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

Abstract

The invention provides a boron-nitrogen-carbon nanosheet enhanced protein composite hydrogel as well as a preparation method and application thereof, and the protein composite hydrogel is prepared from the following raw material components: bovine serum albumin, boron-nitrogen-carbon nanosheets and a coupling agent, the coupling agent is configured to trigger chemical crosslinking of the bovine serum albumin to form a crosslinked network and trigger chemical crosslinking between the bovine serum albumin and the boron-nitrogen-carbon nanosheets to form a crosslinked network. The boron-nitrogen-carbon nanosheet enhanced protein composite hydrogel provided by the invention has good mechanical properties and biological activity, and can play a role in repairing bone defects at bone defect parts, especially bearing bone large defect parts. The preparation method of the boron-nitrogen-carbon nanosheet enhanced protein composite hydrogel provided by the invention is simple and safe, only a small amount of coupling agent is introduced, chemical bond crosslinking between proteins can be triggered, and the prepared hydrogel overcomes the defects of insufficient mechanical strength and insufficient biological activity of traditional hydrogel.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel, a preparation method and an application thereof. Background Art

[0002] The treatment of large bone defects remains a major problem in orthopedic clinical practice. The currently widely used autologous bone transplantation and allogeneic bone transplantation methods have many defects, including limited donors, susceptibility to infection in the donor site, and potential risks of disease transmission. Artificial bone repair materials are considered to be promising to overcome the shortcomings of these traditional transplantation methods. Hydrogels have become an important field of tissue regeneration material research due to their excellent biocompatibility, degradability, and ability to simulate the microenvironment of the extracellular matrix, providing a potential solution to large bone defects. However, traditional medical hydrogels still face some challenges in clinical applications, such as insufficient mechanical strength, failure to meet the mechanical requirements of repair, and insufficient osteoinduction activity, which affect their effect in bone integration. Therefore, the development of a biomedical hydrogel with excellent mechanical strength and good biological activity has important scientific value and clinical significance. Summary of the invention

[0003] The purpose of the present invention is to provide a boron nitrogen carbon nanosheet reinforced protein composite hydrogel, which is a safe and effective mechanically enhanced biomedical active hydrogel, has excellent mechanical properties and biological activity, can meet the repair and regeneration needs of bone tissue, and solves the problems of insufficient mechanical strength of existing medical hydrogels, inability to meet the repair mechanical requirements, and insufficient bone induction activity. In addition, the present invention provides a preparation method and application of the boron nitrogen carbon nanosheet reinforced protein composite hydrogel.

[0004] The first invention of the present invention provides a boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel, which includes the following raw material components: bovine serum albumin (BSA), boron-nitrogen-carbon (BCN) nanosheets and a coupling agent, wherein the coupling agent is configured to trigger the bovine serum albumin to chemically cross-link itself to form a cross-linked network, and to trigger the bovine serum albumin and the boron-nitrogen-carbon nanosheets to chemically cross-link 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 in the porous internal structure of the protein composite hydrogel.

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

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

[0010] S1, preparing a boron nitrogen carbon nanosheet dispersion and a coupling agent solution;

[0011] S2, adding 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 is allowed to stand without foam, add the coupling agent solution and stir evenly to obtain a mixed solution;

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

[0014] In one embodiment, the coupling agent solution includes 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 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1.0-1.5 g / ml, and the solvent is deionized water;

[0017] The concentration range of 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 is in the range of 250-500 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 the bovine serum albumin solution, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, and the N-hydroxysuccinimide solution is 100:(2-3):(2-3).

[0021] The third aspect of the present invention provides the use 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 present invention has the following beneficial effects:

[0023] 1. The boron nitrogen carbon nanosheet-enhanced 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 biological activity and degradability, and can promote the osteogenic differentiation and bone regeneration of bone marrow mesenchymal stem cells. Under the triggering of the coupling agent, the amino and carboxyl groups of the BSA protein undergo an amidation reaction to achieve chemical cross-linking. After the boron nitrogen carbon nanosheet is introduced into the BSA solution as an inorganic active nanomaterial, the carboxyl groups on the nanosheets undergo an amidation reaction with the BSA amino groups to achieve the double network construction of the composite hydrogel.

[0024] 2. The boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel provided by the present invention has good mechanical properties and biological activity, and can repair bone defects, especially large defects in load-bearing bones.

[0025] 3. The method for preparing the protein composite hydrogel enhanced by boron nitrogen carbon nanosheets provided by the present invention is simple and safe, and only a small amount of coupling agent is introduced, which can trigger the chemical bond cross-linking between proteins. The prepared hydrogel improves the defects of insufficient mechanical strength and insufficient biological activity of traditional hydrogels. When used to prepare bone defect materials, the high strength of the protein hydrogel prepared by the present invention can avoid the problems of collapse and rapid degradation, and the inorganic active boron nitrogen carbon nanosheets and BSA protein components can synergistically promote the repair and regeneration of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] Figure 2 is an X-ray photoelectron spectrum (XPS) of the boron nitrogen carbon nanosheets in the boron nitrogen carbon dispersion prepared in Example 2;

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

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

[0030] Figure 51 is the compression strength curve of the BCN@BSA hydrogel prepared in Examples 1-4 and the BSA hydrogel prepared in the comparative example;

[0031] Figure 6 Figure 2 shows the cell proliferation results of the BCN@BSA hydrogels prepared in Examples 1-4 and the BSA hydrogels prepared in the comparative example;

[0032] Figure 7 Cell alkaline phosphatase (ALP activity) expression results of BCN@BSA hydrogels prepared in Examples 1-4 and BSA hydrogels prepared in the comparative example;

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

[0034] Fig. 9 Micro-CT results of the BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example after implantation in the rat skull defect model for 8 weeks. DETAILED DESCRIPTION

[0035] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0036] In this application, the use of "or" means "and / or" unless otherwise stated. In the case of multiple dependent claims, use of "or" refers to more than one of the preceding independent or dependent claims only in the alternative.

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

[0038] In order to solve the problems that the mechanical strength of existing biomedical hydrogels is poor and insufficient to meet the mechanical strength requirements for defect repair, and the bone induction activity is insufficient and not conducive to bone integration, the present invention proposes a boron nitrogen carbon nanosheet reinforced protein composite hydrogel, the boron nitrogen carbon nanosheet reinforced protein composite hydrogel comprises bovine serum albumin, boron nitrogen carbon nanosheets and a coupling agent, under the triggering of the coupling agent, the amino group and the carboxyl group of the BSA protein undergo an amidation reaction to achieve chemical cross-linking, after the boron nitrogen carbon nanosheet as an inorganic active nanomaterial is introduced into the BSA solution, the carboxyl group on the nanosheet and the BSA amino group undergo an amidation reaction to achieve the double network construction of 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 the present invention can avoid the problems of collapse and rapid degradation, and the inorganic active boron nitrogen carbon nanosheet and the BSA protein component can synergistically promote the repair and regeneration of bone defects.

[0039] In an example of preparing a boron nitrogen carbon nanosheet-enhanced protein composite hydrogel, boron nitrogen carbon (BCN) nanosheets are dispersed in deionized water, and the concentration range of the BCN dispersion can be adjusted to 0.2-1.0 mg / ml. BSA powder is dissolved in the BCN dispersion to obtain a BCN@BSA protein composite solution. In the BCN@BSA protein composite solution, the concentration range of BSA protein is 250-500 mg / ml, and the content of BCN nanosheets is set to 0.1-0.8 mg / ml. 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution are prepared, and the concentrations of EDC and NHS are set to 1.0-1.5 g / ml. The EDC solution and the NHS solution are added to the BSA solution respectively and stirred evenly, and the volume ratio of the BSA solution, the EDC solution and the NHS solution is set to 100:(2-3):(2-3), and the stirring time is set to 5-20s. Then the protein solution was added to the polytetrafluoroethylene mold and allowed to stand for 3 minutes to obtain a boron nitrogen carbon nanosheet reinforced protein composite hydrogel. The mechanical properties and osteogenic activity of the protein composite hydrogel of the present invention can be regulated by adjusting these parameters including BCN nanosheet content, BSA protein concentration, EDC and NHS solution volume.

[0040] In the present 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 in the present invention.

[0042] The present invention is further described below through specific examples.

[0043] Example 1

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

[0045] (1) ultrasonically dispersing boron nitrogen carbon powder 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 1 g / ml and NHS solution with a concentration of 1 g / ml, respectively;

[0047] (3) 0.3 ml of the boron nitrogen carbon dispersion was mixed with 2.7 ml of deionized water, and then 1.5 g of BSA powder was added and stirred until the protein was 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 and stir rapidly for 10 s;

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

[0050] (6) Characterize and evaluate the performance of the obtained boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

[0051] Example 2

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

[0053] (1) ultrasonically dispersing boron nitrogen carbon powder 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 1 g / ml and NHS solution with a concentration of 1 g / ml, respectively;

[0055] (3) 0.6 ml of the boron nitrogen carbon dispersion was mixed with 2.4 ml of deionized water, and then 1.5 g of BSA powder was added and stirred until the protein was 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 and stir rapidly for 10 s;

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

[0058] (6) Characterize and evaluate the performance of the obtained boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

[0059] Example 3

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

[0061] (1) ultrasonically dispersing boron nitrogen carbon powder 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 1 g / ml and NHS solution with a concentration of 1 g / ml, respectively;

[0063] (3) 1.2 ml of the boron nitrogen carbon dispersion was mixed with 1.8 ml of deionized water, and then 1.5 g of BSA powder was added and stirred until the protein was 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 and stir rapidly for 10 s;

[0065] (5) The protein complex solution was added into the polytetrafluoroethylene mold and allowed to stand for 3 min to obtain 0.4BCN@BSA hydrogel;

[0066] (6) Characterize and evaluate the performance of the obtained boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

[0067] Example 4

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

[0069] (1) ultrasonically dispersing boron nitrogen carbon powder 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 1 g / ml and NHS solution with a concentration of 1 g / ml, respectively;

[0071] (3) 2.4 ml of the boron nitrogen carbon dispersion was mixed with 0.6 ml of deionized water, and then 1.5 g of BSA powder was added and stirred until the protein was completely dissolved to obtain a protein composite 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 and stir rapidly for 10 s;

[0073] (5) The protein complex solution was added into the polytetrafluoroethylene mold and allowed to stand for 3 min to obtain 0.8BCN@BSA hydrogel;

[0074] (6) Characterize and evaluate the performance of the obtained boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

[0075] Comparative Example

[0076] This comparative example provides a BSA protein hydrogel, which is prepared by the following method: BSA powder is used as a raw material and dissolved in deionized water, and the concentration range of BSA protein is 500mg / ml. 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution are prepared, and the concentrations of EDC and NHS are set to 1.0g / ml. EDC and NHS solutions are added to the BSA solution respectively and stirred evenly. The volume ratio of BSA solution, EDC solution and NHS solution is set to 100:3:3, and the stirring time is set to 10s. The protein solution is then added to a polytetrafluoroethylene mold and allowed to stand for 3min to obtain a BSA protein hydrogel.

[0077] Experimental example

[0078] In this experimental example, the 0.1BCN@BSA hydrogel, 0.2BCN@BSA hydrogel, 0.4BCN@BSA hydrogel, 0.8BCN@BSA hydrogel prepared in the above Examples 1-4, and the BSA protein hydrogel obtained in the comparative example were characterized and their performance was evaluated.

[0079] 1. Characterization of protein composite hydrogels

[0080] See also Figure 1 , is a transmission electron microscope image of the boron nitrogen carbon nanosheets in the boron nitrogen carbon dispersion prepared in Example 2, Figure 1 It can be seen that the boron nitrogen carbon nanosheets in the present invention have an ultrathin nanostructure;

[0081] See also Figure 2 , is the X-ray photoelectron spectrum (XPS) of the boron nitrogen carbon nanosheets in the boron nitrogen carbon dispersion prepared in Example 2, Figure 2 It can be seen that the boron-nitrogen-carbon nanosheet in the present invention is a carbon material co-doped with boron and nitrogen elements.

[0082] See also Figure 3 , are optical photographs and scanning electron microscope (SEM) images of the 0.2BCN@BSA hydrogel prepared in Example 2 and the BSA hydrogel prepared in the comparative example. Figure 3 It can be seen that the interior of the protein hydrogel in the present invention is a porous structure, the boron nitrogen carbon nanosheets are evenly dispersed inside it, and the hydrogel appears black.

[0083] See also Figure 4 , are 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 above 0.2BCN@BSA hydrogel and BSA hydrogel are obtained on a rheological tester). Figure 4 It can be seen that the introduction of boron nitrogen carbon nanosheets can accelerate the gelation time of protein composite hydrogel, and the gelation time can reach 1 minute.

[0084] 1.2 Mechanical properties test of protein composite hydrogel

[0085] The mechanical strength of the hydrogel was tested using a mechanical testing machine. The mechanical properties of the protein composite hydrogel were regulated by changing the content of boron, nitrogen and carbon in the protein composite hydrogel.

[0086] See also Figure 5 , are the compression strength curves of the protein composite hydrogels prepared in Examples 1-4 and the BSA protein hydrogel prepared in the comparative example. Figure 5 It can be seen that the 0.1BCN@BSA hydrogel, 0.2BCN@BSA hydrogel, 0.4BCN@BSA hydrogel, and 0.8BCN@BSA hydrogel prepared in Examples 1-4 all have good compressive strength, and when the BCN content is 0.2 and 0.4 mg / ml, the mechanical properties of the protein composite hydrogel are the best, indicating that the introduction of boron, nitrogen, and carbon can achieve mechanical enhancement of the hydrogel.

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

[0088] First, the effect of protein composite hydrogel on the proliferation of bone marrow mesenchymal stem cells was tested. When the stem cells were cultured in a culture dish to the third generation and the confluence reached 80%, they were digested with trypsin and 8×10 3The cells were inoculated at a density of 100 μg / mL on BSA, 0.1 BCN@BSA, 0.2 BCN@BSA, 0.4 BCN@BSA and 0.8 BCN@BSA hydrogels in a 48-well plate, and the BSA hydrogel group prepared in the comparative example was used as the control group. When the cells were cultured on the hydrogels for 1, 3 and 7 days, the cell proliferation ability was detected by the CCK-8 method.

[0089] Secondly, the effect of protein composite hydrogel on the expression of alkaline phosphatase activity of bone marrow mesenchymal stem cells was detected. When the stem cells were cultured in a culture dish to the third generation and the confluence reached 80%, they were digested with trypsin and 2×10 4 The cells were seeded at a density of 100 μg / mL on BSA, 0.1 BCN@BSA, 0.2 BCN@BSA, 0.4 BCN@BSA and 0.8 BCN@BSA hydrogels in a 24-well plate, and the BSA hydrogel group prepared in the comparative example was used as the control group. The alkaline phosphatase (ALP) expression was used to detect the ALP activity of the cells after 4 and 10 days of culture.

[0090] Combined with the test results of hydrogel on cell proliferation and ALP activity expression, compared with the BSA hydrogel prepared in the comparative example, and the protein composite hydrogel prepared in Example 1, Example 3 and Example 4, the protein composite hydrogel prepared in Example 2 has a more significant promoting effect on cell proliferation and ALP activity expression. On this basis, the effect of the protein composite hydrogel on the expression ability of bone marrow mesenchymal stem cells related to osteogenic genes was further tested. When the stem cells were cultured in a culture dish to the third generation and the degree of confluence reached 80%, they were digested with trypsin and 10×10 4 The cells were inoculated at a density of 100 μg / mL on BSA and 0.2 BCN@BSA hydrogels in a 6-well plate, and the BSA hydrogel group prepared in the comparative ratio was used as the control group. The qPCR method was used to detect the osteogenic differentiation ability of the cells after 7 days of culture.

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

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

[0093] See also Figure 8, are the expression results of cell osteogenic differentiation of the protein composite hydrogel prepared in Example 2 and the BSA protein hydrogel prepared in the comparative example. The results show that when cells are inoculated on the BCN@BSA protein composite hydrogel for 7 days, the expression of osteogenic genes (BMP2, OCN, OPN and Runx2) of the cells can be significantly promoted.

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

[0095] A defect model was constructed in the skull of SD rats, and two types of protein hydrogels were implanted into the defect, respectively. Rats without protein hydrogel implantation served as a blank control group. Samples were collected from the rats 8 weeks after surgery, and the samples were scanned using a Micro-CT device to observe the bone defect repair. Fig. 9 , are the Micro-CT results of 0.2BCN@BSA protein composite hydrogel prepared in Example 2 and BSA hydrogel prepared in the comparative example implanted into the rat skull defect model for 8 weeks. It can be seen from the figure that a small amount of new bone appeared at the defect site of the BSA protein hydrogel group, while a large amount of new bone grew around and inside the defect of the BCN@BSA protein composite hydrogel group. This shows that the BCN@BSA protein composite hydrogel has a more significant effect in promoting bone regeneration.

[0096] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present 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-mentioned embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel, characterized in that: The boron nitrogen carbon nanosheet-enhanced protein composite hydrogel comprises the following raw material components: bovine serum albumin, boron nitrogen carbon nanosheets and a coupling agent, wherein the coupling agent is configured to trigger the bovine serum albumin to chemically cross-link itself to form a cross-linked network, and to trigger the bovine serum albumin and the boron nitrogen carbon nanosheets to chemically cross-link to form a cross-linked network.

2. The boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 1, characterized in that: The mass ratio of the bovine serum albumin, the boron nitrogen carbon nanosheets and the coupling agent is 500:(0.1-0.8):(20-30).

3. The boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 2, characterized in that: The gelling time of the protein composite hydrogel is 50-180s.

4. The boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 2, characterized in that: 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.

5. The boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 1, characterized in that: The coupling agents include 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

6. A method for preparing the boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, preparing a boron nitrogen carbon nanosheet dispersion and a coupling agent solution; S2, adding 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 is allowed to stand without foam, add the coupling agent solution and stir evenly to obtain a mixed solution; S4. Add the mixed solution into a polytetrafluoroethylene mold and let it stand to form a gel spontaneously, thereby obtaining the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel.

7. The method for preparing the boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 6, characterized in that: The coupling agent solution includes a 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution.

8. The method for preparing the boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 7, 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 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1.0-1.5 g / ml, and the solvent is deionized water; The concentration range of 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 is in the range of 250-500 mg / ml.

9. The method for preparing the boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 8, 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.

10. The method for preparing the boron-nitrogen-carbon nanosheet-enhanced protein composite hydrogel according to claim 9, characterized in that: The volume ratio of the bovine serum albumin solution, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, and the N-hydroxysuccinimide solution is 100:(2-3):(2-3).

11. Use of the boron-nitrogen-carbon nanosheet-reinforced protein composite hydrogel according to any one of claims 1 to 5 in preparing bone defect repair materials.

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