A collagen-based hard-core sandwich composite membrane for guiding bone regeneration and its preparation method and application

Abstract

The present invention discloses a collagen-based hard-core sandwich composite membrane for guiding bone regeneration, and its preparation method and application, which belong to the technical field of biomedical materials. The composite membrane of the present invention consists of a hard core and a soft shell. The present invention cross-links and biomimetic mineralizes self-assembled collagen fibers through a dicarboxylic acid ester cross-linking agent and Zr-MOP to form a hard-core cross-linked mineralized collagen fiber membrane, and then wraps it with a soft-shell methacrylated collagen gel, and presses it to obtain the membrane. The soft shell of the composite membrane of the present invention is loose and porous, which is conducive to the adhesion and proliferation of bone cells; the hard core is a dense layer, which can provide better support performance and effectively block epithelial cells and fibroblasts from invading the osteogenic area; the controllable pore structure provided by Zr-MOP can ensure normal material exchange in the defect area. Therefore, the composite membrane of the present invention has high mechanical strength, controllable degradation rate and good biocompatibility, and can play a good barrier role and promote bone regeneration in the process of bone repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a collagen-based hard-core sandwich composite membrane for guiding bone regeneration, and a preparation method and application thereof. Background Art

[0002] Alveolar bone defects caused by factors such as inflammation and trauma are relatively common in oral clinical practice. Insufficient bone mass due to bone defects is also a major challenge and hot issue currently facing clinical dental implants. Guided bone regeneration (GBR) technology is a commonly used method to solve bone defects around implants in clinical practice. This technology uses a barrier membrane to establish a biological barrier between the bone defect area and the gingival soft tissue, preventing the faster-growing soft tissue fibroblasts from invading the bone defect area and providing attachment sites and growth space for slower-growing osteoblasts. Barrier membranes play an important role in guided bone regeneration and are the core of GBR technology. The ideal barrier membrane needs to have good biocompatibility, good support, biodegradability coordinated with the time of bone regeneration, and porosity to ensure the exchange of basic nutrients.

[0003] Based on whether they are degradable, GBR membranes can be divided into non-degradable membranes and biodegradable membranes. Although non-degradable membranes have good mechanical strength and support, they require a second surgery to remove, which increases the risk of inflammation and the burden on patients. Biodegradable membranes can biodegrade while achieving GBR functions and are currently a hot topic of research.

[0004] Collagen is widely used as a raw material for GBR membranes due to its excellent biocompatibility and biodegradability. Currently, the most widely used GBR membrane in clinical practice is the Bio-Gide collagen membrane from the Swiss company Geistlich. It has a unique double-layer structure: one dense layer that blocks the invasion of connective tissue, and the other loose and porous layer that facilitates the attachment and growth of osteoblasts. However, Bio-Gide, as well as most current GBR membranes, still have some drawbacks, such as rapid degradation, poor mechanical properties, and easy membrane exposure. Furthermore, during clinical surgery, GBR membranes with a double-layer structure must distinguish between the front and back sides (with the dense layer facing the gums). However, this can easily cause confusion among doctors (especially after the membrane becomes stained with blood), potentially compromising the effectiveness of GBR surgery. To address the problems of GBR collagen membranes, such as easy degradation, collapse and rupture, and poor mechanical properties, existing technologies generally use chemical cross-linking and biomimetic mineralization to improve their related properties. However, conventional chemical cross-linking agents such as glutaraldehyde have been found to cause inflammation and calcification reactions in the body and are cytotoxic. In addition, traditional mineralized materials (such as hydroxyapatite) have a destructive effect on the pore structure of the membrane, making it difficult to ensure normal material exchange in the bone defect area.

[0005] In summary, the current collagen-based guided bone regeneration barrier membranes still have problems such as poor mechanical strength, rapid degradation time, and the need to distinguish between the front and back sides of the membrane in clinical practice, which makes them inconvenient to use. New preparation technologies and modification strategies are urgently needed to optimize the performance of collagen membranes and improve their practicality. Summary of the Invention

[0006] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a collagen-based hard-core sandwich composite membrane for guided bone regeneration and its preparation method and application, so as to solve the problems of poor mechanical properties of current GBR membranes, rapid degradation rate in vivo, lack of pore structure, and the need to distinguish between the front / back sides of the membrane, which makes it inconvenient to use.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: providing a collagen-based hard-core sandwich composite membrane for guided bone regeneration, comprising a hard core and a soft shell; wherein the core layer of the hard core has a thickness of 0.1-1 mm and is composed of a cross-linked mineralized collagen fiber membrane; the shell layer of the soft shell has a thickness of 0.05-0.1 mm and is composed of a methacrylated collagen gel.

[0008] The present invention provides a method for preparing the collagen-based hard-core sandwich composite membrane for guided bone regeneration, comprising the following steps:

[0009] (1) Preparation of self-assembled collagen fiber gel;

[0010] (2) dispersing the self-assembled collagen fiber gel prepared in step (1) into a suspension, and cross-linking the suspension with a dicarboxylate cross-linking agent for 4-8 hours;

[0011] (3) adding water-soluble metal organic polyhedron (Zr-MOP) to the product obtained in step (2), reacting for 2-4 hours, then centrifuging and washing the salt, resuspending, and filtering to obtain a cross-linked mineralized collagen fiber membrane;

[0012] (4) After the collagen is dissolved, a methacrylic anhydride solution is added and the reaction is carried out for 3-6 hours. After dialysis, a methacrylated collagen solution is obtained. Then, the cross-linked mineralized collagen fiber membrane obtained in step (3) is immersed in the methacrylated collagen solution, the pH of the solution is adjusted to 7.2-7.6, sodium chloride is added, and after incubation, a photoinitiator is added for photocrosslinking to obtain a methacrylated collagen gel;

[0013] (5) The product obtained in step (4) is freeze-dried and pressed to obtain a product.

[0014] Furthermore, the preparation of the self-assembled collagen fiber gel in step (1) specifically includes the following steps: dissolving collagen in a PBS solution containing sodium chloride, adjusting the pH to neutral using a sodium hydroxide solution, and then incubating for 3-4 hours.

[0015] Furthermore, the collagen is fish skin collagen, bovine / pig skin collagen, bovine / pig Achilles tendon collagen, fish skin / fish scale collagen or bovine / pig cartilage collagen.

[0016] Furthermore, the chemical structure of the dicarboxylate crosslinking agent in step (2) is shown in formula (I):

[0017]

[0018] (I)

[0019] Where R is C1-C 10 of the alkyl chain.

[0020] Furthermore, the preparation method of the dicarboxylate crosslinker includes the following steps: adding N-hydroxysulfosuccinimide and dicarboxylic acid to acetone, mixing, adding a dehydrating agent, reacting for 4-8 hours, filtering, collecting the precipitate, washing, and drying to obtain the crosslinker; wherein the mass ratio of N-hydroxysulfosuccinimide to the dicarboxylic acid is 1:0.1-10; and the mass ratio of the dehydrating agent to the N-hydroxysulfosuccinimide is 1:0.1-20.

[0021] Furthermore, the dicarboxylic acid is adipic acid, suberic acid or glutaric acid; and the dehydrating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0022] Furthermore, the preparation method of Zr-MOP in step (3) includes the following steps: adding zirconocene dichloride and amino-p-dibenzoic acid to a mixed solution of water and dimethylacetamide to react for 2-6 hours, filtering and drying to obtain; wherein the mass ratio of zirconocene dichloride to amino-p-dibenzoic acid is 1:0.1-10; and the volume ratio of water to dimethylacetamide is 1:1-20.

[0023] Furthermore, in step (4), the mass ratio of collagen to methacrylic anhydride solution is 1:0.01-0.5; and the collagen is fish skin collagen, bovine / pig skin collagen, bovine / pig Achilles tendon collagen, fish skin / fish scale collagen, or bovine / pig cartilage collagen.

[0024] The present invention also provides a use of the collagen-based hard-core sandwich composite membrane for guided bone regeneration in the preparation of oral bone defect repair materials.

[0025] The present invention has the following beneficial effects:

[0026] (1) The present invention uses self-assembled collagen fibers as building blocks. Self-assembly is a property of collagen that spontaneously aggregates into fibers through non-covalent interactions under specific conditions. Nanoscale collagen fibers can be prepared in this way. Fiber membranes prepared using self-assembled collagen fibers as building blocks have better mechanical properties and degradation resistance than collagen molecular membranes. Furthermore, compared to the electrospinning method commonly used to prepare nanofibers, self-assembly can retain the natural activity of collagen to the greatest extent possible, and the preparation difficulty is much lower than electrospinning, thus having great application potential.

[0027] (2) The present invention uses N-hydroxysulfosuccinimide to activate dicarboxylic acid with good biocompatibility to prepare a dicarboxylic acid ester crosslinker for chemical crosslinking of self-assembled collagen fibers. Compared with existing barrier membranes, the composite membrane of the present invention has a unique "hard core-soft shell" sandwich structure, good biocompatibility, high mechanical strength and suitable degradation rate;

[0028] (3) The present invention uses water-soluble metal organic polyhedrons (Zr-MOP) to perform biomimetic mineralization on collagen self-assembled fibers. Therefore, the composite membrane prepared by the present invention has a controllable pore structure. The water-soluble metal organic polyhedrons are conducive to the reaction and combination with collagen. Moreover, changing the type of metal organic polyhedrons can flexibly control the pore structure of the membrane, which is conducive to the exchange of nutrients at the bone defect site and has a positive effect on bone repair.

[0029] (4) The composite membrane prepared by the present invention can achieve the same GBR effect without distinguishing the front and back sides during clinical use. It is more convenient to use and avoids the negative impact of confusing the front and back sides on GBR.

[0030] (5) The present invention uses methacrylic anhydride to modify collagen, retaining its natural triple helical structure and giving it the property of photocrosslinking, which can be tightly combined with the collagen fiber membrane to form a three-dimensional porous fiber network, which is conducive to the growth and adhesion of bone cells;

[0031] (6) The composite membrane preparation method of the present invention adopts vacuum filtration, natural air drying, freeze drying and mechanical tableting at room temperature. It is simple and easy to operate, has low requirements on equipment, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a surface SEM image of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention;

[0033] Figure 2 This is a cross-sectional SEM image of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention;

[0034] Figure 3 This is the stress-strain curve of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention;

[0035] Figure 4 This is a curve showing the change in mass over time of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention under the action of collagenase;

[0036] Figure 5 The adsorption-desorption curve and pore size distribution curve of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention;

[0037] Figure 6 The figure shows the migration of L929 fibroblasts in the membrane after being cultured on the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention for 3 days. DETAILED DESCRIPTION

[0038] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0039] Example 1:

[0040] A collagen-based hard-core sandwich composite membrane for guided bone regeneration comprises a hard core and a soft shell; the hard core has a core layer thickness of 0.5 mm and is composed of a cross-linked mineralized collagen fiber membrane; the soft shell has a shell layer thickness of 0.08 mm and is composed of methacrylated collagen gel.

[0041] The preparation method comprises the following steps:

[0042] (1) Weigh 10 parts of fish skin collagen and dissolve them in 200 parts of 0.01 M PBS (containing 0.1 M sodium chloride) solution to prepare a collagen solution. Adjust the pH of the solution to 7.4 with 0.1 M sodium hydroxide solution, incubate at 37°C for 4 h, and then mechanically disperse the collagen fibers to obtain a collagen fiber suspension.

[0043] (2) 20 parts of N-hydroxysulfosuccinimide and 15 parts of adipic acid were added to 100 parts of acetone, mixed, and then 25 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added for cross-linking reaction for 4 hours. The precipitate was collected after filtration, washed, and vacuum-dried to obtain a dicarboxylate cross-linking agent; wherein the mass ratio of N-hydroxysulfosuccinimide to dicarboxylic acid was 1:0.1. Its chemical structure is shown in formula (I):

[0044]

[0045] (I)

[0046] Wherein, R is C4H8;

[0047] (3) Zirconocene dichloride and amino-p-dibenzoic acid were added to a mixed solution of water and dimethylacetamide and reacted for 4 h. The mixture was filtered and dried to obtain a water-soluble metal organic polyhedron Zr-MOP. The mass ratio of zirconocene dichloride to amino-p-dibenzoic acid was 1:0.1, and the volume ratio of water to dimethylacetamide was 1:10.

[0048] (4) Add 0.1 parts of the dicarboxylate crosslinker (dissolved in dimethyl sulfoxide (DMSO)) prepared in step (2) to the collagen fiber suspension prepared in step (1), and then add 0.05 parts of the Zr-MOP (dispersed in deionized water) prepared in step (3), and react at 4°C for 2 hours. After the reaction is terminated, the reaction system is centrifuged to obtain a precipitate, which is washed with deionized water and centrifuged three times. The precipitate is filtered using a vacuum filtration pump, and the filtered product is air-dried at room temperature to obtain the "hard core" portion of the collagen membrane, i.e., the cross-linked mineralized collagen fiber membrane;

[0049] (5) Weigh 5 parts of fish skin collagen and dissolve them in 100 parts of 0.1 M acetic acid. Then adjust the pH of the collagen solution to 9 with 1 M sodium hydroxide solution. Then add 0.5 parts of methacrylic anhydride solution and stir to react for 3 hours. Then, dialyze with 0.1 M acetic acid solution for two days to obtain a methacrylated collagen solution.

[0050] (6) Immerse the cross-linked mineralized collagen fiber membrane obtained in step (4) in 10 parts of the methacrylated collagen solution obtained in step (5), adjust the pH of the solution to 7.4, add 0.2 parts of sodium chloride to fully dissolve it, incubate the solution at 37°C for 30 minutes, then add 0.005 parts of VA-086 photoinitiator, place it under an ultraviolet light source for 10 minutes to perform photocrosslinking to obtain a gel, and obtain the "soft shell" part of the composite membrane, i.e., the methacrylated collagen gel;

[0051] (7) The product obtained in step (6) was placed in a -20°C freezer, and then freeze-dried at -60°C using a freeze dryer. Finally, it was pressurized at 5 MPa and 25°C for 15 minutes using a tablet press to obtain a collagen-based hard-core sandwich composite membrane for guided bone regeneration with a dense interior and loose and porous exterior (see Figure 1-2 ).

[0052] Example 2:

[0053] A collagen-based hard-core sandwich composite membrane for guided bone regeneration comprises a hard core and a soft shell; the hard core has a core layer thickness of 1 mm and is composed of a cross-linked mineralized collagen fiber membrane; the soft shell has a shell layer thickness of 0.1 mm and is composed of methacrylated collagen gel.

[0054] The preparation method comprises the following steps:

[0055] (1) Weigh 10 parts of bovine skin collagen and dissolve them in 100 parts of 0.01 M PBS (containing 0.12 M sodium chloride) solution to prepare collagen solution. Adjust the pH of the solution to 7.2 with 0.1 M sodium hydroxide solution, incubate at 37°C for 4 h, and then mechanically disperse the collagen fibers to obtain a collagen fiber suspension.

[0056] (2) 10 parts of N-hydroxysulfosuccinimide and 30 parts of glutaric acid were added to 100 parts of acetone, mixed, and then 20 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added for cross-linking reaction for 8 hours. The precipitate was collected after filtration, washed, and vacuum-dried to obtain a dicarboxylate cross-linking agent; wherein the mass ratio of N-hydroxysulfosuccinimide to dicarboxylic acid was 1:5. Its chemical structure is shown in formula (I):

[0057]

[0058] (I)

[0059] Wherein, R is C3H6;

[0060] (3) Zirconocene dichloride and amino-p-dibenzoic acid were added to a mixed solution of water and dimethylacetamide and reacted for 4 h. The mixture was filtered and dried to obtain a water-soluble metal organic polyhedron Zr-MOP. The mass ratio of zirconocene dichloride to amino-p-dibenzoic acid was 1:1, and the volume ratio of water to dimethylacetamide was 1:20.

[0061] (4) Add 0.05 parts of the dicarboxylate crosslinker (DMSO-dissolved) prepared in step (2) to the collagen fiber suspension prepared in step (1), and then add 0.1 parts of the Zr-MOP (deionized water-dispersed) prepared in step (3), and react at 4°C for 2 hours. After the reaction is terminated, the reaction system is centrifuged to obtain a precipitate, which is washed with deionized water and centrifuged three times. The precipitate is filtered using a vacuum filtration pump, and the filtered product is air-dried at room temperature to obtain the "hard core" portion of the collagen membrane, i.e., the cross-linked mineralized collagen fiber membrane;

[0062] (5) Weigh 5 parts of fish skin collagen and dissolve them in 50 parts of 0.1 M acetic acid. Then adjust the pH of the collagen solution to 9 with 1 M sodium hydroxide solution. Then add 0.1 parts of methacrylic anhydride solution and stir to react for 3 hours. Then, dialyze with 0.1 M acetic acid solution for two days to obtain a methacrylated collagen solution.

[0063] (6) Immerse the cross-linked mineralized collagen fiber membrane obtained in step (4) in 20 parts of the methacrylated collagen solution obtained in step (5), adjust the pH of the solution to 7.2, add 0.5 parts of sodium chloride to fully dissolve it, incubate the solution at 37°C for 30 minutes, then add 0.001 parts of VA-086 photoinitiator, place it under an ultraviolet light source for 10 minutes to perform photocrosslinking to obtain a gel, and obtain the "soft shell" part of the composite membrane, i.e., the methacrylated collagen gel;

[0064] (7) The product obtained in step (6) was placed in a membrane frozen at -20°C, then freeze-dried at -60°C using a freeze dryer, and finally pressurized at 10 MPa and 30°C for 10 minutes using a tablet press to obtain a collagen-based hard-core sandwich composite membrane for guided bone regeneration that was dense on the inside and loose and porous on both sides of the outside.

[0065] Example 3:

[0066] A collagen-based hard-core sandwich composite membrane for guided bone regeneration comprises a hard core and a soft shell; the hard core has a core layer thickness of 0.5 mm and is composed of a cross-linked mineralized collagen fiber membrane; the soft shell has a shell layer thickness of 0.08 mm and is composed of methacrylated collagen gel.

[0067] The preparation method comprises the following steps:

[0068] (1) Weigh 20 parts of pig skin collagen and dissolve them in 150 parts of 0.01 M hydrochloric acid (containing 0.1 M sodium chloride) solution to prepare a collagen solution. Adjust the pH of the solution to 7.6 with 3 M trishydroxyaminomethane solution, incubate at 37°C for 4 h, and then mechanically disperse the collagen fibers to obtain a collagen fiber suspension.

[0069] (2) 40 parts of N-hydroxysulfosuccinimide and 10 parts of suberic acid were added to 100 parts of acetone, mixed, and then 60 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added for cross-linking reaction for 8 hours. The precipitate was collected after filtration, washed, and vacuum-dried to obtain a dicarboxylic acid ester cross-linking agent; wherein the mass ratio of N-hydroxysulfosuccinimide to dicarboxylic acid was 1:10. Its chemical structure is shown in formula (I):

[0070]

[0071] (I)

[0072] Where R is C6H 12 ;

[0073] (3) Adding zirconocene dichloride and amino-p-dibenzoic acid to a mixed solution of water and dimethylacetamide to react for 4 hours, filtering and drying to obtain a water-soluble metal organic polyhedron Zr-MOP; wherein the mass ratio of zirconocene dichloride to amino-p-dibenzoic acid is 1:10; and the volume ratio of water to dimethylacetamide is 1:40.

[0074] (4) Add 0.3 parts of the dicarboxylate crosslinker (dissolved in dimethyl sulfoxide (DMSO)) prepared in step (2) to the collagen fiber suspension prepared in step (1), and then add 0.15 parts of the Zr-MOP (dispersed in deionized water) prepared in step (3), and react at 4°C for 2 hours. After the reaction is terminated, the reaction system is centrifuged to obtain a precipitate, which is washed with deionized water and centrifuged three times. The precipitate is filtered using a vacuum filtration pump, and the filtered product is air-dried at room temperature to obtain the "hard core" part of the collagen membrane, i.e., the cross-linked mineralized collagen fiber membrane;

[0075] (5) Weigh 20 parts of fish skin collagen and dissolve them in 100 parts of 0.1 M acetic acid. Then adjust the pH of the collagen solution to 9 with 1 M sodium hydroxide solution. Then add 1 part of methacrylic anhydride solution and stir to react for 3 hours. Then, dialyze with 0.1 M acetic acid solution for two days to obtain a methacrylated collagen solution.

[0076] (6) Immerse the cross-linked mineralized collagen fiber membrane obtained in step (4) in 20 parts of the methacrylated collagen solution obtained in step (5), adjust the pH of the solution to 7.6, add 1 part of sodium chloride to fully dissolve it, incubate the solution at 37°C for 60 minutes, then add 0.01 parts of VA-086 photoinitiator, and place it under an ultraviolet light source for 20 minutes to perform photocrosslinking to obtain a gel, thereby obtaining the "soft shell" part of the composite membrane, i.e., the methacrylated collagen gel;

[0077] (7) The product obtained in step (6) was placed in a membrane frozen at -20°C, then freeze-dried at -60°C using a freeze dryer, and finally pressurized at 10 MPa and 35°C for 15 minutes using a tablet press to obtain a collagen-based hard-core sandwich composite membrane for guided bone regeneration that was dense on the inside and loose and porous on both sides of the outside.

[0078] Test example:

[0079] (1) Stress-strain curve of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention: The composite membrane prepared in Example 1 was cut into strips with a length of 30 mm and a width of 5 mm. The upper and lower ends were clamped on the fixture of a universal testing machine and the tensile strength test was performed to obtain the stress-strain curve of the composite membrane prepared in Example 1. Figure 3It can be seen that the maximum tensile strength of the composite membrane is about 35 MPa, and the tensile strength of the commercial Bio-Gide is about 5 MPa, indicating that the mechanical properties of the composite membrane prepared by the preparation method of the present invention can be effectively improved.

[0080] (2) Curve of the change of mass of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention under the action of collagenase over time: the composite membrane was cut into 1*1 cm discs, placed in a 24-well plate, 2 mL of 15 U / mL collagenase solution was added, and incubated at 37°C for 1, 3, 5 and 7 h, respectively. Then, the membranes were taken out, washed, dried and weighed, and compared with the original mass to obtain a curve of the change of membrane mass fraction over the enzyme action time. Figure 4 CCFM is unmineralized collagen membrane, MCCFM is mineralized cross-linked fiber membrane, 4%, 8%, 12% represent the proportion of Zr-MOP added. Figure 4 It can be seen that after mineralization and cross-linking treatment, the resistance of the composite membrane to enzymatic degradation is significantly improved, indicating that the use of cross-linking modification and biomimetic mineralization can significantly improve the stability of the composite membrane and effectively prolong the degradation time of the composite membrane.

[0081] (3) Adsorption-desorption curve and pore size distribution curve of the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention: The composite membrane was cut into pieces, 1 g of the composite membrane was weighed and placed in a measuring tube, and the membrane specific surface area and pore size were measured using a BET fully automatic specific surface area analyzer to obtain its adsorption-desorption curve and pore size distribution curve. Figure 5 It can be seen that the average pore size of the composite membrane is about 45 nm, which is a mesoporous structure and can achieve good material exchange.

[0082] (4) The migration of L929 fibroblasts in the collagen-based hard-core sandwich composite membrane for guided bone regeneration prepared in Example 1 of the present invention after culturing for 3 days: First, the membrane was sterilized by irradiation, and then the membrane was cut into 1*1 cm discs and placed in a laser confocal microplate. L929 mouse fibroblasts cultured to the logarithmic growth phase were inoculated on the surface of the composite membrane at a cell density of 5000 cells / sample. After 3 days, the cells were stained and the migration of cells in the cross section of the composite membrane was observed to determine the cell barrier performance of the membrane. Figure 6 As shown in Figure B, the maximum depth of cell migration in the cross section after 3 days was about 0.1 mm, which was comparable to the commercial collagen membrane in the control group (see Figure 6 This shows that the composite membrane prepared in Example 1 has good cell barrier function and can effectively play a barrier role in guided bone regeneration.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A collagen-based hard-core sandwich composite membrane for guided bone regeneration, characterized in that: It comprises a hard core and a soft shell; wherein the core layer of the hard core is 0.1-1 mm thick and is composed of a cross-linked mineralized collagen fiber membrane; the shell layer of the soft shell is 0.05-0.1 mm thick and is composed of a methacrylated collagen gel; The method for preparing the collagen-based hard-core sandwich composite membrane for guided bone regeneration comprises the following steps: (1) Preparation of self-assembled collagen fiber gel; (2) dispersing the self-assembled collagen fiber gel prepared in step (1) into a suspension, and cross-linking the suspension with a dicarboxylate cross-linking agent for 4-8 hours; (3) adding water-soluble metal organic polyhedron to the product obtained in step (2), reacting for 2-4 hours, then centrifuging and washing the salt, resuspending, and filtering to obtain a cross-linked mineralized collagen fiber membrane; (4) After the collagen is dissolved, a methacrylic anhydride solution is added and the reaction is carried out for 3-6 hours. After dialysis, a methacrylated collagen solution is obtained. Then, the cross-linked mineralized collagen fiber membrane obtained in step (3) is immersed in the methacrylated collagen solution, the pH of the solution is adjusted to 7.2-7.6, sodium chloride is added, and after incubation, a photoinitiator is added for photocrosslinking to obtain a methacrylated collagen gel; (5) The product obtained in step (4) is freeze-dried and pressed to obtain a product.

2. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 1, characterized in that: The preparation of the self-assembled collagen fiber gel described in step (1) specifically includes the following steps: dissolving collagen in a PBS solution containing sodium chloride, adjusting the pH to neutral using a sodium hydroxide solution, and then incubating for 3-4 hours.

3. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 2, characterized in that: The collagen is fish skin collagen, cow skin collagen or pig skin collagen.

4. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 1, characterized in that: The chemical structure of the dicarboxylate cross-linking agent in step (2) is shown in formula (I): （Ⅰ） Where R is C1-C 10 of the alkyl chain.

5. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 4, characterized in that: The preparation method of the dicarboxylate crosslinker comprises the following steps: adding N-hydroxysulfosuccinimide and dicarboxylic acid to acetone, mixing, adding a dehydrating agent, reacting for 4-8 hours, filtering, collecting the precipitate, washing, and drying to obtain the crosslinker; wherein the mass ratio of the N-hydroxysulfosuccinimide to the dicarboxylic acid is 1:0.1-10; and the mass ratio of the dehydrating agent to the N-hydroxysulfosuccinimide is 1:0.1-20.

6. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 5, characterized in that: The dicarboxylic acid is adipic acid, suberic acid or glutaric acid; and the dehydrating agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

7. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 1, characterized in that: The preparation method of the water-soluble metal organic polyhedron in step (3) comprises the following steps: adding dichlorozirconocene and amino-p-dibenzoic acid to a mixed solution of water and dimethylacetamide for reaction for 2-6 hours, filtering and drying to obtain the water-soluble metal organic polyhedron; wherein the mass ratio of the dichlorozirconocene to the amino-p-dibenzoic acid is 1:0.1-10; and the volume ratio of the water to the dimethylacetamide is 1:1-20.

8. The collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 1, characterized in that: The mass ratio of the collagen to the methacrylic anhydride solution in step (4) is 1:0.01-0.5; the collagen is fish skin collagen, cow skin collagen or pig skin collagen.

9. Use of the collagen-based hard-core sandwich composite membrane for guided bone regeneration according to claim 1 in the preparation of oral bone defect repair materials.

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