Preparation method and application of side-branch modified Gelma hydrogel

Abstract

The present invention is applicable to the technical field of bone defect repair, and provides a preparation method and application of a collateral-modified Gelma hydrogel. The present invention performs collateral modification on the Gelma hydrogel by Fibrinogen, successfully improving the mechanical properties and cell adhesion of the hydrogel scaffold. At the same time, the loading of NELL‑1 protein significantly improves the scaffold's ability to repair bone defects. The Fibrinogen‑Gelma / NELL‑1 hydrogel scaffold constructed by the present invention has shown ideal mechanical properties in relevant characterizations, and has been shown to play a key role in the repair of bone tissue defects in in vitro and in vivo experiments, and has greatly promoted the expression of osteogenesis-related genes. Therefore, the present invention is expected to be applied to clinical treatments related to bone defect repair, such as maxillary sinus bone augmentation and guided bone regeneration (GBR), in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of bone defect repair, and in particular relates to a preparation method and application of a collateral-modified Gelma hydrogel. Background Art

[0002] Bone tissue engineering, as one of the primary approaches for reconstructing bone tissue and repairing bone defects, has led to the development of a variety of materials with excellent physical properties for promoting stem cell-mediated osteogenesis. Hydrogels, as an emerging scaffold for bone tissue engineering, possess physical properties similar to those of the extracellular matrix (ECM), effectively mimicking the biological microenvironment of damaged tissue, resulting in a high degree of biomimetic properties and excellent biocompatibility. Furthermore, hydrogels possess high water content and porosity, which ensure the smooth exchange of metabolic waste generated during bone defect repair, promoting rapid repair. Consequently, they are attracting increasing attention in bone tissue engineering, biopharmaceuticals, and clinical treatments. However, due to their simple structure, most single-network hydrogels often suffer from deficiencies such as insufficient mechanical properties, uncontrollable spatial structure, poor cell adhesion, and limited ability to promote bone regeneration. These limitations, to a certain extent, limit their widespread application as biomaterials. Therefore, developing hydrogels with ideal mechanical properties, controllable spatial structure, and strong cell adhesion has become a major challenge in bone tissue engineering.

[0003] In order to develop smart hydrogels with both good mechanical properties and strong cell adhesion, one of the strategies is to introduce growth factors, proteins, drugs or other biomolecules that are beneficial to bone repair by changing the structure of a single network hydrogel. The existing methods for changing the structure of a single network hydrogel include hybrid hydrogels produced by simple reversible molecular entanglement or physical bond connection, and hydrogels cross-linked by forming irreversible covalent bonds with the incorporated components. Compared with the reversible introduction method, the method of grafting functional groups into the hydrogel to introduce new functional groups to react and produce different cross-linking points can obtain stronger mechanical properties. When different functional groups or macromolecules penetrate each other to form a double-network double-cross-linked hydrogel with a complex spatial structure, the mechanical strength of the hydrogel can be controlled by adjusting the content of the grafted groups and macromolecules. In addition, this type of hydrogel can also greatly improve the adhesion of the hydrogel through covalent bonds, thereby improving cell adhesion and proliferation.

[0004] Methacrylamide-based gelatin Gelma is a photocrosslinked hydrogel material made by chemically modifying methacrylic anhydride and gelatin. Gelma hydrogel containing a water-soluble photoinitiator can produce crosslinks when exposed to 405nm ultraviolet (UV). The cross-linked hydrogel can maintain its stable physical form at body temperature and has excellent structural stability. Fibrinogen not only has excellent biocompatibility, good cell adhesion, degradability and low immunogenicity, but also has the ability to activate the Wnt / β-catenin osteogenic signaling pathway. In addition, by adjusting the degree and concentration of Gelma methacrylate and the content of introduced fibrinogen, the mechanical properties of the hydrogel scaffold can be controlled.

[0005] While ensuring that the hydrogel scaffold has ideal mechanical strength and biocompatibility, the ability to promote bone defect repair is also one of the key indicators for evaluating bone tissue engineering biomaterials. Nell-1 protein is a new secretory protein that promotes bone differentiation. It is preferentially expressed in neural crest-derived tissues and therefore has a specific effect on craniofacial tissue development and regeneration. Nell-1 protein has been shown to have excellent performance in promoting bone and cartilage regeneration, and can promote cranial suture closure, craniofacial bone and dental pulp regeneration. At the same time, Nell-1 protein also plays a key role in the osteogenic differentiation of various stem cells such as adipose-derived mesenchymal stem cells. It can activate the Wnt / β-catenin pathway, increase the nuclear accumulation of β-catenin, and then stimulate the regulation of Runx-2 transcription factors, thereby greatly promoting osteoblast differentiation and bone regeneration. To this end, we propose a method for preparing a branch-modified Gelma hydrogel and its application. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of a side-branch modified Gelma hydrogel and its application, aiming to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a side-branch modified Gelma hydrogel comprises the following steps:

[0009] Step 1: Take 5g of type A pigskin gelatin and 50ml of PBS, add 5g of type A pigskin gelatin to 50ml of PBS, place in a 50°C water bath and stir continuously for 1-2 hours until the type A pigskin gelatin is completely dissolved to obtain a gelma hydrogel precursor solution with a concentration of 10% w / v;

[0010] Step 2: Place the Gelma hydrogel precursor solution in a 50°C water bath and continue stirring. At the same time, add 8 ml of methacrylic anhydride dropwise to the Gelma hydrogel precursor solution and continue stirring for 3 hours.

[0011] Step 3: Add 300 ml of 40°C PBS solution to the Gelma hydrogel precursor solution. The reaction gradually stops with the addition of PBS.

[0012] Step 4: Transfer the Gelma hydrogel precursor solution after the reaction is stopped to a dialysis membrane, and dialyze the Gelma hydrogel precursor aqueous solution in distilled water at 40° C. for 2 weeks;

[0013] Step 5: Filter the dialyzed Gelma hydrogel precursor solution using a filter membrane;

[0014] Step 6: Centrifuge the filtered Gelma hydrogel precursor solution at room temperature;

[0015] Step 7: The supernatant obtained after centrifugation was aspirated and transferred to a 50 ml test tube, and then stored in a -80 ° C refrigerator for 0.5 h;

[0016] Step 8: The reaction product is taken out of the refrigerator and placed in a freeze dryer. After freeze drying for 1 week, a white porous foam-like Gelma hydrogel precursor is obtained;

[0017] Step 9. Weigh 100 mg of Gelma hydrogel precursor and 10 mg of fibrinogen and dissolve them in 500 μl of sterile NELL-1 protein solution containing 0.01% w / v. Mix the two precursor solutions thoroughly to obtain 1 ml of GFN hydrogel precursor solution with a Gelma concentration of 10% w / v and a fibrinogen concentration of 1% w / v. Add 1 mg of LAP photoinitiator and irradiate at 405 nm for 30 s for photopolymerization and crosslinking to obtain Fibrinogen-Gelma / NELL-1 hydrogel.

[0018] Furthermore, in the step 2, 8 ml of methacrylic anhydride was slowly and dropwise added to the Gelma hydrogel precursor solution at a rate of 0.5 ml / min.

[0019] Furthermore, in step 4, during the dialyzing of the hydrogel precursor aqueous solution in distilled water at 40° C. for 2 weeks, the distilled water was replaced every 8 hours in the first week and every half a day in the second week.

[0020] Furthermore, in step 4, the molecular weight cut-off of the dialysis membrane used for dialysis is 14000KD.

[0021] Furthermore, in step five, the pore diameter of the filter membrane is 0.22 μm.

[0022] Furthermore, in step six, the centrifugal speed is 6000 rpm and the centrifugal time is 5 min.

[0023] A method for preparing a side-branch modified Gelma hydrogel is provided. The prepared Fibrinogen side-branch modified Gelma hydrogel carrying NELL-1 protein is used in clinical treatment related to bone defect repair.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention achieves side branch modification of the hydrogel by forming CS covalent bonds between fibrinogen and gelma hydrogel, thereby significantly improving its mechanical strength. At the same time, the mechanical properties of the hydrogel can be controlled by regulating the amount of fibrinogen incorporated. In addition, the introduction of fibrinogen promotes cell adhesion on the surface of the hydrogel material to a certain extent, effectively avoiding the defect of poor cell adhesion on the hydrogel surface.

[0026] 2. The present invention loads NELL-1 protein into the Fibrinogen-Gelma hydrogel scaffold, and activates the key Wnt / β-catenin pathway in the osteogenesis repair process through the cooperation of NELL-1 protein and Fibrinogen, thereby greatly improving the ability of the Fibrinogen-Gelma scaffold to promote bone tissue regeneration.

[0027] 3. The Fibrinogen-Gelma / NELL-1 hydrogel scaffold constructed in this invention has demonstrated ideal mechanical properties in relevant characterizations. Furthermore, in vitro and in vivo experiments have demonstrated its key role in bone defect repair, significantly promoting the expression of osteogenesis-related genes. Therefore, this invention has the potential for future applications in clinical treatments related to bone defect repair, such as maxillary sinus bone augmentation and guided bone regeneration (GBR). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Flowchart of the present invention.

[0029] Figure 2 The present invention is a flow chart of bone defect repair.

[0030] Figure 3 (A) is a scanning electron micrograph of the synthesized Gelma hydrogel; (B) is a scanning electron micrograph of the Fibrinogen-Gelma hydrogel; (C) is a scanning electron micrograph of the Fibrinogen-Gelma / Nell-1 hydrogel.

[0031] Figure 4is the elastic modulus of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0032] Figure 5 is the compression modulus of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0033] Figure 6 Swelling curves of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0034] Figure 7 Degradation curves of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0035] Figure 8 Release curves of Gelma / Nell-1 (GN) and Fibrinogen-Gelma / NELL-1 (GFN).

[0036] Figure 9 Fourier transform infrared spectra of Gelma (G), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0037] Figure 10 These are the cell activity test results of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0038] Figure 11 These are the live / dead cell fluorescence staining results of Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN) after seven days of cell culture.

[0039] Figure 12(A) is the fluorescence staining of Col-1 protein expression after 7 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN); (B) is the quantitative results of the fluorescence intensity of Col-1 protein expression after 7 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0040] Figure 13 (A) is the fluorescence staining of OCN protein expression after 7 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN); (B) is the quantitative results of fluorescence intensity of OCN protein expression after 7 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0041] Figure 14 These are the quantitative results of the gene expression of osteogenic-related genes Runx-2, OPN, OCN, BMP-2, VEGF and COL-1 proteins after 7 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0042] Figure 15 These are the quantitative results of the gene expression of osteogenic-related genes Runx-2, OPN, OCN, BMP-2, VEGF and COL-1 proteins after 14 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0043] Figure 16(A) shows the alkaline phosphatase expression staining of each group after 14 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN); (B) shows the quantitative results of alkaline phosphatase expression staining of each group after 14 days of osteogenic induction by Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN).

[0044] Figure 17 Quantitative results of the gene expression of osteogenic-related genes Runx-2 (A), OPN (B), OCN (C), BMP-2 (D), COL-1 (E) and VEGF (F) proteins after Gelma (G), Gelma / Nell-1 (GN), Fibrinogen-Gelma (GF) and Fibrinogen-Gelma / NELL-1 (GFN) were implanted into rabbits for eight weeks.

[0045] Figure 18 Figure 1 shows the results of He and Masson staining of Gelma (G), Fibrinogen-Gelma (GF), and Fibrinogen-Gelma / NELL-1 (GFN) grafts implanted into the rabbit maxillary sinus 8 weeks after implantation. The images show augmented sinus membrane (M), new bone (NB), basic bone (BB), and connective tissue (CT). DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0048] An embodiment of the present invention provides a method for preparing a side-branch modified Gelma hydrogel, comprising the following steps:

[0049] Step 1: Accurately weigh 5 g of type A pigskin gelatin and 50 ml of phosphate-buffered saline (PBS). Place 5 g of type A pigskin gelatin in 50 ml of PBS and stir continuously in a 50°C water bath for 1-2 hours until the type A pigskin gelatin is completely dissolved to obtain a gelma hydrogel precursor solution with a concentration of 10% w / v.

[0050] Step 2: Place the Gelma hydrogel precursor solution in a 50°C water bath and continue stirring. At the same time, add 8 ml of methacrylic anhydride dropwise to the Gelma hydrogel precursor solution and continue stirring for 3 hours.

[0051] Step 3: Add 300 ml of 40°C PBS solution to the Gelma hydrogel precursor solution. The reaction gradually stops with the addition of PBS.

[0052] Step 4: Transfer the Gelma hydrogel precursor solution after the reaction is stopped to a dialysis membrane, and dialyze the Gelma hydrogel precursor aqueous solution in distilled water at 40° C. for 2 weeks;

[0053] Step 5: Filter the dialyzed Gelma hydrogel precursor solution using a filter membrane;

[0054] Step 6: Centrifuge the filtered Gelma hydrogel precursor solution at room temperature;

[0055] Step 7: The supernatant obtained after centrifugation was aspirated and transferred to a 50 ml test tube, and then stored in a -80 ° C refrigerator for 0.5 h;

[0056] Step 8: The reaction product is taken out of the refrigerator and placed in a freeze dryer. After freeze drying for 1 week, a white porous foam-like Gelma hydrogel precursor is obtained;

[0057] Step 9. Weigh 100 mg of Gelma hydrogel precursor and 10 mg of fibrinogen and dissolve them in 500 μl of sterile NELL-1 protein solution containing 0.01% w / v. Mix the two precursor solutions thoroughly to obtain 1 ml of GFN hydrogel precursor solution with a Gelma concentration of 10% w / v and a fibrinogen concentration of 1% w / v. Add 1 mg of LAP photoinitiator and irradiate at 405 nm for 30 s for photopolymerization and crosslinking to obtain Fibrinogen-Gelma / NELL-1 hydrogel.

[0058] As a preferred embodiment of the present invention, in the step 2, 8 ml of methacrylic anhydride is slowly and dropwise added to the Gelma hydrogel precursor solution at a rate of 0.5 ml / min.

[0059] As a preferred embodiment of the present invention, in step 4, the hydrogel precursor aqueous solution is dialyzed in distilled water at 40° C. for 2 weeks, with the distilled water replaced every 8 hours in the first week and every half a day in the second week to remove excess methacrylic anhydride, harmful substances, and other impurities.

[0060] As a preferred embodiment of the present invention, in step 4, the molecular weight cut-off of the dialysis membrane used for dialysis is 14000 KD.

[0061] As a preferred embodiment of the present invention, in step five, the pore diameter of the filter membrane is 0.22 um.

[0062] As a preferred embodiment of the present invention, in step six, the centrifugal speed is 6000 rpm and the centrifugal time is 5 min.

[0063] A method for preparing a side-branch modified Gelma hydrogel provided in one embodiment of the present invention provides a NELL-1 protein-loaded Fibrinogen side-branch modified Gelma hydrogel for use in clinical treatments related to bone defect repair.

[0064] like Figure 3 As shown in the figure, scanning electron microscopy results show that the pore size of the Fibrinogen-Gelma hydrogel is smaller and the porosity is higher than that of the Gelma hydrogel, which may be caused by the binding of Fibrinogen to the Gelma surface. Secondly, compared with the scanning electron microscopy image of the Fibrinogen-Gelma hydrogel, the scanning electron microscopy of the Fibrinogen-Gelma / NELL-1 hydrogel shows that its surface is scattered with small particle-like protrusions, indicating that the NELL-1 protein has successfully adhered to the surface of the Fibrinogen-Gelma hydrogel.

[0065] like Figure 4 As shown, the elastic modulus of the GFN group was significantly improved compared with the G, GN, and GF groups, indicating that the GFN group improved the mechanical strength of G to the greatest extent relative to the GN and GF groups.

[0066] like Figure 5 As shown in the figure, the compression modulus of the GFN group was significantly improved compared with the G, GN, and GF groups, indicating that the GFN group improved the mechanical strength of G to the greatest extent relative to the GN and GF groups.

[0067] like Figure 6 As shown, the swelling ratio of the GFN group was significantly reduced compared with the G, GN, and GF groups, indicating that the GFN group improved the stability of G to the greatest extent relative to the GN and GF groups.

[0068] like Figure 7As shown, compared with the G, GN, and GF groups, the GFN group had the highest degradation rate within the same period of time, indicating that the GFN group had the strongest degradation ability in vitro compared with the GN and GF groups, which to a certain extent ensured the biosafety of the scaffold.

[0069] like Figure 8 As shown, the GN group had a defect in the sudden release of NELL-1 protein, while the GFN group showed a sustained release effect. This feature ensures that the NELL-1 protein can better perform its function of promoting bone repair in vivo.

[0070] 1. In vitro experiments

[0071] like Figure 10 As shown, compared with the G, GN, and GF groups, the absorbance of the GFN group was the highest at 450 nm, indicating that the GFN group was most conducive to cell proliferation.

[0072] like Figure 11 As shown, compared with the G, GN, and GF groups, the GFN group had the most surviving cells, indicating that it had the least toxicity to cells.

[0073] like Figure 12 As shown in the figure, Col-1, as one of the key osteogenesis-related proteins, plays an important role in the bone repair process. As can be seen from the figure, a large amount of COL-1 in the GFN group is present in the cell nucleus. Compared with the G, GN and GF groups, the GFN group significantly promoted the expression of COL-1 protein.

[0074] like Figure 13 As shown in the figure, OCN protein, as one of the key osteogenesis-related proteins, plays an important role in the bone repair process. As can be seen from the figure, OCN in the GFN group is present in large quantities in the cell cytoplasm. Compared with the G, GN, and GF groups, the GFN group significantly promoted the expression of OCN protein.

[0075] like Figure 14 As shown in Figure 2, the gene expression of Runx-2, OPN, OCN, BMP-2, VEGF and COL-1 proteins determines the effect of bone regeneration to a certain extent. Figure 14 It can be seen that compared with the G, GN and GF groups, the GFN group significantly promoted the expression of Runx-2, OPN, OCN, BMP-2, VEGF and COL-1 proteins. In this regard, the GFN group showed the most significant effect in promoting osteogenesis.

[0076] like Figure 15 As shown in Figure 2, the gene expression of Runx-2, OPN, OCN, BMP-2, VEGF and COL-1 proteins determines the effect of bone regeneration to a certain extent. Figure 15As can be seen, the gene expression trends of Runx-2, OPN, OCN, BMP-2, VEGF, and COL-1 proteins after 14 days of osteogenesis induction were basically consistent with the gene expression trends after 7 days. Compared with G, GN, and GFN, the GFN group significantly promoted the expression of Runx-2, OPN, OCN, BMP-2, VEGF, and COL-1 proteins, showing the most significant effect in promoting osteogenesis.

[0077] like Figure 16 As shown in the results, ALP is a protein expressed in the early stage of osteogenesis. The GFN group showed better results than the G, GN and GF groups in both qualitative and quantitative alkaline phosphatase staining.

[0078] 2. In vivo experiments

[0079] like Figure 17 As shown in the results, the gene expression of Runx-2, OPN, OCN, BMP-2, COL-1, and VEGF proteins determines the effect of bone regeneration to a certain extent. Compared with G, GN, and GFN, the GFN group significantly promoted the expression of Runx-2, OPN, OCN, BMP-2, COL-1, and VEGF proteins after eight weeks of implantation in rabbits, demonstrating the most significant effect of promoting bone formation.

[0080] like Figure 18 As shown, the volume of new bone formation in the GFN group was the largest compared with the G and GF groups eight weeks after implantation.

[0081] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for preparing a side-branch modified Gelma hydrogel, characterized in that: The following steps are involved: Step 1: Take 5g of type A pigskin gelatin and 50ml of PBS, add 5g of type A pigskin gelatin to 50ml of PBS, place in a 50℃ water bath and stir continuously for 1-2h until the type A pigskin gelatin is completely dissolved to obtain a gelma hydrogel precursor solution with a concentration of 10% w / v; Step 2: Place the Gelma hydrogel precursor solution in a 50°C water bath and continue stirring. At the same time, add 8 ml of methacrylic anhydride dropwise to the Gelma hydrogel precursor solution and continue stirring for 3 hours. Step 3: Add 300 ml of 40°C PBS solution to the Gelma hydrogel precursor solution. The reaction gradually stops with the addition of PBS. Step 4: Transfer the Gelma hydrogel precursor solution after the reaction is stopped to a dialysis membrane, and dialyze the Gelma hydrogel precursor aqueous solution in distilled water at 40° C. for 2 weeks; Step 5: Filter the dialyzed Gelma hydrogel precursor solution using a filter membrane; Step 6: Centrifuge the filtered Gelma hydrogel precursor solution at room temperature; Step 7: The supernatant obtained after centrifugation was aspirated and transferred to a 50 ml test tube, and then stored in a -80 ° C refrigerator for 0.5 h; Step 8: The reaction product is taken out of the refrigerator and placed in a freeze dryer. After freeze drying for 1 week, a white porous foam-like Gelma hydrogel precursor is obtained; Step 9. Weigh 100 mg of Gelma hydrogel precursor and 10 mg of fibrinogen and dissolve them in 500 μl of sterile NELL-1 protein solution containing 0.01% w / v. Mix the two precursor solutions thoroughly to obtain 1 ml of GFN hydrogel precursor solution with a Gelma concentration of 10% w / v and a fibrinogen concentration of 1% w / v. Add 1 mg of LAP photoinitiator and irradiate at 405 nm for 30 s for photopolymerization and cross-linking to obtain Fibrinogen-Gelma / NELL-1 hydrogel.

2. The method for preparing the side-branch modified Gelma hydrogel according to claim 1, wherein: In the step 2, 8 ml of methacrylic anhydride was slowly and dropwise added to the Gelma hydrogel precursor solution at a rate of 0.5 ml / min.

3. The method for preparing the side-branch modified Gelma hydrogel according to claim 1, wherein: In the fourth step, the hydrogel precursor aqueous solution was dialyzed in distilled water at 40° C. for 2 weeks, with the distilled water being replaced every 8 hours in the first week and every half a day in the second week.

4. The method for preparing the side-branch modified Gelma hydrogel according to claim 3, wherein: In the step 4, the molecular weight cut-off of the dialysis membrane used for dialysis is 14000 KD.

5. The method for preparing the side-branch modified Gelma hydrogel according to claim 1, wherein: In the step 5, the pore diameter of the filter membrane is 0.22 μm.

6. The method for preparing the side-branch modified Gelma hydrogel according to claim 1, wherein: In step 6, the centrifugal speed is 6000 rpm and the centrifugal time is 5 min.

7. Use of a NELL-1 protein-loaded Fibrinogen-coated Gelma hydrogel prepared by the method for preparing a lateral-branch modified Gelma hydrogel according to any one of claims 1 to 6 in preparing a drug for promoting bone defect repair.

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