A sericin / bacterial cellulose / nano-hydroxyapatite composite gel and a preparation method and application thereof
By cross-linking sericin, bacterial cellulose and aminated nano-hydroxyapatite, a bone repair material with excellent biocompatibility and mechanical properties was prepared, which solved the problem of insufficient biocompatibility and mechanical properties of existing materials and realized the support for bone tissue regeneration.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2026-03-10
AI Technical Summary
Existing bone repair materials are inadequate in terms of biocompatibility, mechanical properties, and degradation rate, making it difficult to meet the needs of bone tissue regeneration.
A composite gel was prepared by combining sericin, bacterial cellulose and aminated nano-hydroxyapatite through a cross-linking reaction. This combined the advantages of natural materials, enhanced mechanical properties, and regulated degradation rate.
The prepared composite gel exhibits excellent biocompatibility, good mechanical strength, and a suitable degradation rate, promoting osteoblast proliferation and differentiation and providing stable three-dimensional support for bone tissue regeneration.
Smart Images
Figure CN120420517B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of high polymer materials, in particular, relates to a silk sericin / bacterial cellulose / nano-hydroxyapatite composite gel and a preparation method and application thereof. BACKGROUND
[0002] An ideal bone repair / bone filling material should have excellent biocompatibility, low immunogenicity, a suitable degradation rate and good bone induction properties. Gel materials, such as collagen, chitosan, hyaluronic acid and the like, contain various biological active sites and signal molecules, are easy to interact with receptors on the cell surface, activate intracellular signaling pathways, promote the adhesion, proliferation and differentiation of bone cells, and accelerate bone tissue regeneration. In addition, the three-dimensional network structure of the gel is closer to the microstructure of natural bone tissue, which provides a favorable growth environment for cells, and cells are easy to migrate and proliferate inside the gel, inducing bone tissue reconstruction.
[0003] Silk sericin is a natural protein extracted from silk, has good biocompatibility, and can promote the proliferation and differentiation of bone cells, fibroblasts and the like. Silk sericin can be degraded by biological enzymes in vivo, and the degradation rate matches the speed of bone tissue repair, and the degradation products are small molecular substances such as amino acids and polypeptides, which can be metabolized and absorbed by the human body, and will not have adverse effects on the human body. Therefore, the silk sericin gel has multiple advantages as a bone tissue repair material. However, the silk sericin gel has poor physical and mechanical properties due to the high content of hydrophilic amino acids and the main flexible structure of secondary structure with random coil and beta-turn. Bacterial cellulose is a natural polymer material, does not contain other impurities and toxins, and its nanoscale fiber network structure is similar to the structure of natural bone matrix, which can provide a good template and support for bone tissue regeneration. In particular, bacterial cellulose has high strength and toughness, can be compounded with silk sericin to enhance the mechanical properties of the silk sericin gel, while retaining the biological activity and other advantages of silk sericin. Hydroxyapatite is a thermodynamically stable calcium phosphate mineral, is the main component of the natural bone mineralization matrix in the human body, has no immunogenicity risk, and also has bone integration and bone induction properties, but it has the problems of insufficient toughness and difficulty in forming a gel when used alone. SUMMARY
[0004] The purpose of the present application is to construct a gel that meets the performance characteristics of a bone repair / bone filling material, and a silk sericin / bacterial cellulose / nano-hydroxyapatite composite gel and a preparation method and application thereof are provided.
[0005] In order to achieve the above technical problems, the present application adopts the following technical solutions:
[0006] One of the purposes of the present application is to provide a sericin / bacterial cellulose / nano-hydroxyapatite composite gel; which is made of sericin, bacterial cellulose, amino-nano-hydroxyapatite and cross-linking agent, is mixed uniformly by sericin solution and bacterial cellulose dispersion liquid, activated by calcium carbonate induction, then amino-nano-hydroxyapatite and cross-linking agent are added, and the composite gel is obtained by cross-linking; wherein the mass ratio of bacterial cellulose to sericin is 1:(1-5), and the mass ratio of amino-nano-hydroxyapatite to sericin is 1:(2-10); the preparation method of the composite gel is carried out according to the following steps:
[0007] Step 1, the bacterial cellulose is swelled in deionized water at room temperature, frozen for at least 12h, then broken to obtain bacterial cellulose dispersion liquid;
[0008] Step 2, the sericin solution and the bacterial cellulose dispersion liquid are uniformly mixed, calcium carbonate is added, and low-speed stirring is induced to activate, then amino-nano-hydroxyapatite and cross-linking agent are added, and after uniform stirring, the gel is formed in the environment of 0-4℃.
[0009] Further limitation, according to the composite gel of claim 1, the sericin solution is prepared according to the following steps: clean raw silk (or cocoon) is added to deionized water for boiling for 2h-6h, filtered and separated, and concentrated to the required concentration.
[0010] Further limitation, the clean raw silk (or cocoon) is added to deionized water in the proportion of 1:(10-20) of raw silk (or cocoon) to deionized water.
[0011] Further limitation, the mass concentration of sericin in the sericin solution is 6%-12%.
[0012] Further limitation, the bacterial cellulose is formed by static culture of Xylophilus annosus, then soaked in 0.1M sodium hydroxide at 80℃ for 4h, and washed with deionized water for more than 3 times to obtain.
[0013] Further limitation, the amino-nano-hydroxyapatite is prepared according to the following steps:
[0014] Nano-hydroxyapatite is dispersed in anhydrous ethanol in the proportion of 1%-5% of mass concentration, and ultrasonic treatment is carried out for 5-10min under the condition of power 30-50mW to form a uniformly dispersed suspension;
[0015] Then, polyethyleneimine was added at a mass ratio of nano-hydroxyapatite to polyethyleneimine of 1:(0.5-5). The mixture was stirred slowly in an ice bath for 12-24 hours. The precipitate was centrifuged at 5000-10000 rpm for at least 30 minutes. The supernatant was removed, and the mixture was washed with deionized water and centrifuged three times. The mixture was then frozen at -20℃ for at least 12 hours and freeze-dried for 36-48 hours to obtain aminated nano-hydroxyapatite.
[0016] Among them, the molecular weight of nano-hydroxyapatite is 502.31, the purity is >97%, and the particle size is less than 100nm; the molecular weight of polyethyleneimine is 1.8~25kDa.
[0017] Further specifying, in step 1, the swelling time is 4h to 12h.
[0018] Further specifying, in step 1, the freezing temperature is -20℃ to -80℃.
[0019] Further specifying, in step 1, a high-speed shear homogenizer is used for crushing, and the crushing time is 10,000 rpm to 20,000 rpm for 30 min to 60 min.
[0020] Further specifying, in step 2, calcium carbonate is added at a mass concentration of 0.2% to 0.6%, and activation is induced at a stirring speed of 60 rpm to 200 rpm for 5 min to 10 min.
[0021] Further specifying, in step 3, the crosslinking agent used is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the amount used is 20% to 40% of the sericin protein content. The mixed solution after adding the crosslinking agent is slowly stirred at 4°C for 30 min to 2 h.
[0022] Furthermore, the use of the sericin / bacterial cellulose / nanohydroxyapatite composite gel of the present invention is also provided, wherein the composite gel is used as a bone repair material for the proliferation and / or osteogenic differentiation of bone marrow mesenchymal stem cells.
[0023] The present invention has the following beneficial effects:
[0024] Maintaining the spatial structure of the bone defect site is crucial during bone defect repair. Hydrogels, due to their high hydrophilicity, generate osmotic pressure, which resists stress at the bone defect site, preventing the surrounding tissue from overfilling the defect area and providing a stable three-dimensional space for bone regeneration. This also facilitates the growth, proliferation, and differentiation of bone cells within this specific space. Existing biodegradable natural polymer gels, such as collagen, sodium hyaluronate, sodium alginate, and chitosan, while possessing strong hydrophilicity and good gel shape retention, degrade too rapidly in vivo, making it difficult to provide sustained support for bone regeneration. Synthetic polymer gels, such as polylactic acid-glycolic acid and polylactic acid, allow for the control of physicochemical properties and degradation rates; however, their degradation products pose a risk of inducing aseptic inflammation in vivo, and their in vivo cell compatibility is inferior to that of natural materials.
[0025] This invention uses highly hydrophilic natural sericin as the main gel material, supplemented with natural bacterial cellulose to enhance the mechanical properties of sericin. The resulting composite gel exhibits excellent biocompatibility, promoting the proliferation and differentiation of osteocytes and fibroblasts, and its in vivo degradation rate matches the bone tissue repair rate. Furthermore, based on the fact that natural bone is essentially a composite material composed of collagen and hydroxyapatite, this invention modifies nano-hydroxyapatite by amylation and crosslinks it with sericin and bacterial cellulose. Through this composite approach of natural organic and inorganic materials, a novel gel material with excellent biocompatibility, high hydrophilicity, good mechanical strength, and high osteogenic activity is developed, which is beneficial for promoting bone tissue repair and reconstruction.
[0026] To further understand the features and technical content of this invention, please refer to the following detailed description and accompanying drawings. However, the accompanying drawings are for reference and illustration only and are not intended to limit the invention. Attached Figure Description
[0027] Figure 1 The purified bacterial cellulose membrane prepared in Example 1;
[0028] Figure 2 The image shown is a scanning electron microscope image of the aminated nano-hydroxyapatite prepared in Example 1.
[0029] Figure 3 This is a 50x magnified scanning electron microscope image of the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in Example 1;
[0030] Figure 4 This is a 150x magnified scanning electron microscope image of the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in Example 1;
[0031] Figure 5The stress-strain curve of the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in Example 1;
[0032] Figure 6 These are scanning electron microscope images of the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in Example 2 before and after degradation;
[0033] Figure 7 The degradation rates of the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in Example 2 at 3, 7, and 14 days are shown.
[0034] Figure 8 These are laser confocal images of bone marrow mesenchymal stem cells grown in a sericin / bacterial cellulose / nanohydroxyapatite composite gel for 1, 3, and 5 days, as shown in Example 2.
[0035] Figure 9 The cell survival rate of bone marrow mesenchymal stem cells in Example 2 after 1, 3, and 5 days of proliferation in sericin / bacterial cellulose / nanohydroxyapatite composite gel;
[0036] Figure 10 The images show alkaline phosphatase (ALP) staining of bone marrow mesenchymal stem cells cultured in a sericin / bacterial cellulose / nanohydroxyapatite composite gel for 7 and 14 days, as shown in Example 2. Detailed Implementation
[0037] Those skilled in the art can refer to the content of this document to appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.
[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, which can be obtained commercially by those skilled in the art.
[0039] Example 1: The preparation of the sericin / bacterial cellulose / nanohydroxyapatite composite gel in this example was achieved through the following steps:
[0040] (1) Preparation of bacterial cellulose: Acetobacter xylinum was statically cultured in a medium (containing 0.3% tryptone, 0.5% yeast extract, and 2.5% mannitol) at 30℃ until a uniform membrane was formed. The cellulose membrane was then removed, treated in 0.1M sodium hydroxide at 80℃ for 4 hours, and washed three times with deionized water to obtain a pure bacterial cellulose membrane, such as... Figure 1 As shown, the bacterial cellulose membrane was immersed in deionized water and swollen for 4 hours. Then, it was transferred to a -20°C freezer and frozen for 12 hours. Finally, it was homogenized using a high-speed shear homogenizer at 20,000 rpm for 30 minutes to obtain a uniform bacterial cellulose dispersion.
[0041] (2) Preparation of aminated nano-hydroxyapatite: Nano-hydroxyapatite was dispersed in anhydrous ethanol to prepare a 1% (w / w) suspension, which was then ultrasonically treated for 10 min using a 30 mW ultrasonic disperser. Polyethyleneimine with a molecular weight of 1.8 kDa was added, with a mass ratio of nano-hydroxyapatite to polyethyleneimine of 1:0.5. The mixture was slowly stirred in an ice bath for 12 h. After the reaction, the mixture was centrifuged at 5000 rpm for 30 min, the supernatant was removed, and the mixture was washed with deionized water. This process was repeated three times to obtain aminated nano-hydroxyapatite. Figure 2 As shown in the figure, the aminated nano-hydroxyapatite is uniformly dispersed, and its size is not significantly different from that before modification.
[0042] (3) Preparation of sericin solution: Weigh 100g of raw silkworm silk, wash it with deionized water, wring it dry, and add it to 1L of deionized water. Boil for 2 hours. When the water volume is insufficient to completely submerge the raw silk, add an appropriate amount of deionized water until the raw silk is completely submerged. After the boiling treatment, filter out the fibroin fibers with gauze to obtain a pure sericin solution. The mass concentration of the obtained sericin solution was measured to be approximately 4.0% by drying and weighing. The sericin solution was then concentrated to 6% in a 60℃ oven.
[0043] (4) Add bacterial cellulose dispersion to the sericin solution, with a bacterial cellulose to sericin mass ratio of 1:1. After stirring evenly, add calcium carbonate to a final concentration of 0.6% and stir at 60 rpm for 10 min. Then add aminated nano-hydroxyapatite, with a hydroxyapatite to sericin mass ratio of 1:2. After stirring evenly, add crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the amount of which is 40% of the total mass of sericin. The blend solution is slowly stirred at 4℃ for 30 min, and then transferred to a 0℃ refrigerator to stand and form a composite gel.
[0044] (5) The formed gel was frozen at -20℃ for 24 hours, and then freeze-dried for 48 hours to obtain a dried sericin / bacterial cellulose / nanohydroxyapatite composite gel. The morphology of the gel was observed under a scanning electron microscope, such as... Figure 3 and 4 As shown, the gel exhibits a uniformly interconnected pore structure, with nano-hydroxyapatite evenly distributed on the pore wall surface. The average pore size is approximately 723 μm, and the porosity is as high as 68%.
[0045] The mechanical properties of the composite gel prepared by the method of this embodiment were tested: The gel was cut into cylindrical shapes with a diameter of 10 mm and a height of 12 mm, and the compressive mechanical properties of the gel were tested using a texture analyzer. An initial stress of 0.03 N, a deformation rate of 10 mm / min, a load of 25 N, and a deformation of 70% were set, and the compressive modulus was calculated using the slope of the initial segment of the stress-strain curve. The formula for calculating the compressive strength (σ) of the gel is as follows: σ = 4P / (πd 2 In the formula, P is the compressive strength (N); d is the sample diameter (mm). The compressive elastic recovery rate (η) of the gel is calculated as follows: η = (L0 - L1) / L, where L0 is the displacement (mm) at the target distance in the second cycle; L1 is the displacement (mm) at the beginning of the second cycle; and L is the target displacement (mm) for each cycle. The stress-strain curve is shown below. Figure 5 As shown, calculations revealed that the compressive modulus of the composite gel is approximately 5.76 kPa, and when the gel is subjected to 70% deformation, its compressive strength is 34.1 kPa, with a compressive elastic recovery rate of 75.3%. This indicates that the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in this embodiment possesses excellent mechanical properties, meeting the mechanical strength requirements for bone repair / bone filling gel materials.
[0046] Example 2: The preparation of the sericin / bacterial cellulose / nanohydroxyapatite composite gel in this example was achieved through the following steps:
[0047] (1) Preparation of bacterial cellulose: Acetobacter xylinum was statically cultured in a medium (containing 0.3% tryptone, 0.5% yeast extract, and 2.5% mannitol) at 30℃ until a uniform membrane was formed. The cellulose membrane was then removed, treated in 0.1M sodium hydroxide at 80℃ for 4 hours, and washed three times with deionized water to obtain a pure bacterial cellulose membrane, such as... Figure 1 As shown, the bacterial cellulose membrane was immersed in deionized water and swollen for 12 hours. Then, it was transferred to a -80°C freezer and frozen for 24 hours. Finally, it was homogenized using a high-speed shear homogenizer at 10,000 rpm for 60 minutes to obtain a uniform bacterial cellulose dispersion.
[0048] (2) Preparation of aminated nano-hydroxyapatite: Nano-hydroxyapatite was dispersed in anhydrous ethanol to prepare a 5% (w / w) suspension, which was then ultrasonically treated for 5 min using a 50 mW ultrasonic disperser. Polyethyleneimine with a molecular weight of 25 kDa was added, with a mass ratio of nano-hydroxyapatite to polyethyleneimine of 1:5. The mixture was stirred slowly in an ice bath for 24 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 30 min, the supernatant was removed, and the mixture was washed with deionized water. The process was repeated three times to obtain aminated nano-hydroxyapatite.
[0049] (3) Preparation of sericin solution: Weigh 100g of raw silkworm silk, wash it with deionized water, wring it dry, and add it to 2L of deionized water. Boil for 4 hours. When the water volume is insufficient to completely submerge the raw silk, add an appropriate amount of deionized water until the raw silk is completely submerged. After the boiling treatment, filter out the silk fibers with gauze to obtain a pure sericin solution. The mass concentration of the obtained sericin solution was measured to be approximately 5.2% by drying and weighing. The sericin solution was then concentrated to 12% in a 60℃ oven.
[0050] (4) Add bacterial cellulose dispersion to the sericin solution, with a bacterial cellulose to sericin mass ratio of 1:5. After stirring evenly, add calcium carbonate to a final concentration of 0.2%, and stir at 200 rpm for 5 min. Then add aminated nano-hydroxyapatite, with a hydroxyapatite to sericin mass ratio of 1:10. After stirring evenly, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a crosslinking agent, with an addition amount of 20% of the total sericin mass. The blend solution is slowly stirred at 4℃ for 2 h, and then transferred to a 0℃ freezer to stand and form a composite gel. Freeze the formed gel at -40℃ for 24 h, and then freeze-dry for 48 h to obtain a dried sericin / bacterial cellulose / nano-hydroxyapatite composite gel.
[0051] The degradation behavior of the composite gel prepared by the method in this embodiment was as follows: A phosphate buffer solution (PBS) containing 5000 U / L lysozyme was prepared, and the dried composite gel was completely immersed in the PBS buffer at 37°C to test its degradation performance. The gel was removed after 3, 7, and 14 days of degradation, freeze-dried, weighed, and its morphology before and after degradation was observed under a scanning electron microscope. The results are as follows. Figure 6 As shown, before degradation, the gel maintains a complete porous structure with uniform pore size and neat arrangement. After 3 and 7 days of degradation, the porous structure of the gel is gradually destroyed, and some pores collapse. After 14 days of degradation, the overall structure of the gel is significantly damaged, with pore walls fracturing. Furthermore, the degradation rate of the composite gel is calculated using the following formula: Degradation rate = (W0 - W...) t ) / W0×100%, where W0 is the dry weight of the composite gel before degradation, Wt The degradation rate is the dry weight of the composite gel after degradation to a specific time, as shown in the figure. Figure 7 As shown, degradation was not significant on day 3, with a degradation rate of about 6%; by day 7, about 17% of the gel had been degraded; and by day 14, the gel degradation rate was about 33%. This indicates that the degradation rate of the composite gel is relatively slow, matching the bone repair speed, and is suitable as a bone repair / bone filling material.
[0052] Proliferation of bone marrow mesenchymal stem cells in the composite gel prepared by the method in this embodiment: The dried sericin / bacterial cellulose / nanohydroxyapatite composite gel was cut into circular pieces with a diameter of 1.5 cm and a thickness of 2 mm, and then... 60 Sterilized by Co-γ irradiation (15 kGy), the cells were immersed in sterile MEMα medium for 3 days and then transferred to 12-well plates. Bone marrow mesenchymal stem cells in the logarithmic growth phase were harvested and cultured at a concentration of 1 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 1 cells / well into 12-well plates and incubated at 37°C in a 5% CO2 incubator for 2 hours, followed by replenishment of 1 mL of complete culture medium. Cell growth morphology in the gel material was observed under a laser confocal microscope, and the results are as follows: Figure 8 As shown, bone marrow mesenchymal stem cells adhere tightly to the gel material, extend pseudopodia, and proliferate well.
[0053] After co-culturing cells with gel for 1, 3, and 5 days, the culture medium was aspirated, and the cells were washed with PBS solution. 450 μL of MEMα medium and 50 μL of CCK-8 reagent were added to each well, and the cells were incubated for 2 hours. The culture medium was then transferred to centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. 100 μL of the supernatant was collected and transferred to a 96-well plate. The absorbance (OD) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of serum-free medium and CCK-8 mixture) / (OD value of blank control group - OD value of serum-free medium and CCK-8 mixture) × 100%. The results are shown below. Figure 9 As shown, bone marrow mesenchymal stem cells proliferated well in the composite gel, with a cell survival rate exceeding 100%, exhibiting higher proliferative activity compared to the blank culture plate. This indicates that the sericin / bacterial cellulose / nanohydroxyapatite composite gel prepared in this invention is beneficial for promoting the adhesion and proliferation of bone marrow mesenchymal stem cells.
[0054] Osteogenic differentiation of bone marrow mesenchymal stem cells in the composite gel prepared by the method in this embodiment: After co-culturing bone marrow mesenchymal stem cells with sericin / bacterial cellulose / nanohydroxyapatite composite gel for 7 and 14 days, the culture medium was aspirated, the samples were gently washed three times with PBS solution, and the samples were stained using an alkaline phosphatase staining kit. The staining was observed under an inverted microscope, and the results are as follows.Figure 10 As shown. ALP staining utilizes the blue-purple color produced by alkaline phosphatase substrates under the catalysis of alkaline phosphatase; the deeper the color, the higher the alkaline phosphatase activity, indicating a higher degree of osteogenic differentiation of bone marrow mesenchymal stem cells. From Figure 10 As can be seen, when bone marrow mesenchymal stem cells differentiated in the composite gel material for 7 days, about half of the gel surface area was blue-purple; when differentiated for 14 days, the blue-purple color covered about 4 / 5 of the material. This indicates that the stem cells had a high degree of osteogenic differentiation in the gel, and that the gel material is beneficial for promoting stem cell differentiation and osteogenic repair.
[0055] The contents not described in detail in this specification are well-known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be defined by the claims.
Claims
1. A sericin / bacterial cellulose / nano-hydroxyapatite composite gel, characterized by, The composite gel is prepared by mixing sericin solution and bacterial cellulose dispersion, activating by calcium carbonate, adding amino-functionalized nano-hydroxyapatite and cross-linking agent, and cross-linking the mixture.
2. The composite gel of claim 1, wherein, The sericin solution is prepared by the following steps: clean raw silk or cocoon is added into deionized water in a mass ratio of 1: (10-20) and boiled for 2-6 hours, then filtered and concentrated to the desired concentration.
3. The composite gel of claim 1, wherein The mass concentration of sericin is 6%-12%.
4. The composite gel of claim 1, wherein, The bacterial cellulose is obtained by static culture of Acetobacter xylinum, soaking in 0.1 M NaOH at 80℃ for 4 hours, and washing with deionized water for at least 3 times.
5. The composite gel of claim 1, wherein The amino-functionalized nano-hydroxyapatite is prepared by the following steps: Nano-hydroxyapatite is dispersed in anhydrous ethanol in a mass concentration of 1%-5%, and ultrasonic treatment is performed for 5-10 minutes at a power of 30-50 mW to form a uniform suspension; Then, polyethyleneimine is added in a mass ratio of 1: (0.5-5) to the nano-hydroxyapatite, and slowly stirred in an ice bath for 12-24 hours; centrifugal precipitation is performed at a speed of 5000-10000 rpm for at least 30 minutes, and the supernatant is removed; the precipitate is washed with deionized water and centrifuged for 3 times; freezing is performed at -20℃ for at least 12 hours, and freeze-drying is performed for 36-48 hours to obtain the amino-functionalized nano-hydroxyapatite; The molecular weight of the nano-hydroxyapatite is 502.31, the purity is > 97%, and the particle size is less than 100 nm; the molecular weight of the polyethyleneimine is 1.8-25 kDa.
6. The method for preparing a composite gel according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: Step 1: the bacterial cellulose is swelled in deionized water at room temperature, frozen for at least 12 hours, and then broken to obtain a bacterial cellulose dispersion; Step 2: the sericin solution and the bacterial cellulose dispersion are uniformly mixed, and calcium carbonate is added for activation by low-speed stirring; Step 3: amino-functionalized nano-hydroxyapatite and cross-linking agent are added, and the mixture is stirred uniformly and then placed in an environment at 0-4℃ to form a gel.
7. The method of claim 6, wherein, The swelling time is 4-12 hours, and the freezing temperature is -20℃ to -80℃.
8. The method of claim 6, wherein, The high-speed shearing homogenizer is used for breaking, and the breaking is performed at a speed of 10000-20000 rpm for 30-60 minutes.
9. The method of claim 6, wherein, The mass concentration of calcium carbonate is 0.2%-0.6%, and the activation is performed by stirring at a speed of 60-200 rpm for 5-10 minutes; the cross-linking agent is 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, and the usage amount is 20%-40% of the mass of sericin.
10. The composite gel according to any one of claims 1 to 5 or prepared by the method according to any one of claims 6 to 9 for use as a bone repair material for the proliferation and / or osteogenic differentiation of mesenchymal stem cells.