Boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel, preparation method and application thereof

Abstract

The application provides a boron-nitrogen-carbon nanosheet reinforced protein composite hydrogel, a preparation method and application thereof, and the protein composite hydrogel comprises the following raw material components: bovine serum albumin, boron-nitrogen-carbon nanosheets and a coupling agent, the coupling agent is configured to trigger the bovine serum albumin to be chemically cross-linked by itself to form a cross-linked network, and trigger the bovine serum albumin and the boron-nitrogen-carbon nanosheets to be chemically cross-linked to form a cross-linked network. The boron-nitrogen-carbon nanosheet reinforced protein composite hydrogel has good mechanical properties and biological activity, and can repair bone defects, especially large defects of load-bearing bones. The preparation method of the boron-nitrogen-carbon nanosheet reinforced protein composite hydrogel is simple and safe, only a small amount of coupling agent is introduced, the chemical bond cross-linking between proteins can be triggered, and the prepared hydrogel improves the defects of insufficient mechanical strength and insufficient biological activity of traditional hydrogels.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogels, their preparation methods, and applications. Background Technology

[0002] The treatment of large bone defects remains a major challenge in orthopedic clinical practice. Currently widely used autologous and allogeneic bone grafting methods have several drawbacks, including limited donor sites, susceptibility to infection at the donor site, and potential risks of disease transmission. Artificial bone repair materials are considered promising solutions to overcome these shortcomings of traditional transplantation methods. Hydrogels, due to their excellent biocompatibility, biodegradability, and ability to mimic the extracellular matrix microenvironment, have become an important area of ​​research in tissue regeneration materials, offering a potential solution for large bone defects. However, traditional medical hydrogels still face some challenges in clinical applications, such as insufficient mechanical strength, inability to meet repair mechanical requirements, and insufficient osteoinductive activity, affecting their effectiveness in osseointegration. Therefore, developing a biomedical hydrogel with excellent mechanical strength and good bioactivity has significant scientific and clinical value. Summary of the Invention

[0003] The present invention aims to provide a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel. This boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel is a safe and effective mechanically enhanced biomedical active hydrogel with excellent mechanical properties and bioactivity, capable of meeting the needs of bone tissue repair and regeneration. It solves the problems of insufficient mechanical strength, inability to meet repair mechanical requirements, and insufficient osteoinductive activity of existing medical hydrogels. Furthermore, the present invention provides a method for preparing this boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel and its applications.

[0004] The first invention provides a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, which comprises the following raw material components: bovine serum albumin (BSA), boron-nitrogen-carbon (BCN) nanosheets, and a coupling agent. The coupling agent is configured to trigger chemical cross-linking of the boron serum albumin itself to form a cross-linked network, and to trigger chemical cross-linking between the boron serum albumin and the boron-nitrogen-carbon nanosheets to form a cross-linked network.

[0005] In one embodiment, the mass ratio of bovine serum albumin, boron-nitrogen-carbon nanosheets and coupling agent is 500:(0.1-0.8):(20-30).

[0006] In one embodiment, the gelation time of the protein composite hydrogel is 50-180 s.

[0007] In one embodiment, the protein composite hydrogel has a porous structure, and the boron-nitrogen-carbon nanosheets are uniformly dispersed within the porous internal structure of the protein composite hydrogel.

[0008] In one embodiment, the coupling agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0009] A second aspect of this invention provides a method for preparing the above-mentioned boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, comprising the following steps:

[0010] S1. Prepare boron-nitrogen-carbon nanosheet dispersion and prepare coupling agent solution;

[0011] S2. Add bovine serum albumin powder to the boron-nitrogen-carbon nanosheet dispersion to obtain a bovine serum albumin solution;

[0012] S3. After the bovine serum albumin solution has settled and no more foam has formed, add the coupling agent solution and stir until homogeneous to obtain a mixed solution.

[0013] S4. Add the mixed solution into a polytetrafluoroethylene mold and let it stand to spontaneously form a gel, thus obtaining the protein composite hydrogel.

[0014] In one embodiment, the coupling agent solution comprises a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution.

[0015] In one embodiment, in step S1: the concentration range of the prepared boron-nitrogen-carbon nanosheet dispersion is 0.2-1.0 mg / ml, and the dispersant is deionized water;

[0016] The concentration range of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1.0-1.5 g / ml, and the solvent is deionized water;

[0017] The concentration range of the N-hydroxysuccinimide solution is 1.0-1.5 g / ml, and the solvent is deionized water;

[0018] In step S2, the concentration of bovine serum albumin in the bovine serum albumin solution ranges from 250 to 500 mg / ml.

[0019] In one embodiment, in step S2, the boron-nitrogen-carbon nanosheet dispersion is diluted with deionized water to 0.1-0.8 mg / ml, and then bovine serum albumin powder is added to obtain a bovine serum albumin solution.

[0020] In one embodiment, the volume ratio of bovine serum albumin solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, and N-hydroxysuccinimide solution is 100:(2-3):(2-3).

[0021] The third aspect of this invention provides the application of the above-mentioned boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel in the preparation of bone defect repair materials.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. The boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel provided by the present invention comprises boron-nitrogen-carbon nanosheets, bovine serum albumin (BSA), and a coupling agent. Bovine serum albumin has excellent bioactivity and degradability, and can promote osteogenic differentiation and bone regeneration of bone marrow mesenchymal stem cells. Under the triggering of the coupling agent, BSA protein undergoes an amidation reaction of its own amino and carboxyl groups to achieve chemical cross-linking. After boron-nitrogen-carbon nanosheets, as an inorganic active nanomaterial, are introduced into the BSA solution, the carboxyl groups on the nanosheets undergo an amidation reaction with the amino groups of BSA to achieve the construction of a double network of the composite hydrogel.

[0024] 2. The boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel provided by the present invention has good mechanical properties and bioactivity, and can play a role in repairing bone defects, especially large defects in weight-bearing bones.

[0025] 3. The method for preparing boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogels provided by this invention is simple and safe. It introduces only a small amount of coupling agent, which can trigger cross-linking of chemical bonds between proteins. The prepared hydrogel overcomes the shortcomings of traditional hydrogels, such as insufficient mechanical strength and inadequate bioactivity. When used to prepare bone defect materials, the high strength of the protein hydrogel prepared by this invention can prevent collapse and rapid degradation. Furthermore, the inorganic active boron-nitrogen-carbon nanosheets and BSA protein components can synergistically promote the repair and regeneration of bone defects. Attached Figure Description

[0026] Figure 1 This is a transmission electron microscope image of boron-nitrogen-carbon nanosheets in the boron-nitrogen-carbon dispersion prepared in Example 2;

[0027] Figure 2 The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of boron-nitrogen-carbon nanosheets in the boron-nitrogen-carbon dispersion prepared in Example 2.

[0028] Figure 3 Optical photographs and scanning electron microscope (SEM) images of the 0.2 BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example;

[0029] Figure 4 The rheological curves are those of the 0.2 BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example.

[0030] Figure 5These are the compressive strength curves of the BCN@BSA hydrogels prepared in Examples 1-4 and the BSA hydrogels prepared in the comparative examples;

[0031] Figure 6 Cell proliferation results of BCN@BSA hydrogels prepared in Examples 1-4 and BSA hydrogels prepared in comparative examples;

[0032] Figure 7 The cell alkaline phosphatase (ALP) expression results of BCN@BSA hydrogels prepared in Examples 1-4 and BSA hydrogels prepared in the comparative examples are shown in the figure.

[0033] Figure 8 The expression results of osteogenic differentiation of BCN@BSA hydrogel prepared in Example 2 and BSA hydrogel prepared in the comparative example;

[0034] Figure 9 Micro-CT results of BCN@BSA hydrogel prepared in Example 2 and BSA hydrogel prepared in the comparative example after implantation into a rat skull defect model for 8 weeks. Detailed Implementation

[0035] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0036] In this application, unless otherwise stated, the use of "or" means "and / or". In the case of multiple dependent claims, "or" is used only in alternatives to refer to more than one of the aforementioned independent or dependent claims.

[0037] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, and related fields.

[0038] To address the problems of insufficient mechanical strength in existing biomedical hydrogels to meet the mechanical strength requirements for defect repair and insufficient osteoinductive activity hindering bone integration, this invention proposes a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel. This hydrogel comprises bovine serum albumin (BSA), boron-nitrogen-carbon nanosheets, and a coupling agent. Under the triggering of the coupling agent, the amino and carboxyl groups of the BSA protein undergo amidation, achieving chemical cross-linking. The boron-nitrogen-carbon nanosheets, as an inorganic active nanomaterial, are introduced into the BSA solution, where the carboxyl groups on the nanosheets undergo amidation with the amino groups of the BSA, thus constructing a dual-network structure for the composite hydrogel. When used to prepare bone defect materials, the high strength of the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel prepared by this invention can prevent collapse and rapid degradation. Furthermore, the inorganic active boron-nitrogen-carbon nanosheets and the BSA protein components can synergistically promote the repair and regeneration of bone defects.

[0039] In one example of preparing boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogels, boron-nitrogen-carbon (BCN) nanosheets were dispersed in deionized water, with the concentration of the BCN dispersion adjusted to 0.2-1.0 mg / ml. BSA powder was dissolved in the BCN dispersion to obtain a BCN@BSA protein composite solution. In the BCN@BSA protein composite solution, the concentration of BSA protein ranged from 250-500 mg / ml, and the content of BCN nanosheets was set to 0.1-0.8 mg / ml. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solutions were prepared, with EDC and NHS concentrations set to 1.0-1.5 g / ml. The EDC and NHS solutions were added separately to the BSA solution and stirred until homogeneous. The volume ratio of BSA, EDC, and NHS solutions was set to 100:(2-3):(2-3), and the stirring time was set to 5-20 s. The protein solution was then added to a polytetrafluoroethylene mold and allowed to stand for 3 minutes to obtain a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel. Adjusting these parameters, including BCN nanosheet content, BSA protein concentration, and the volumes of EDC and NHS solutions, allows for the regulation of the mechanical properties and osteogenic activity of the protein composite hydrogel of this invention.

[0040] In this invention, two coupling agents, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), are used in combination to promote the formation of amide bonds in protein hydrogels.

[0041] The BCN@BSA hydrogel in the following examples refers to the boron-nitrogen-carbon nanosheet-reinforced protein hydrogel of the present invention.

[0042] The present invention will be further illustrated below through specific embodiments.

[0043] Example 1

[0044] This embodiment provides a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, which is prepared by the following method:

[0045] (1) The boron nitrogen carbon powder was ultrasonically dispersed in deionized water to obtain a boron nitrogen carbon dispersion with a concentration of 1.0 mg / ml;

[0046] (2) EDC powder and NHS powder were dissolved in deionized water to obtain EDC solution with a concentration of 1g / ml and NHS solution with a concentration of 1g / ml, respectively.

[0047] (3) Mix 0.3 ml of boron nitrogen carbon dispersion with 2.7 ml of deionized water, then add 1.5 g of BSA powder and stir until the protein is completely dissolved to obtain a protein complex solution (BCN@BSA) with a BSA concentration of 500 mg / ml and a BCN content of 0.1 mg / ml.

[0048] (4) Add 30 μl of EDC solution and 30 μl of NHS solution to the protein complex solution respectively, and stir rapidly for 10 s;

[0049] (5) Add the protein composite solution into the polytetrafluoroethylene mold and let it stand for 3 min to obtain 0.1 BCN@BSA hydrogel;

[0050] (6) The protein composite hydrogel reinforced with boron, nitrogen and carbon nanosheets was characterized and its performance was evaluated.

[0051] Example 2

[0052] This embodiment provides a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, which is prepared by the following method:

[0053] (1) The boron nitrogen carbon powder was ultrasonically dispersed in deionized water to obtain a boron nitrogen carbon dispersion with a concentration of 1.0 mg / ml;

[0054] (2) EDC powder and NHS powder were dissolved in deionized water to obtain EDC solution with a concentration of 1g / ml and NHS solution with a concentration of 1g / ml, respectively.

[0055] (3) Mix 0.6 ml of boron nitrogen carbon dispersion with 2.4 ml of deionized water, then add 1.5 g of BSA powder and stir until the protein is completely dissolved to obtain a protein complex solution (BCN@BSA) with a BSA concentration of 500 mg / ml and a BCN content of 0.2 mg / ml.

[0056] (4) Add 30 μl of EDC solution and 30 μl of NHS solution to the protein complex solution respectively, and stir rapidly for 10 s;

[0057] (5) Add the protein composite solution into the polytetrafluoroethylene mold and let it stand for 3 min to obtain 0.2 BCN@BSA hydrogel;

[0058] (6) The protein composite hydrogel reinforced with boron, nitrogen and carbon nanosheets was characterized and its performance was evaluated.

[0059] Example 3

[0060] This embodiment provides a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, which is prepared by the following method:

[0061] (1) The boron nitrogen carbon powder was ultrasonically dispersed in deionized water to obtain a boron nitrogen carbon dispersion with a concentration of 1.0 mg / ml;

[0062] (2) EDC powder and NHS powder were dissolved in deionized water to obtain EDC solution with a concentration of 1g / ml and NHS solution with a concentration of 1g / ml, respectively.

[0063] (3) Mix 1.2 ml of boron nitrogen carbon dispersion with 1.8 ml of deionized water, then add 1.5 g of BSA powder and stir until the protein is completely dissolved to obtain a protein complex solution (BCN@BSA) with a BSA concentration of 500 mg / ml and a BCN content of 0.4 mg / ml.

[0064] (4) Add 30 μl of EDC solution and 30 μl of NHS solution to the protein complex solution respectively, and stir rapidly for 10 s;

[0065] (5) Add the protein composite solution into the polytetrafluoroethylene mold and let it stand for 3 min to obtain 0.4 BCN@BSA hydrogel;

[0066] (6) The protein composite hydrogel reinforced with boron, nitrogen and carbon nanosheets was characterized and its performance was evaluated.

[0067] Example 4

[0068] This embodiment provides a boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, which is prepared by the following method:

[0069] (1) The boron nitrogen carbon powder was ultrasonically dispersed in deionized water to obtain a boron nitrogen carbon dispersion with a concentration of 1.0 mg / ml;

[0070] (2) EDC powder and NHS powder were dissolved in deionized water to obtain EDC solution with a concentration of 1g / ml and NHS solution with a concentration of 1g / ml, respectively.

[0071] (3) Mix 2.4 ml of boron nitrogen carbon dispersion with 0.6 ml of deionized water, then add 1.5 g of BSA powder and stir until the protein is completely dissolved to obtain a protein complex solution (BCN@BSA) with a BSA concentration of 500 mg / ml and a BCN content of 0.8 mg / ml.

[0072] (4) Add 30 μl of EDC solution and 30 μl of NHS solution to the protein complex solution respectively, and stir rapidly for 10 s;

[0073] (5) Add the protein composite solution into the polytetrafluoroethylene mold and let it stand for 3 min to obtain 0.8 BCN@BSA hydrogel;

[0074] (6) The protein composite hydrogel reinforced with boron, nitrogen and carbon nanosheets was characterized and its performance was evaluated.

[0075] Comparative Example

[0076] This comparative example provides a BSA protein hydrogel prepared using the following method: BSA powder was dissolved in deionized water, with a BSA protein concentration ranging from 500 mg / ml. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) solutions were prepared, with EDC and NHS concentrations set to 1.0 g / ml. The EDC and NHS solutions were added separately to the BSA solution and stirred until homogeneous. The volume ratio of BSA, EDC, and NHS solutions was set to 100:3:3, and the stirring time was set to 10 s. The protein solution was then poured into a polytetrafluoroethylene mold and allowed to stand for 3 min to obtain the BSA protein hydrogel.

[0077] Experimental Example

[0078] This experiment characterizes and evaluates the performance of the 0.1BCN@BSA hydrogels, 0.2BCN@BSA hydrogels, 0.4BCN@BSA hydrogels, 0.8BCN@BSA hydrogels, and the BSA protein hydrogels obtained in the comparative examples prepared in Examples 1-4 above.

[0079] 1. Characterization of protein-composite hydrogels

[0080] See Figure 1 This is a transmission electron microscope (TEM) image of boron-nitrogen-carbon nanosheets in the boron-nitrogen-carbon dispersion prepared in Example 2. Figure 1 It is known that the boron-nitrogen-carbon nanosheets in this invention have an ultrathin nanostructure;

[0081] See Figure 2 The image shown is an X-ray photoelectron spectrum (XPS) of boron-nitrogen-carbon nanosheets in the boron-nitrogen-carbon dispersion prepared in Example 2. Figure 2 It is known that the boron-nitrogen-carbon nanosheets in this invention are carbon materials co-doped with boron and nitrogen.

[0082] See Figure 3 These are optical photographs and scanning electron microscope (SEM) images of the 0.2 BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example. Figure 3 As can be seen, the protein hydrogel in this invention has a porous structure inside, with boron, nitrogen, and carbon nanosheets uniformly dispersed inside, which makes the hydrogel appear black.

[0083] See Figure 4 The figures show the rheological curves of the 0.2BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example (the rheological curves of the 0.2BCN@BSA hydrogel and the BSA hydrogel were obtained on a rheometer). Figure 4 It is known that the introduction of boron-nitrogen-carbon nanosheets can accelerate the gelation time of protein composite hydrogels, which can reach 1 minute.

[0084] 1.2 Mechanical property testing of protein composite hydrogels

[0085] The mechanical strength of the hydrogel was tested using a mechanical testing machine. The mechanical properties of the protein-polymer hydrogel were controlled by altering the boron, nitrogen, and carbon content in the hydrogel.

[0086] See Figure 5 The figures show the compressive strength curves of the protein composite hydrogels prepared in Examples 1-4 and the BSA protein hydrogels prepared in the comparative examples. Figure 5 It can be seen that the 0.1BCN@BSA hydrogels, 0.2BCN@BSA hydrogels, 0.4BCN@BSA hydrogels, and 0.8BCN@BSA hydrogels prepared in Examples 1-4 all have good compressive strength. Furthermore, the protein composite hydrogels exhibit the best mechanical properties when the BCN content is 0.2 and 0.4 mg / ml, indicating that the introduction of boron, nitrogen, and carbon can enhance the mechanical properties of the hydrogels.

[0087] 1.3 In vitro osteogenic differentiation capacity test of protein composite hydrogel

[0088] First, the effect of protein-composite hydrogels on the proliferative capacity of bone marrow mesenchymal stem cells was examined. Stem cells were cultured in culture dishes to passage 3 and reach 80% confluence. They were then digested with trypsin and subjected to a reaction at 8 × 10⁻⁶ ppm. 3Cells were seeded at appropriate densities on BSA, 0.1 BCN@BSA, 0.2 BCN@BSA, 0.4 BCN@BSA, and 0.8 BCN@BSA hydrogels in 48-well plates, with the BSA hydrogel group used as a control. Cell proliferation was assessed using the CCK-8 assay at 1, 3, and 7 days of culture on the hydrogels.

[0089] Secondly, the effect of protein-composite hydrogels on the expression of alkaline phosphatase activity in bone marrow mesenchymal stem cells was examined. Stem cells were cultured in culture dishes to passage 3 and reach 80% confluence. They were then digested with trypsin and subjected to a 2×10⁻⁶ hydrogel. 4 Cells were seeded at appropriate densities on BSA, 0.1 BCN@BSA, 0.2 BCN@BSA, 0.4 BCN@BSA, and 0.8 BCN@BSA hydrogels in 24-well plates, with the BSA hydrogel group prepared in the comparative proportion serving as the control group. Alkaline phosphatase expression (ALP) was used to detect ALP activity in cells after 4 and 10 days of culture.

[0090] Based on the results of the hydrogel's detection of cell proliferation and ALP activity expression, compared with the BSA hydrogel prepared in the comparative example and the protein composite hydrogels prepared in Examples 1, 3, and 4, the protein composite hydrogel prepared in Example 2 showed a more significant promoting effect on cell proliferation and ALP activity expression. Furthermore, the effect of the protein composite hydrogel on the expression capacity of osteogenic genes in bone marrow mesenchymal stem cells was investigated. When stem cells were cultured to passage 3 and reached 80% confluence in culture dishes, they were digested with trypsin and subjected to a 10×10⁻⁶ solution. 4 Cells were seeded at appropriate densities on BSA and 0.2 BCN@BSA hydrogels in 6-well plates, with the BSA hydrogel group prepared in comparison serving as the control group. The osteogenic differentiation capacity of the cells after 7 days of culture was detected using qPCR.

[0091] See Figure 6 The results show the cell proliferation of the protein composite hydrogels prepared in Examples 1-4 and the BSA protein hydrogel prepared in the comparative example. The results show that, compared with the BSA protein hydrogel, the cells have a more significant proliferation ability when cultured on the BCN@BSA protein composite hydrogel for 1, 3 and 7 days.

[0092] See Figure 7 The results show the expression of alkaline phosphatase (ALP) in cells using the protein composite hydrogels prepared in Examples 1-4 and the BSA protein hydrogels prepared in the comparative example. The results indicate that BCN@BSA protein composite hydrogels can significantly promote ALP activity expression when cells are cultured for 4 and 10 days.

[0093] See Figure 8The results show the expression of osteogenic differentiation of cells in the protein composite hydrogel prepared in Example 2 and the BSA protein hydrogel prepared in the comparative example. The results show that BCN@BSA protein composite hydrogel can significantly promote the expression of osteogenic genes (BMP2, OCN, OPN and Runx2) after 7 days of cell seeding.

[0094] 1.4 In vivo bone defect repair capacity test of protein composite hydrogel

[0095] A skull defect model was constructed in SD rats, and two types of protein hydrogels were implanted into the defects. Rats without protein hydrogel implantation served as a blank control group. Eight weeks post-surgery, samples were harvested from the rats and scanned using a Micro-CT scanner to observe the bone defect repair process. (See also...) Figure 9 The figures show the Micro-CT results of 8 weeks after implantation of 0.2 BCN@BSA protein composite hydrogel prepared in Example 2 and BSA hydrogel prepared in the comparative example into a rat skull defect model. As can be seen from the figures, a small amount of new bone appeared at the defect site in the BSA protein hydrogel group, while a large amount of new bone grew both around and inside the defect in the BCN@BSA protein composite hydrogel group. This indicates that the BCN@BSA protein composite hydrogel has a more significant effect on promoting bone regeneration.

[0096] The above description discloses only preferred embodiments of the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to make good use of the invention. The present invention is limited only by the claims and their full scope and equivalents.

[0097] Under the guidance of the present invention and the above embodiments, those skilled in the art will readily foresee that all the raw materials or their equivalents, processing methods or their equivalents listed or exemplified in the present invention can achieve the present invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve the present invention. Examples are not listed one by one here.

Claims

1. A boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel, characterized in that, The boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel comprises the following raw material components: bovine serum albumin, boron-nitrogen-carbon nanosheets, and a coupling agent. The coupling agent is configured to trigger the bovine serum albumin to undergo chemical cross-linking to form a cross-linked network, and to trigger the bovine serum albumin to undergo chemical cross-linking with the boron-nitrogen-carbon nanosheets to form a cross-linked network. In this process, bovine serum albumin undergoes amidation of its amino and carboxyl groups under the triggering of a coupling agent, thereby achieving chemical cross-linking. The carboxyl groups on boron nitrogen carbon nanosheets undergo amidation of the amino groups on bovine serum albumin to achieve the construction of a dual-network composite hydrogel. The mass ratio of bovine serum albumin, boron-nitrogen-carbon nanosheets, and coupling agent is 500:(0.1-0.8):(20-30); The gelation time of the protein composite hydrogel is 50-180s; The protein composite hydrogel has a porous structure, and the boron-nitrogen-carbon nanosheets are uniformly dispersed in the porous internal structure of the protein composite hydrogel. The coupling agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

2. A method for preparing the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 1, characterized in that, Includes the following steps: S1. Prepare boron-nitrogen-carbon nanosheet dispersion and prepare coupling agent solution; S2. Add bovine serum albumin powder to the boron-nitrogen-carbon nanosheet dispersion to obtain a bovine serum albumin solution; S3. After the bovine serum albumin solution has settled and no foam has formed, add the coupling agent solution and stir until homogeneous to obtain a mixed solution. S4. Add the mixed solution into a polytetrafluoroethylene mold and let it stand to spontaneously form a gel, thus obtaining the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

3. The method for preparing the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 2, characterized in that, The coupling agent solution includes a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution.

4. The method for preparing the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 3, characterized in that, In step S1: the concentration range of the prepared boron-nitrogen-carbon nanosheet dispersion is 0.2-1.0 mg / ml, and the dispersant is deionized water; The concentration range of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1.0-1.5 g / ml, and the solvent is deionized water; The concentration range of the N-hydroxysuccinimide solution is 1.0-1.5 g / ml, and the solvent is deionized water; In step S2, the concentration of bovine serum albumin in the bovine serum albumin solution ranges from 250 to 500 mg / ml.

5. The method for preparing the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 4, characterized in that, In step S2, the boron-nitrogen-carbon nanosheet dispersion is diluted with deionized water to 0.1-0.8 mg / ml, and then bovine serum albumin powder is added to obtain a bovine serum albumin solution.

6. The method for preparing the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 5, characterized in that, The volume ratio of bovine serum albumin solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, and N-hydroxysuccinimide solution was 100:(2-3):(2-3).

7. The application of the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to claim 1 in the preparation of bone defect repair materials.

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