Enzymatic polypeptide-based hydrogel and preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to an enzymatic polypeptide-based hydrogel as well as a preparation method and application thereof. The enzymatic polypeptide-based hydrogel comprises gelatin, bioactive polypeptides and TGase enzyme, and the gelatin and the bioactive polypeptides are connected through the TGase enzyme; the amino acid sequence of the bioactive polypeptide is Q x GRGDS, wherein x is an integer selected from 1-5. The hydrogel improves the mechanical strength and the bioactivity through a physical-chemical double network structure, eliminates the interference of metal ions, realizes the directional differentiation regulation of osteoblasts, and effectively promotes the proliferation and mineralization of the osteoblasts. The enzymatic polypeptide hydrogel has the characteristics of easy synthesis and control, good biocompatibility and low biological toxicity, and has great application prospect in bone tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an enzyme-catalyzed polypeptide-based hydrogel, its preparation method, and its application. Background Technology

[0002] Bone defect repair has always been a significant challenge in clinical orthopedics. Traditional treatments for bone defect repair include autologous bone grafting and allogeneic bone grafting, but both have significant limitations: autologous grafting can lead to donor site complications such as bleeding and infection; while allogeneic grafting faces a risk of immune rejection as high as 30%, accompanied by potential disease transmission risks. In recent years, bioactive materials have shown promising applications in bone defect repair. However, existing bioactive materials still suffer from insufficient biocompatibility and difficulties in effectively coordinating mechanical properties with biological functions in practical applications. For example, commonly used metal and ceramic implants are prone to causing foreign body reactions in the body, hindering the bone integration process; and byproducts generated during the degradation of certain polymers may inhibit the activity of surrounding osteoblasts, further affecting new bone formation; materials such as polyetheretherketone (PEEK) have strong surface inertness and lack osteogenic bioactivity. Materials with excellent bio-inducible activity (such as collagen-based hydrogels) have weak mechanical properties, with their storage modulus generally below 10 kPa, making it difficult to provide sufficient structural support in load-bearing areas and severely limiting their clinical applications.

[0003] Peptide hydrogels have become a research hotspot in bone tissue engineering due to their excellent biocompatibility and extracellular matrix-like properties. For example, the Fmoc-FF / Fmoc-R physically crosslinked hydrogel system has a storage modulus of up to 6 kPa and promotes cell adhesion and activity to some extent. Alginate-peptide hybrid hydrogel systems rely on ionic chemical crosslinking; however, the introduced metal ions may interfere with the cellular microenvironment, particularly adversely affecting osteoblast mineralization signaling pathways. Although the above approaches have improved the mechanical properties and biological functions of hydrogels to some extent, they generally lack the ability to dynamically respond to the complex enzymatic microenvironment during bone repair. Summary of the Invention

[0004] The purpose of this invention is to provide an enzyme-catalyzed polypeptide-based hydrogel, its preparation method, and its application, thereby overcoming the shortcomings of the prior art and developing a bone repair material with good biocompatibility, suitable mechanical strength, and high osteogenic activity. This material has great application prospects in bone defect repair.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an enzyme-catalyzed polypeptide-based hydrogel, comprising gelatin, a bioactive polypeptide, and a TGase enzyme, wherein the gelatin and the bioactive polypeptide are linked by the TGase enzyme. The amino acid sequence of the bioactive polypeptide is Q. x GRGDS, where x is an integer selected from 1 to 5.

[0006] Q is the single-letter code for glutamine (Fmoc-Gln-OH), G is the single-letter code for glycine (Fmoc-Gly-OH), R is the single-letter code for arginine, D is the single-letter code for aspartic acid, and S is the single-letter code for serine (Fmoc-L-Ser(Trt)-OH). x can be any integer from 1, 2, 3, 4, or 5.

[0007] Gelatin forms the basis of the physical cross-linked network that gives rise to hydrogels, containing a large amount of TGase substrates (-NH2). Bioactive polypeptide Q x The glutamine (Gln, Q) residues in GRGDS can serve as a specific substrate for microbial transglutaminase (TGase) to undergo enzymatic cross-linking reactions. GRGDS is a classic cell adhesion peptide sequence that can be specifically recognized by integrin receptors on the cell surface, thereby promoting cell adhesion, spreading, proliferation, and differentiation. Microbial transglutaminase (TGase) catalyzes the formation of a covalent isopeptide bond between the γ-amide group of the glutamine (Q) residue and gelatin -NH2. In this system, one end of the TGase acts on the Q sequence of the bioactive peptide, and the other end acts on the lysine residue in the gelatin, thereby achieving a combination of "physical cross-linking" and "chemical cross-linking" and integrating "biological activity".

[0008] In some other embodiments, both the gelatin and the bioactive polypeptide contain TGase reaction sites; The amino acid sequence of the bioactive polypeptide is Q2GRGDS.

[0009] This specific amino acid sequence of gelatin and bioactive peptides can synergistically enhance mechanical strength (storage modulus ≥1 kPa) and bioactivity through a physical-chemical dual network structure, eliminate metal ion interference, and achieve targeted differentiation regulation of osteoblasts.

[0010] In some other embodiments, the concentration ratio of the gelatin to the bioactive polypeptide is (32-100):2.81; and the concentration of the TGase enzyme is 8.0-15.0 U / mL.

[0011] By regulating the concentrations of gelatin, bioactive peptides, and TGase enzymes, the peptide-based hydrogel aims to regulate cell behavior such as pre-osteoblast differentiation, and promotes cell differentiation by adjusting the mechanical strength and bioactive sites in the hydrogel.

[0012] In a second aspect, the present invention provides a method for preparing the enzyme-catalyzed polypeptide-based hydrogel described in the first aspect, comprising the following steps: Gelatin was dissolved in Tris-HCl buffer solution, and the mixture was stirred, heated, and allowed to stand to obtain a gelatin solution. The bioactive peptide solution and the gelatin solution were mixed, and TGase enzyme solution and Ca2+ were added. 2+ Enzyme-catalyzed polypeptide-based hydrogels are obtained by incubation with a solution.

[0013] This preparation method offers low-cost hydrogel synthesis, and the bioactive peptides are dissolved in a buffer solution with a pH close to physiological pH. The incubated compound hydrogels exhibit more thorough mixing and stability.

[0014] In some other embodiments, the heating temperature is 40-50°C, the stirring time is 20-60 min, and the settling time is 24-72 h.

[0015] Specifically, the heating treatment temperature is 40, 45 or 50°C, the stirring time is 20, 30, 40, 50 and 60 min, and the standing time is 24, 30, 36, 40, 48, 50, 60 or 72 h.

[0016] Preferably, the heating temperature is 45°C, the stirring time is 40 min, and the settling time is 48 h.

[0017] In some other embodiments, the concentration of the gelatin solution is 32-100 mg / mL, the concentration of the bioactive peptide solution is 2.81 mg / mL, and the mixing volume ratio of the gelatin solution and the bioactive peptide solution is 1:(1-2); the solution is a Tris-HCl buffer solution.

[0018] Specifically, the concentration of the gelatin solution is 32, 64, 80, or 100 mg / mL, the concentration of the bioactive peptide solution is 2.81 mg / mL, and the volume ratio of the gelatin solution to the bioactive peptide solution is 1:1, 1:1.5, or 1:2.

[0019] In some other embodiments, the concentration of the TGase enzyme solution is 8.0-15.0 U / mL, and the Ca... 2+ The concentration of the dependent solution is 0.8-0.12 mM; The incubation temperature is 35-40℃, and the time is 18-48h.

[0020] The TGase enzyme solution comprises dithiothreitol (DTT), TGase, and Tris-HCl buffer solution, wherein the mass ratio of DTT to TGase is (2-4)×10⁻⁶. -3 The concentration of the TGase enzyme solution is 10.0-15.0 U / mL.

[0021] Specifically, the concentration of the TGase enzyme solution is 10, 11, 12, 13, 14 or 15 U / mL, the incubation temperature is 35, 37, 38 or 40℃, and the incubation time is 18, 24, 30, 36, 40 or 48 h.

[0022] Preferably, the concentration of the TGase enzyme solution is 10.0 U / mL, the incubation temperature is 37°C, and the incubation time is 24 h.

[0023] Thirdly, the present invention provides the application of the enzyme-catalyzed polypeptide-based hydrogel described in the first aspect in the repair of bone defects.

[0024] In some other embodiments, the bone defect repair involves osteoblast mineralization, proliferation, and differentiation.

[0025] Fourthly, the present invention provides a bone defect repair material, comprising the enzyme-catalyzed polypeptide-based hydrogel described in the first aspect.

[0026] The beneficial effects of this invention are: (1) This invention addresses the three major bottlenecks in existing biomaterials for bone defect repair: biocompatibility defects, mechanical-biological functional decoupling, and lack of dynamic response capability. By constructing an enzyme-specifically triggered all-bio-derived polypeptide hydrogel system, the following breakthroughs have been achieved: obtaining a polypeptide hydrogel with good biocompatibility and eliminating interference from metal ions and synthetic polymer degradation products; achieving synergy between mechanical and biological activity: the innovative design of a physical-chemical dual-network cross-linking structure can precisely control the biological activity and mechanical strength of the hydrogel.

[0027] (2) The bioactive enzyme-catalyzed polypeptide-based hydrogel of the present invention can promote cell adhesion, proliferation, migration and differentiation, and plays a vital role in the regeneration of bone tissue. At the same time, the TGase enzyme solution provides the hydrogel with sufficient mechanical strength to match the natural hard tissue, and overcomes the shortcomings of traditional extracellular matrix scaffolds with single, uneven composition and few bioactive sites. A polypeptide-based hydrogel with specific mechanical strength and bioactive sites is prepared. By regulating the assembly unit, the polypeptide-based hydrogel is endowed with viscoelastic properties similar to those of real tissue.

[0028] (3) This invention achieves the purpose of regulating cell behavior such as pre-osteoblast differentiation by adjusting the bioactive sequence and concentration in the hydrogel, and promotes cell differentiation by adjusting the mechanical strength and bioactive sites in the hydrogel. Attached Figure Description

[0029] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 Transmission electron microscopy (TEM) images of Q2GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) in Example 1 of this invention without the addition of TGase. Figure 2 Transmission electron microscopy (TEM) images of Q2GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) after the addition of TGase in Example 1 of this invention; Figure 3 This is a comparison of the mechanical strength of Q2GRGDS (2.81 mg / mL) and gelatin of different concentrations (32, 64, 80, 100 mg / mL) after the addition of TGase in Example 1 of this invention. Figure 4 This is a graph showing the mineralization capacity of Q2GRGDS (2.81 mg / mL) and different concentrations of gelatin (32, 64, 80, 100 mg / mL) on MC3T3-E1 cells after the addition of TGase in Example 1 of this invention. Figure 5 This is a graph showing the cytotoxicity of Q2GRGDS (2.81 mg / mL) and different concentrations of gelatin (32, 64, 80, 100 mg / mL) on MC3T3-E1 cells after the addition of TGase in Example 1 of this invention. Figure 6 Frequency scans of Q3GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) after the addition of TGase. Figure 7 The graph shows the cytotoxicity of Q3GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) on MC3T3-E1 cells after the addition of TGase. Detailed Implementation

[0031] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products; parts not described can be achieved by referring to existing technologies.

[0032] This invention addresses the limitations of existing peptide hydrogels in precisely controlling cell-selective adhesion behavior in time and space, and in programmatically releasing pro-mineralization active factors according to the repair process. Therefore, there is an urgent need to develop an enzyme-specifically triggered hydrogel system to overcome the technical bottleneck of precise material-cell signal modulation.

[0033] The main raw materials and experimental equipment required for this invention are shown in Tables 1 and 2.

[0034] Table 1 List of Experimental Instruments

[0035] Table 2 List of reagents and consumables used in the experiment

[0036] The amino acid sequence of the polypeptide in this invention is Q. 1-5 GRGDS.

[0037] The preparation method of enzyme-catalyzed polypeptide-based hydrogels is as follows: 1. Peptide molecule Q 1-5 Preparation of GRGDS (1) Synthesis: The peptide was synthesized using a solid-phase synthesis method. The resin was swollen in a DCM for 30 min, and then the swollen resin was transferred to a microwave reactor. The target amino acid sequence of the peptide molecule was Q. 1-5 GRGDS involves sequentially adding amino acids for a reaction. The first amino acid is attached to the resin via a dehydration condensation reaction. Then, the carboxyl group of the second amino acid is activated by an activator. The activated carboxyl group undergoes a dehydration condensation reaction with the amino group of the first amino acid already attached to the resin to form a peptide bond. This process continues until the target sequence is synthesized, resulting in a target polypeptide sequence attached to the resin. (2) Cleavage: The target polypeptide sequence synthesized in step (1) with resin was stirred at room temperature for 4 h to cleave the target polypeptide sequence from the solid resin. The cleavage agent was a mixture of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5. Then, the cleaved polypeptide solution was collected and the excess liquid in the bottle was removed by rotary evaporator to obtain the polypeptide solid. (3) Purification: The target peptide was precipitated with ice-cold ether and then centrifuged with ice-cold ether in a centrifuge at 8000 rpm and 4℃ for 10 min until the pH of the supernatant of the peptide was neutral, thus obtaining the target peptide. (4) Freeze-drying: Dissolve the target peptide in ultrapure water, then place it in a -80℃ freezer overnight, and finally freeze-dry it in a freeze dryer for 60 h to obtain the peptide product Q. 1-5 GRGDS polypeptide powder.

[0038] 2. Preparation of enzyme-catalyzed peptide-based hydrogels Q 1-5 GRGDS peptide powder was dissolved in Tris-HCl buffer to prepare Q 1-5 GRGDS peptide solution. Add 0.62 mg DTT and 0.2 g TGase to 2 mL of Tris-HCl buffer solution and dissolve thoroughly to obtain TGase stock solution.

[0039] Gelatin was slowly dissolved in Tris-HCl buffer solution, and the resulting solution was obtained by heating and stirring.

[0040] Q 1-5 GRGDS and gelatin solution of equal volume were mixed, and TGase enzyme stock solution and Ca were added. 2+ The TGase enzyme concentration was adjusted to 10.0 U / mL, and the mixture was incubated at 37°C for 24 h to obtain the enzymatically catalyzed polypeptide-based hydrogel.

[0041] Example 1 An enzymatically catalyzed polypeptide-based hydrogel prepared from polypeptide molecules Q2GRGDS and gelatin solution includes the following steps: 1. Preparation of Q2GRGDS for polypeptide molecules (1) Synthesis: The peptide was synthesized using solid-phase synthesis. The resin was swollen in DCM for 30 min, and then the swollen resin was transferred to a reactor. Amino acids were added in the order of Q2GRGDS, the target amino acid sequence of the peptide molecule. The first amino acid was attached to the resin through a dehydration condensation reaction. Then, the carboxyl group of the second amino acid was activated by activating agents (HBTU and HOBt). The activated carboxyl group and the amino group of the first amino acid attached to the resin underwent a dehydration condensation reaction to form a peptide bond until the target sequence was synthesized, and the target peptide sequence with resin was obtained. (2) Cleavage: The target polypeptide sequence synthesized in step (1) with resin was stirred at room temperature for 4 h using about 5 mL of cleavage agent (composed of 95% TFA, 2.5% ultrapure water and 2.5% triisopropylsilane) to cleave the target polypeptide sequence from the solid resin; then, the cleaved polypeptide solution was collected and the excess liquid in the bottle was removed by rotary evaporator to obtain the polypeptide solid. (3) Purification: The target peptide was precipitated with ice-cold ether and then centrifuged with ice-cold ether in a centrifuge at 8000 rpm and 4℃ for 10 min until the pH of the supernatant of the peptide was neutral, thus obtaining the target peptide. (4) Freeze-drying: Dissolve the target peptide in ultrapure water, then place it in a -80℃ freezer overnight, and finally freeze-dry it in a freeze dryer for 60 h to obtain the peptide product, namely Q2GRGDS peptide powder.

[0042] 2. Preparation of enzyme-catalyzed peptide-based hydrogels Q2GRGDS peptide powder was dissolved in Tris-HCl buffer to prepare solutions with concentrations ranging from 0.45 to 3.67 mM. Gelatin solids were dissolved in Tris-HCl buffer, and gelatin solutions were prepared by heating and stirring to obtain concentrations of 32, 64, 80, and 100 mg / mL. 0.62 mg of DTT and 0.2 g of TGase were added to 2 mL of Tris-HCl buffer and dissolved thoroughly to obtain a TGase stock solution.

[0043] Mix Q2GRGDS with gelatin in equal volume, add 45 μL of TGase enzyme stock solution and Ca 2+ The TGase enzyme concentration was adjusted to 10.0 U / mL, and the mixture was incubated at 37°C for 24 h to obtain the enzymatically catalyzed polypeptide-based hydrogel.

[0044] Performance testing 1. Morphological and structural characterization of enzyme-catalyzed peptide-based hydrogels The prepared enzyme-catalyzed peptide-based hydrogel was characterized by TEM morphology: The hydrogel sample was measured using a JEOL JEM-200UHR electron microscope with an accelerating voltage of 120 kV. The specific steps were as follows: 20 μL of the enzyme-catalyzed peptide-based hydrogel was dropped onto a clean 400-mesh copper grid. After the copper grid dried, it was negatively stained with 2% (w / v) uranium acetate for 6 min. Excess staining solution was then absorbed from the edge of the copper grid with filter paper. After drying, TEM imaging was performed, and the measurement results are shown below. Figure 1 and Figure 2 As shown.

[0045] Figure 1Transmission electron microscopy (TEM) images of Q2GRGDS and gelatin solutions without the addition of TGase (scale bar: 500 nm). Figure 2 Transmission electron microscopy (TEM) image of the sample after adding TGase to Q2GRGDS and gelatin solution (scale bar: 200 nm). From Figure 1 and Figure 2 As can be seen, the mixture of peptide Q2GRGDS and gelatin solution exhibits a nanofiber structure. Upon addition of TGase enzyme, fine fibers are generated.

[0046] 2. Determination of the strength of enzyme-catalyzed peptide-based hydrogels Rheological characterization of enzyme-catalyzed peptide-based hydrogels: The mechanical properties of the enzyme-catalyzed hydrogels were tested using a Haake rheometer. A Haake rheometer with a diameter of 35 mm, a taper of 2°, a test temperature of 25℃, and a test slit size of 0.105 nm was used for frequency and stress scanning. First, a stress scan was performed at a frequency of 1 Hz, with a stress scan range of 0.01%–100%. The linear viscoelastic region of the sample was determined by the stress scan. Then, a different sample was used for frequency scanning. The test results are shown below. Figure 3 As shown. Figure 3 This is a comparison of the mechanical strength of Q2GRGDS and gelatin samples at different concentrations (32, 64, 80, 100 mg / mL) after the addition of TGase. Figure 3 It was found that the addition of Q2GRGDS to the gelatin solution did not form an inverted, supportable hydrogel; however, the addition of TGase resulted in the formation of a hydrogel with a mechanical strength of 1.2 kPa (Q2GRGDS: 2.81 mg / mL, gelatin: 64 mg / mL). This demonstrates that the addition of TGase can induce a cross-linking reaction between the gelatin solution and Q2GRGDS, significantly improving the strength of the hydrogel.

[0047] 3. Effects of enzyme-catalyzed polypeptide-based hydrogels on osteoblast mineralization The ability of the hydrogel to mineralize osteoblasts was investigated using a quantitative alizarin red staining assay: Matrix mineralization was determined using alizarin red staining. After culturing in osteogenic induction medium for 21 days, cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. The cells were then washed twice with PBS. Next, in 96-well plates, the prepared cells were stained with 200 μL of 2% (w / v) alizarin red S (ARS) solution (pH 4.3) at 37 °C for approximately 1 hour. After washing twice with PBS, the cells were observed under a fluorescence microscope.

[0048] To quantitatively determine the yield of matrix precipitate, 200 μL of 10% (w / v) hexadecylpyridine chloride monohydrate solution (2 g of hexadecylpyridine chloride monohydrate dissolved in 20 mL of PB solution, with a final pH of 7.0) was added to a 96-well plate, and cells were pre-seeded on the peptide hydrogel. After incubation at 37 °C for 1 hour, the supernatant was collected, and the absorbance was measured at 570 nm using an M2 e microplate reader (M2 e). The experimental results are as follows: Figure 4 As shown.

[0049] Figure 4 This image shows the mineralization capacity of Q2GRGDS (2.81 mg / mL) and different concentrations of gelatin (32, 64, 80, 100 mg / mL) on MC3T3-E1 cells after the addition of TGase. Figure 4 It is known that enzymatic hydrogels can significantly enhance the mineralization ability of osteoblasts.

[0050] 4. Effects of enzyme-catalyzed peptide-based hydrogels on cell growth Cytotoxicity of hydrogels: The toxicity of peptide-based hydrogels and the morphology of cells in hydrogels were detected using the Calcein / PI live / dead staining assay.

[0051] Calcein-AM powder was first dissolved in DMSO to prepare a stock solution, then diluted with PBS to 1 mg / mL and stored at -20°C in the dark. PI solution was diluted with ultrapure water to 1 mg / mL and stored at -20°C. Gels were seeded into 96-well plates and incubated at 37°C for 24 h. Cells were then seeded onto the gel surface and cultured in a cell culture incubator for 24 h. The culture medium from both the experimental and control groups was aspirated, and the gel and cells were washed with PBS. Then, 200 μL of Calcein-AM / PI dye solution diluted with PBS was added, resulting in final concentrations of 1 μg / mL for Calcein-AM and 5 μg / mL for PI solution. The 96-well plates were then incubated in a cell culture incubator for 30 min. Afterward, the plates were removed, washed with PBS, and the results were observed and recorded using a fluorescence inverted microscope. The results are shown below. Figure 5 As shown. By Figure 5 As can be seen, the enzymatic hydrogel images show only green fluorescent live cells and no red fluorescent dead cells, indicating that the hydrogel has low cytotoxicity and can support the proliferation and growth of osteoblasts, thus exhibiting good cell compatibility.

[0052] Example 2 Unlike Example 1, the enzyme-catalyzed polypeptide-based hydrogel prepared by QGRGDS and gelatin solution was prepared using the same method as in Example 1.

[0053] Example 3 Unlike Example 1, the enzyme-catalyzed polypeptide-based hydrogel prepared from Q3GRGDS and gelatin solution was prepared using the same method as in Example 1.

[0054] Figure 6 The image shows frequency scans of Q3GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) after the addition of TGase. As can be seen from the image, the addition of TGase can induce a cross-linking reaction between gelatin and Q3GRGDS, significantly improving the strength of the hydrogel.

[0055] Figure 7 This image shows the cytotoxicity of Q3GRGDS (2.81 mg / mL) and gelatin solution (64 mg / mL) on MC3T3-E1 cells after the addition of TGase. Figure 7 As can be seen, the enzymatic hydrogel images show only green fluorescent live cells and no red fluorescent dead cells, indicating that the hydrogel has low cytotoxicity.

[0056] Table 3 Comparison of bioactivity and mechanical strength properties

[0057] As shown in Table 3, the number of Qs increases the mechanical strength and bioactivity of the hydrogel. However, when the number of Qs is increased to 3, there is no significant change in the mechanical strength and bioactivity of the hydrogel. This may be related to the reaction efficiency between the two.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enzyme-catalyzed polypeptide-based hydrogel, characterized in that, It includes bioactive peptides, gelatin, and TGase enzyme, wherein the bioactive peptides and gelatin are linked by TGase enzyme; The amino acid sequence of the bioactive polypeptide is Q. x GRGDS, where x is an integer selected from 1 to 5.

2. The enzyme-catalyzed polypeptide-based hydrogel according to claim 1, characterized in that, Both the gelatin and the bioactive polypeptide contain TGase reaction sites; The amino acid sequence of the bioactive polypeptide is Q2GRGDS.

3. The enzyme-catalyzed polypeptide-based hydrogel according to claim 1, characterized in that, The concentration ratio of gelatin to bioactive polypeptide is 32-100:2.81; the concentration of TGase enzyme is 8-15.0 U / mL.

4. A method for preparing an enzyme-catalyzed polypeptide-based hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: Gelatin was slowly dissolved in Tris-HCl buffer solution, and the solution was prepared by stirring, heating and standing. Mix the gelatin solution and the bioactive peptide solution, then add the TGase enzyme solution and Ca. 2+ Enzyme-catalyzed polypeptide-based hydrogels are obtained by incubation with a solution.

5. The method for preparing the enzyme-catalyzed polypeptide-based hydrogel according to claim 4, characterized in that, The heating treatment temperature is 40-50℃, the stirring time is 20-60 min, and the standing time is 24-72 h.

6. The method for preparing the enzyme-catalyzed polypeptide-based hydrogel according to claim 4, characterized in that, The concentration of the gelatin solution is 32-100 mg / mL, the concentration of the bioactive peptide solution is 2.81 mg / mL, and the mixing volume ratio of the bioactive peptide solution and gelatin is 1:(1-2); the bioactive peptide solution is a Tris-HCl buffer solution of the bioactive peptide.

7. The method for preparing the enzyme-catalyzed polypeptide-based hydrogel according to claim 4, characterized in that, The concentration of the TGase enzyme solution is 8.0-15.0 U / mL, and the Ca 2+ The concentration of the dependent solution is 0.8-1.2 mM; The incubation temperature is 35-40℃, and the time is 18-48h.

8. The application of an enzyme-catalyzed polypeptide-based hydrogel according to any one of claims 1-3 in bone defect repair.

9. The application according to claim 8, characterized in that, The bone defect repair involves osteoblast proliferation, mineralization, and differentiation.

10. A bone defect repair material, characterized in that, Including the enzymatic polypeptide-based hydrogel according to any one of claims 1-3.

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