces of 0.5cm*0.5cm*0.5cm, wash with high-pressure water gun to remove blood and bone marrow residues, and use TritonX-100, H 2 O 2 Degreasing and decellularizing in sequence; grinding into 100 mesh particles with a grinder, removing non-collagen impurities with 0.1 mol / L NaOH, and decalcifying with 0.6 mol / L hydrochloric acid; adjusting the pH of the decalcified bone powder particles to 1.8, adding 1 mol / L acetic acid and pepsin according to 10% of the cortical bone mass, and gelling treatment on a shaker at room temperature for 2 days; then centrifuging the acid and enzymatic hydrolyzed samples, taking the precipitate, adjusting the pH to 6.2, and salting out for 24 hours, dialysis with acetic acid and deionized water overnight to prepare bone matrix gel, freeze-drying, and preparing a film; (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 cross-linking agent, cross-linked at room temperature for 12 h, and washed with PBS and deionized water 3 times / h to prepare a porous sponge scaffold.

[0025] Experimental example: 1. Microscopic morphology characterization: The surface microstructure of the porous sponge scaffold loaded with vitamin C microspheres prepared in Example 1 was characterized by scanning electron microscopy (SEM). Figure 1 It can be seen that the porous sponge scaffold presents a porous structure with a pore size of 50μm-150μm. In addition, it can be easily observed from the scanning electron microscope image that there is good physical adhesion between the chitosan microspheres loaded with vitamin C and the sponge scaffold. The distribution and aggregation of some microspheres were observed on the surface of the scaffold, and the particle size of the microspheres was 20μm-80μm.

[0026] 2. Cell proliferation: BMSCs cells were cultured on the sponge scaffolds prepared in Examples 1-3 and Comparative Examples 1-4 for 3 days, and piezoelectric stimulation was performed every day. After 3 days, CCK-8 reagent was used to evaluate the effect of the scaffolds on BMSC cell proliferation.

[0027] The calculation formula of cell proliferation rate (RGR) is: Cell proliferation rate = (OD value of experimental group - OD value of control group) / OD value of control group × 100% 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 / quantity of cells.

[0028] 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 for data normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. P<0.05 was considered statistically significant. The measurement results are shown in Table 1: Table 1

[0029] 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 piezoelectric stimulation was performed 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, according to the definition of the enzyme activity unit, the alkaline phosphatase (ALP) activity in the sample was calculated (enzyme activity definition: 1 μmol PNPP produced by hydrolyzing PNP per minute per milligram of protein at 37°C is defined as 1 enzyme activity unit). A standard curve was drawn based on the detection value of the standard product, 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; V1----added sample volume, 0.01mL; T----reaction time, 15min; D---dilution factor, undiluted is 1; Cpr----sample protein concentration (BCA result), mg / mL; ΔA=Adetermination-Ablank.

[0030] 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 for data normality test. For data that met the normal distribution, the t-test was used to compare the mean differences between the two groups. P<0.05 was considered statistically significant. The measurement results are shown in Table 2: Table 2

[0031] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a porous sponge scaffold loaded with vitamin C microspheres, characterized in that: Follow these steps: (1) Take fresh cortical bone, remove soft tissue, cut the cortical bone into small pieces, rinse with a high-pressure water gun to remove blood and bone marrow residues, use TritonX-100 and H2O2 to degrease and decellularize in turn; grind into particles with a particle size of 80-120 using a grinder, remove non-collagen impurities with 0.05-0.15 mol / L NaOH, and decalcify with 0.4-0.8 mol / L hydrochloric acid; adjust the pH of the decalcified bone powder particles to 1.5-2.0, add 0.5-1.5 mol / L acetic acid and 8-12% of the weight of the cortical bone with pepsin, and gelate on a shaker at room temperature for 1-3 days; then centrifuge the acid and enzymatic hydrolysis samples, take the precipitate, adjust the pH to 6.0-6.5, and then salt out for 12-36 hours, and dialyze with acetic acid and deionized water overnight to make a 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 into 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 into 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, and freeze-drying to form a film; (4) Soak the film prepared in step (3) in a 0.03-0.07 mol / L MES solution for 20-40 min, add EDC and NHS to the MES solution at a molar ratio of 4:1 to prepare an 8-12 mmol / L cross-linking agent, cross-link at room temperature for 10-14 h, and wash with PBS and deionized water 2-4 times / h, respectively, 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 bone matrix gel.

7. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The BMP protein is one or more of BMP2 protein, BMP4 protein, BMP7 protein and BMP13 protein.

8. The method for preparing the porous sponge scaffold loaded with vitamin C microspheres according to claim 1, characterized in that: The BMP protein is a mixed protein of BMP2 protein and BMP13 protein in a mass ratio of 1:1.

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