A method for preparing a composite nanofiber bone scaffold

Abstract

This invention discloses a method for preparing a composite nanofiber bone scaffold, belonging to the field of medical engineering technology. First, a bacterial cellulose (BC) membrane is prepared. BC membrane powder is then prepared by pre-cooling with liquid nitrogen and mechanical grinding, and subsequently dispersed in a polydopamine solution to obtain a BC polydopamine solution. The prepared decellularized submucosal layer (SIS) of the biological matrix membrane is incubated overnight in the BC polydopamine solution, and finally freeze-dried to obtain the composite nanofiber bone scaffold. This invention provides a method for preparing a composite bone scaffold using polydopamine, which has excellent film-forming properties and biocompatibility, does not hinder cell growth and differentiation, and combines BC and SIS in a green synthesis process with simple operation.

Description

Technical Field

[0001] This invention relates to the field of medical engineering technology, and more specifically to a method for preparing a composite nanofiber bone scaffold. Background Technology

[0002] Bone health remains a significant concern for many, and bone diseases are often caused by trauma, infection, arthritis, tumors, osteoporosis, and osteonecrosis. Over the past few decades, various treatments have been developed to restore the structure and function of damaged bone, such as autologous bone grafting, xenografting, and artificial replacements. However, due to defects such as transplant rejection, bone regeneration remains a major challenge in clinical practice. Artificial biomaterials based on synthetic materials such as ceramics, polymers, and metals offer advantages over autologous and allogeneic transplants in treating bone defects. However, natural materials, with their biocompatibility, chemical functionality, biodegradability, cost-effectiveness, and mass production capabilities, have shown potential in practical applications in tissue engineering. Bacterial cellulose (BC) not only possesses a three-dimensional nanonetwork structure similar to bone 3D extracellular matrix proteins, but also exhibits unique properties such as biocompatibility, blood compatibility, mechanical strength, high water absorption and retention, and non-immunogenicity. In particular, its high water absorption and retention are beneficial for supplying nutrients to the cells within the scaffold for growth. Furthermore, BC possesses excellent mechanical properties in a wet state and can serve as a biotemplate for the synthesis of hydroxyapatite (HA). BC can rapidly induce HA synthesis in simulated body fluids. The synthesized HA exhibits natural osteoconductivity and osteointegration capabilities. Osteoconductivity provides a scaffold for blood vessel ingrowth and new bone formation, while osteointegration enables direct contact between the scaffold and bone tissue scaffold without fibrous connective tissue interface, preventing loosening and failure after implantation. These characteristics indicate that it is a promising biomaterial for bone tissue engineering. However, the small pore size (0.05–5 μm) of the BC nanofiber membrane network limits cell permeability and nutrient transport, and the lack of antibacterial properties of BC affects postoperative infection control and the healing rate of postoperative wounds.

[0003] BC nanofibers mimic the characteristics of natural collagen fibers in shape and molecular structure, exhibiting superior mechanical properties compared to collagen. However, the latter demonstrates a higher cell differentiation capacity than BC membranes. Cell differentiation capacity determines the healing rate at the injury site. Loading multiple cell growth factors can promote cell differentiation in bone scaffolds, but adding exogenous cytokines may lead to cytotoxicity, genotoxicity, and mutagenic effects. Therefore, endowing the implant itself with excellent cell differentiation capacity and antibacterial properties is a highly effective means of promoting postoperative healing and preventing postoperative infection.

[0004] The submucosa SIS matrix membrane derived from porcine small intestine is primarily composed of collagen, which mimics the shape and molecular structure of bone collagen. It is mainly composed of orderly arranged type I collagen fibers, forming a three-dimensional nanonetwork structure similar to bone 3D extracellular matrix proteins. In terms of antibacterial properties, SIS contains antimicrobial peptides, exhibiting activity against both Gram-positive and Gram-negative bacteria, thus inhibiting bacterial contamination. It also possesses antimicrobial activity and is biodegradable in vivo. Furthermore, SIS exhibits excellent bioactivity, containing mucopolysaccharides, proteoglycans, glycoproteins, heparin, fibronectin, and various functional growth factors, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and nerve growth factor. These factors promote cell adhesion, proliferation, and differentiation, without the risk of cytotoxicity, genotoxicity, or mutagenicity from the addition of exogenous cytokines. As a carrier of growth factors, SIS can accelerate the healing process at injury sites, making it suitable as a tissue engineering scaffold material. The porous microstructure of SIS, with pores ranging from 20 μm to 30 μm, facilitates nutrient transport and oxygen diffusion, essential for cell survival and proliferation. Completely decellularized SIS has been proven non-immunogenic in animal experiments and clinical applications. The reabsorbability of SIS supports rapid cell infiltration and inward growth into host tissue, inducing angiogenesis and promoting tissue regeneration by recruiting endothelial cells and endothelial progenitor cells. SIS possesses a naturally designed architecture, providing physical support to accommodate cells and support three-dimensional (3D) cell growth, thereby promoting 3D tissue formation and generating desired tissue substitutes. Transplantation of human bone marrow mesenchymal stem cells into the SIS stromal membrane significantly enhances their therapeutic potential, and like BC, SIS serves as a biotemplate for HA synthesis, rapidly inducing HA synthesis in simulated body fluids, showing great potential in bone regeneration tissue engineering applications.

[0005] Inspired by the adhesion proteins of clams, dopamine (3,4-dihydroxyphenylethylamine) has been found to self-polymerize under alkaline pH conditions, forming a highly adhesive film on almost all organic and inorganic substrates. The adhesion behavior of dopamine to substrate surfaces stems from its catechol and amino functional groups. This structure allows for covalent and non-covalent interactions with organic-inorganic surfaces, resulting in the strong adhesion of polydopamine films to all materials. Polydopamine films exhibit excellent film-forming properties and biocompatibility, without hindering cell growth and differentiation. By combining polydopamine with BC powder and SIS, the small pore size (0.05–5 μm) structure of the BC nanofiber membrane, which restricts cell permeation and nutrient transport, is broken down. This not only leverages the high water absorption and retention capacity and excellent wet dynamics of BC but also overcomes the lack of antibacterial properties of BC. Furthermore, SIS carries multiple cell growth factors that promote cell adhesion, proliferation, and differentiation, eliminating concerns about the potential cytotoxicity, genotoxicity, and mutagenic effects of adding exogenous cell growth factors. Both BC and SIS are biological templates synthesized from HA, and their combination has the potential to enhance osteoconduction and osteointegration in bone scaffolds. The prepared BC / SIS composite nanofiber bone scaffold shows promising application prospects in bone substitute materials.

[0006] In summary, how to provide a method for preparing BC / SIS composite nanofiber bone scaffolds is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing a composite nanofiber bone scaffold. The technical problem to be solved by the present invention is to provide a method for preparing a composite bone scaffold by green synthesis of polydopamine, which has excellent film-forming properties and biocompatibility and does not hinder cell growth and differentiation, combined with BC and SIS, with a simple operation process.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a composite nanofiber bone scaffold includes the following steps:

[0010] Step 1: Inoculate the activated bacterial strain into the seed culture medium to obtain the seed liquid. Inoculate the seed liquid into the fermentation medium to obtain the BC membrane. Take out the BC membrane, wash it, and set it aside for later use.

[0011] Step 2: Freeze-dry the BC membrane cleaned in Step 1, pre-cool it with liquid nitrogen, grind it into powder, sterilize it, and obtain BC membrane powder;

[0012] Step 3: Adjust the pH of the dopamine solution to 10 to form a polydopamine solution;

[0013] Step 4: The BC membrane powder is ultrasonically dispersed in the polydopamine solution to obtain a polydopamine solution of BC;

[0014] Step 5: Take the proximal jejunum of the pig, remove the connective tissue and residual impurities in the intestine, then remove the intestinal epithelial cells, and then remove the serosa and smooth muscle tissue to obtain the submucosal tissue of the small intestine.

[0015] Step 6: Immerse the submucosal tissue of the small intestine in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution is 20%; then rinse with PBS for 15 minutes, twice each time; rinse with sterile water twice, 15 minutes each time; freeze-encapsulate and sterilize to obtain the biological matrix membrane SIS.

[0016] Step 7: Place the biomatrix membrane SIS in the polydopamine solution of BC overnight, and then freeze-dry to obtain a composite nanofiber bone scaffold.

[0017] Furthermore, the bacterial strain is Acetobacter xylinum.

[0018] Furthermore, the bacterial strain is Acetobacter xylinum ATCC 23767.

[0019] Furthermore, the seed culture medium comprises the following components at the following mass concentrations: glucose 20 g / L, yeast extract 5 g / L, peptone 5 g / L, citric acid 1 g / L and disodium hydrogen phosphate 5 g / L, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0020] The fermentation medium comprises the following components at the following mass concentrations: glucose 20 g / L, yeast extract 5 g / L, peptone 5 g / L, citric acid 1 g / L and disodium hydrogen phosphate 5 g / L, with a pH of 7; the fermentation medium is sterilized at 121°C for 20 min before use.

[0021] Furthermore, the fermentation process conditions in step 1 are as follows: inoculum size 8-10%, temperature 28℃, and time 8 days.

[0022] Furthermore, the specific cleaning operation in step 1 is as follows: under the condition of 65-80℃, the BC membrane is treated with 0.1-0.5mol / L NaOH solution for 1-2 hours to obtain a translucent milky white BC membrane, and then rinsed with deionized water until neutral.

[0023] Furthermore, the freeze-drying process conditions in step 2 are: -50℃, vacuum degree 5~20, and duration 12h.

[0024] Furthermore, in step 3, the concentration of the dopamine solution is 1 mg / mL, the pH of the solution is adjusted to 10 using 1 mol / L NaOH solution, and the reaction time is 15 hours to obtain a polydopamine solution.

[0025] Furthermore, in step 4, the mass ratio of the BC film powder to the polydopamine solution is (1-4):1.

[0026] Furthermore, the specific operation of step 5 is as follows: take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0027] Furthermore, the bio-matrix membrane SIS described in step 7 is... Immerse in 200–500 μL of BC polydopamine solution for 12–16 h.

[0028] Furthermore, the freeze-drying process conditions in step 7 are: -50℃, vacuum degree 5~20, and time duration 12h.

[0029] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0030] The preparation process of this invention is green and environmentally friendly, simple to operate, requires no large equipment, and consumes little energy. Production costs are low, and it is easy to industrialize the production of finished products.

[0031] In this invention, BC membrane powder is a natural biomaterial. By combining a biocompatible polydopamine membrane with a SIS biomaterial matrix membrane, a three-dimensional nanofiber network structure mimicking bone is achieved. This breaks down the small pore size (0.05–5 μm) structure of the BC nanofiber membrane, which restricts cell permeability and nutrient transport. Furthermore, it leverages the high water absorption and retention capacity and excellent wet mechanical properties of BC while overcoming the lack of antibacterial activity. SIS also carries multiple cell growth factors that promote cell adhesion, proliferation, and differentiation, eliminating concerns about the potential cytotoxicity, genotoxicity, and mutagenic effects of adding exogenous cell growth factors. Both BC and SIS serve as biological templates for inducing HA synthesis, which endows the BC / SIS composite nanofiber bone scaffold with osteoconduction and osteointegration potential. Thus, an antibacterial biological bone scaffold with a three-dimensional nanofiber network structure, high water absorption, good wet mechanical properties, and osteoinduction, osteoconduction, and osteointegration potential is obtained. This invention provides a new pathway for natural material composite bone scaffolds and has significant practical implications. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0033] Figure 1 This is a schematic diagram illustrating the fabrication of the composite nanofiber bone scaffold of the present invention;

[0034] Figure 2 The images show the SEM morphology and pore size distribution of the three-dimensional nanofiber network and hierarchical micropore structure of the composite nanofiber bone scaffold prepared in Example 1 of this invention. A represents the bone scaffold; B is a magnified view of a portion of the scaffold in Figure A (the red box shows a magnified view of the local three-dimensional nanofiber network); C represents the pore size distribution of the BC membrane. Figure 2 C is derived from Wen Xiaoshuang's master's thesis at Donghua University [Construction of Three-Dimensional Cell Culture Substrate by Bacterial Cellulose Composite Porous Support and Its Performance Evaluation]; D is the pore size distribution of the bone scaffold.

[0035] Figure 3 The FITR results for the composite nanofiber bone scaffold prepared in Example 1 of this invention are shown below (BC is the FITR spectrum of bacterial cellulose; SIS is the FITR spectrum of the small intestinal mucosal layer; BC / SIS is the FITR spectrum of the BC / SIS composite bone scaffold).

[0036] Figure 4 The XRD results of the composite nanofiber bone scaffold prepared in Example 2 of this invention before and after mineralization are shown.

[0037] Figure 5 The images show the SEM morphology of the composite nanofiber bone scaffold prepared in Example 2 of this invention before and after mineralization, where A is the bone scaffold before mineralization and B is the bone scaffold after mineralization.

[0038] Figure 6 The water absorption performance of the composite nanofiber bone scaffold prepared in Example 2 of the present invention is shown, wherein 1-4 represent SIS scaffold, BC scaffold, BC / SIS scaffold and mineralized BC / SIS scaffold in sequence.

[0039] Figure 7 The wet mechanical properties (A-strain; B-mechanical strength) of the composite nanofiber bone scaffold prepared in Example 3 of this invention;

[0040] Figure 8 The results of live cell staining after 1 day of culture of bone marrow mesenchymal stem cells (MSCs) in the composite nanofiber bone scaffold prepared in Example 4 of this invention are shown. In this example, A represents MSC culture without bone scaffold; and B represents MSC co-culture with bone scaffold.

[0041] Figure 9 The antibacterial properties of the composite nanofiber bone scaffold prepared in Example 5 of the present invention are shown, wherein A is the BC scaffold and B is the composite bone scaffold. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0044] Example 1

[0045] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0046] (1) The activated Acetobacter xylinum ATCC 23767 (purchased from Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into the seed culture medium to obtain the seed liquid. The seed liquid was then inoculated into the fermentation culture medium to obtain the bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0047] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0048] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0049] The fermentation process conditions were: inoculum size 9%, temperature 28℃, and time 8 days.

[0050] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0051] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0052] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0053] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0054] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0055] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0056] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0057] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then, it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0058] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0059] Biological matrix membrane SIS Immerse in 400 μL of the final solution from step (4) for 16 h.

[0060] The freeze-drying process conditions are: -50℃, vacuum degree 20, and time duration 12h.

[0061] Figure 1 This is a schematic diagram illustrating the preparation steps and process of the composite nanofiber bone scaffold of the present invention.

[0062] Figure 2 SEM morphology and pore size distribution of the three-dimensional nanofiber network and multi-level micropore structure of the composite nanofiber bone scaffold prepared in Example 1. Figure 2 The three-dimensional nanofiber network structure, hierarchical microporous structure, and pore size distribution of the composite nanofiber bone scaffold are shown, and compared with... Figure 2The pore size of the BC membrane in C is significantly improved, which is beneficial for the transport of nutrients and metabolism of bone scaffold cells.

[0063] Example 2

[0064] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0065] (1) The activated Acetobacter xylinum ATCC 23767 strain (purchased from Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into seed culture medium to obtain seed liquid. The seed liquid was then inoculated into fermentation culture medium to obtain bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0066] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0067] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0068] The fermentation process conditions were: 8% inoculum, 28℃ temperature, and 8 days.

[0069] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0070] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0071] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0072] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0073] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0074] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0075] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0076] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then, it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0077] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0078] Biological matrix membrane SIS Immerse in 500 μL of the final solution from step (4) for 16 h.

[0079] The freeze-drying process conditions are: -50℃, vacuum degree 5, and time duration 12h.

[0080] Mineralization method of composite scaffold: 10×SBF simulated body fluid (10 times the concentration of simulated body fluid) was used. The sample mass was weighed before soaking and recorded as W0. The material was soaked at 50g / mL for 6h, and the solution was changed every 2h. 3 samples were replicated.

[0081] Determination of water absorption rate of composite scaffold: Three samples from each group were placed in ultrapure water for 24 hours, and then weighed (W). w After freeze-drying, weigh (W) d ).

[0082] Water absorption rate = [(W w -W d ) / W d ×100%.

[0083] Figure 4 The XRD patterns of the composite nanofiber bone scaffold prepared in Example 2 before and after mineralization are shown.

[0084] Figure 5 SEM images of the composite nanofiber bone scaffold prepared in Example 2 before and after mineralization (A - bone scaffold before mineralization, B - bone scaffold after mineralization); the mineralized bone scaffold was observed to have uniformly deposited HA (reference). Figure 4 Mineral layer.

[0085] Figure 6The water absorption properties of the composite nanofiber bone scaffold prepared in Example 2 are shown. The water absorption rate of the composite nanofiber bone scaffold is approximately four times that of the SIS scaffold. The good water absorption properties of the bone scaffold are beneficial for supplying nutrients to its internal cells.

[0086] Example 3

[0087] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0088] (1) The activated Acetobacter xylinum ATCC 23767 strain (purchased from Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into seed culture medium to obtain seed liquid. The seed liquid was then inoculated into fermentation culture medium to obtain bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0089] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0090] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0091] The fermentation process conditions were: 10% inoculum, 28℃ temperature, and 8 days.

[0092] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0093] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0094] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0095] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0096] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0097] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0098] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0099] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0100] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0101] Biological matrix membrane SIS Immerse in 500 μL of the final solution from step (4) for 16 h.

[0102] The freeze-drying process conditions are: -50℃, vacuum degree 20, and time duration 12h.

[0103] Determination of wet mechanical properties of composite scaffold: Three samples from each group were placed in ultrapure water for 24 hours. The wet mechanical properties of the scaffold were tested using a universal testing machine with a tensile speed of 2 mm / min and a sensor of 100 N.

[0104] Figure 7 The wet mechanical properties (A-strain; B-mechanical strength) of the composite nanofiber bone scaffold prepared in Example 3 are shown. The tensile strength of the composite scaffold is nearly twice that of the SIS scaffold when the strain is lower than that of the SIS scaffold.

[0105] Example 4

[0106] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0107] (1) The activated Acetobacter xylinum ATCC 23767 strain (purchased from Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into seed culture medium to obtain seed liquid. The seed liquid was then inoculated into fermentation culture medium to obtain bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0108] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0109] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0110] The fermentation process conditions were: 10% inoculum, 28℃ temperature, and 8 days.

[0111] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0112] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0113] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0114] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0115] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0116] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0117] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0118] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then, it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0119] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0120] Biological matrix membrane SIS Immerse in 500 μL of the final solution from step (4) for 16 h.

[0121] The freeze-drying process conditions are: -50℃, vacuum degree 20, and time duration 12h.

[0122] Resuscitated bone marrow mesenchymal stem cells (MSCs) were cultured in DMEM medium containing 10% (v / v) fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. The medium was changed twice a week. After 7 days, cells were harvested, and monolayers were separated using trypsin-ethylamine tetraacetic acid (EDTA) to prepare a monolayer cell suspension. Sterilized composite bone scaffolds were soaked in cell culture overnight the day before, and then placed in 24-well plates the next day. Viable cell counts were performed using a hematology counter, and the cells were diluted to 1×10⁶ cells / mL with DMEM medium containing 10% FBS. 6 MSCs were seeded at a density of [number] cells / ml in 24-well plates without bone scaffolds. A single-celled plate served as a control. After 24 hours of culture, dead and live cells were stained using the acridine orange-ethidium bromide double staining kit. The cells were then observed using a fluorescence inverted microscope. Results are shown in [figure missing]. Figure 8 .

[0123] Figure 8 The results of live cell staining after 1 day of co-culturing the composite nanofiber bone scaffold prepared in Example 4 with bone marrow mesenchymal stem cells (MSCs) (A - MSCs cultured without bone scaffold; B - MSCs co-cultured with bone scaffold). The results show that the cells on the scaffold grew well.

[0124] Example 5

[0125] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0126] (1) The activated Acetobacter xylinum ATCC 23767 strain (purchased from Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into seed culture medium to obtain seed liquid. The seed liquid was then inoculated into fermentation culture medium to obtain bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0127] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0128] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0129] The fermentation process conditions were: 10% inoculum, 28℃ temperature, and 8 days.

[0130] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0131] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0132] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0133] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0134] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0135] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0136] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0137] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then, it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0138] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0139] Biological matrix membrane SIS Immerse in 500 μL of the final solution from step (4) for 16 h.

[0140] The freeze-drying process conditions are: -50℃, vacuum degree 20, and time duration 12h.

[0141] To determine the antibacterial properties of the bone scaffold using the agar well diffusion method, bacteria were cultured for 24 hours, and then the bacterial solutes were retained in physiological serum until a concentration equal to 0.5 McFarland (1.5 × 10⁻⁶) was obtained. 8 CFU ml -1 The bacterial suspension was then cultured on agar medium. Sterilized BC (control group) and composite nanofiber bone scaffold (experimental group) were placed on bacterial-coated plates and incubated at 37°C aerobic conditions for 24 hours to observe their antibacterial effect. *Escherichia coli* AATCC 11303 (Gram-negative) was used for the antibacterial test. Results are shown below. Figure 9 .

[0142] Figure 9 The antibacterial properties of the composite nanofiber bone scaffold prepared in Example 5 (A-BC membrane; B-bone scaffold) are shown. An antibacterial ring is visible around the composite nanofiber bone scaffold, while no antibacterial ring is visible around the BC scaffold. This indicates that the composite scaffold acquired a certain antibacterial ability after being combined with SIS.

[0143] Example 6

[0144] A composite nanofiber bone scaffold, the preparation method of which includes the following steps:

[0145] (1) The activated Acetobacter xylinum ATCC 23767 strain (Shanghai Hewu Biotechnology Co., Ltd.) was inoculated into the seed culture medium to obtain the seed liquid. The seed liquid was then inoculated into the fermentation culture medium to obtain the bacterial cellulose membrane. The bacterial cellulose membrane was then removed and washed.

[0146] The seed culture medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7; the seed culture medium is sterilized at 121°C for 20 min before use.

[0147] The fermentation medium consists of: 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, 1 g / L citric acid and 5 g / L disodium hydrogen phosphate, with a pH of 7. The fermentation medium is sterilized at 121°C for 20 min before use.

[0148] The fermentation process conditions were: 10% inoculum, 28℃ temperature, and 8 days.

[0149] The specific cleaning procedure is as follows: treat with 0.5 mol / L NaOH solution at 80℃ for 2 hours to obtain a translucent milky white bacterial cellulose membrane, and then rinse with deionized water until neutral.

[0150] (2) Freeze-dry the bacterial cellulose membrane obtained in step (1), pre-cool it with liquid nitrogen, grind it into powder with an agate mortar, and sterilize it at 121°C for 20 minutes before use.

[0151] The freeze-drying process conditions are: -50℃, vacuum degree 5-20, and time duration 12h.

[0152] (3) Adjust the pH of the dopamine solution to 10 to induce polymerization and form a polydopamine solution.

[0153] The concentration of dopamine solution was 1 mg / mL. The pH of the solution was adjusted to 10 using 1 mol / L NaOH solution, and the reaction time was 15 hours to obtain polydopamine solution.

[0154] (4) The bacterial cellulose membrane powder from step (2) is ultrasonically dispersed in the polydopamine solution prepared in step (3), with a mass ratio of bacterial cellulose membrane powder to polydopamine solution of 1:1.

[0155] (5) Take 10-18cm of the proximal jejunum of a healthy adult pig, remove excess connective tissue and residual impurities in the intestine, then decellularize with physiological saline to remove intestinal epithelial cells; then remove the serosa and smooth muscle tissue by mechanical scraping and hydration to obtain the submucosal tissue of the small intestine.

[0156] (6) The small intestinal submucosal tissue obtained in step (5) was immersed in an ethanol solution of 0.1% peracetic acid for 2 hours, wherein the concentration of the ethanol solution was 20%. Then, it was rinsed with PBS for 15 minutes, twice each time; rinsed with sterile water for 15 minutes, twice each time; frozen and encapsulated, and sterilized with ethylene oxide to obtain the biological matrix membrane SIS.

[0157] (7) The biomatrix membrane SIS obtained in step (6) was placed in the polydopamine solution of bacterial cellulose prepared in step (4) overnight, and then freeze-dried to obtain a composite nanofiber bone scaffold.

[0158] Biological matrix membrane SIS Immerse in 500 μL of the final solution from step (4) for 16 h.

[0159] The freeze-drying process conditions are: -50℃, vacuum degree 20, and time duration 12h.

[0160] The cytokine content on the composite bone scaffold was detected by ELISA. 100 mg of the composite bone scaffold was cut into small pieces, moistened in PBS, frozen in liquid nitrogen, then ground into a homogenate. The homogenate was centrifuged at 3000 rpm for 5 min with a radius of 10 cm, and the supernatant was collected. The ELISA kit was followed to detect the levels of growth factors VEGF and TGF-β. The results are shown in Table 1.

[0161] Table 1. Content of naturally loaded cell growth factors in the composite nanofiber bone scaffold

[0162]

[0163] Table 1 shows that in Example 6, the bone scaffold, through BC-composite SIS, acquired naturally loaded cytokines. The presence of these factors gave it the potential to promote cell differentiation, which is beneficial to improving the healing speed of the injury site.

[0164] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0165] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a composite nanofibrous bone scaffold, characterized by: The method comprises the following steps: Step 1: inoculating the activated bacterial strain into a seed culture medium to obtain a seed liquid, inoculating the seed liquid into a fermentation culture medium to obtain a BC membrane, taking out the BC membrane, washing the BC membrane, and reserving the BC membrane for later use; Step 2: freeze-drying the BC membrane after washing in step 1, pre-cooling the BC membrane in liquid nitrogen, grinding the BC membrane into powder, sterilizing the BC membrane, and obtaining a BC membrane powder; Step 3: adjusting a dopamine solution to have a pH value of 10 to form a polydopamine solution; Step 4: ultrasonically dispersing the BC membrane powder in the polydopamine solution to obtain a BC polydopamine solution; Step 5: taking a pig proximal jejunum, removing connective tissue and intestinal residual impurities, then removing intestinal epithelial cells, and then removing serosal and smooth muscle tissue to obtain a small intestinal submucosa tissue; Step 6: immersing the small intestinal submucosa tissue in a 0.1% peroxoacetic acid ethanol solution for 2 hours, wherein the concentration of the ethanol solution is 20%; then washing the small intestinal submucosa tissue in PBS for 15 minutes twice; then washing the small intestinal submucosa tissue in sterile water for 15 minutes twice; then freeze-packaging the small intestinal submucosa tissue, sterilizing the small intestinal submucosa tissue, and obtaining a biological matrix membrane SIS; Step 7: placing the biological matrix membrane SIS in the BC polydopamine solution overnight, then freeze-drying the biological matrix membrane SIS to obtain a composite nanofiber bone scaffold. The bacterial strain is Acetobacter xylinum.

2. The method of claim 1, wherein the composite nanofiber bone scaffold is prepared by the steps of: The seed culture medium comprises the following components in the following mass concentrations: 20 g / L of glucose, 5 g / L of yeast powder, 5 g / L of peptone, 1 g / L of citric acid, and 5 g / L of sodium phosphate dibasic, and has a pH value of 7; 3. The method of claim 1, wherein the composite nanofibrous bone scaffold is prepared by electrospinning. The fermentation culture medium comprises the following components in the following mass concentrations: 20 g / L of glucose, 5 g / L of yeast powder, 5 g / L of peptone, 1 g / L of citric acid, and 5 g / L of sodium phosphate dibasic, and has a pH value of 7. The process conditions for fermentation in step 1 are as follows: an inoculation amount of 8-10%, a temperature of 28 DEG C, and a time of 8 days.

4. The method of claim 1, wherein the composite nanofibrous bone scaffold is prepared by electrospinning. The specific operation for washing in step 1 is as follows: treating the BC membrane in a 0.1-0.5 mol / L NaOH solution at 65-80 DEG C for 1-2 hours to obtain a semi-transparent, milky-white BC membrane, and then washing the BC membrane in deionized water until the BC membrane is neutral.

5. The method of claim 1, wherein the composite nanofibrous bone scaffold is prepared by electrospinning. In step 3, the concentration of the dopamine solution is 1 mg / mL, a 1 mol / L NaOH solution is used to adjust the pH value of the solution to 10, and the reaction time is 15 hours to obtain a polydopamine solution.

6. The method of claim 1, wherein the composite nanofibrous bone scaffold is prepared by electrospinning. In step 4, the mass ratio of the BC membrane powder to the polydopamine solution is (1-4):

1.

7. The method of claim 1, wherein the composite nanofibrous bone scaffold is prepared by electrospinning. In step 7, the biological matrix membrane SIS is immersed in 200-500 μL of the BC polydopamine solution in a φ24 mm manner for 12-16 hours.

8. The method for preparing a composite nanofiber bone scaffold as described in claim 1, characterized in that, ​

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