Preparation method and application of biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid

Abstract

The present invention provides a preparation method of a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid. The method comprises the following steps: reacting carboxymethyl-β-cyclodextrin (CM-β-CD), EDC·HCl, NHS, and VPM-pm26 TGF-β1 with gelatin to obtain β-CD-g-NHS covalently coupled to a polypeptide; reacting γ-PGA, EDC, NHS, adipic acid dihydrazide, and 1-adamantane acetic acid to obtain Ad-γ-PGA-ADH; and physically crosslinking the covalently coupled β-CD-g-NHS to Ad-γ-PGA-ADH to obtain a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid. The double-crosslinked hydrogel has excellent biocompatibility and biodegradability, and also possesses the characteristics of synthetic hydrogels, such as optimizable mechanical strength and easy modification.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method and application of a biomimetic double-crosslinked hydrogel based on gelatin and gamma-polyglutamic acid. Background Art

[0002] Cartilage is a specialized tissue lacking blood vessels, nerves, and lymphatic vessels. Cartilage degeneration caused by joint injury is currently one of the leading causes of disability. Currently, clinical treatments for cartilage injury primarily include allogeneic cartilage transplantation, autologous chondrocyte transplantation, and artificial joint replacement prostheses. However, most treatment options are invasive, terminal, and only address the symptoms of the damaged cartilage, not the underlying cause. Furthermore, the implants used can wear out over time, potentially leading to postoperative complications.

[0003] Hydrogels are a system composed of a three-dimensional network of hydrophilic polymers and water as a medium. Therefore, biomimetic hydrogels are applicable to the field of cartilage regeneration technology. Therefore, the biomimetic double-crosslinked hydrogel (β-CD-Gelatin-NHS / Ad-γ-PGA-ADH) based on gelatin and polyglutamic acid (γ-PGA) can effectively solve this problem due to its excellent mechanical and self-healing properties. Summary of the Invention

[0004] In view of the above-mentioned defects of the prior art, the object of the present invention is to provide a preparation method and application of a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid.

[0005] A method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid comprises the following steps:

[0006] S10. Preparation of gelatin intermediate:

[0007] S11, dissolving carboxymethyl-β-cyclodextrin (CM-β-CD) in pure water at room temperature and stirring to obtain a carboxymethyl-β-cyclodextrin solution;

[0008] S12, add EDC·HCl and NHS to the solution obtained in S11, and stir at pH 4.75-5 for 1 h;

[0009] S13. Dissolve gelatin in pure water and stir thoroughly at 40°C;

[0010] S14, slowly adding the gelatin solution obtained in S13 dropwise to the solution obtained in S12 to react for 24 hours. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the first cross-linked monomer intermediate of β-CD-Gelatin;

[0011] S20. Preparation of gelatin cross-linked product:

[0012] S21, dissolve β-CD-Gelatin in pure water, stir evenly and add EDC·HCl to activate for 1h.

[0013] S22, adding an appropriate amount of NHS to the solution obtained in S21 and reacting for 24 hours. The solution was dialyzed for 3 days, centrifuged to remove the precipitate, and lyophilized to obtain the β-CD-Gelatin-NHS product;

[0014] S23, adding the VPM-pm26 TGF-β1 polypeptide to 1 wt% β-CD-g-NHS, reacting at 4°C for 5 h, then titrating the solution to a neutral pH with NaOH, dialyzing the solution against pre-chilled ultrapure water for 3 days, and then lyophilizing to obtain the polypeptide covalently coupled to β-CD-g-NHS;

[0015] S30, Preparation of γ-polyglutamic acid intermediate:

[0016] S31, dissolve γ-polyglutamic acid (γ-PGA) in pure water and stir thoroughly to mix;

[0017] S32, adding appropriate amounts of EDC and NHS to the solution obtained in step S31, and reacting at room temperature for 2 h;

[0018] S33, adding an appropriate amount of adipic acid dihydrazide (ADH) to the solution obtained in step S32, reacting at a pH of 5.5 for 24 hours, dialyzing the reaction solution after the reaction, and freeze-drying to obtain the second cross-linking intermediate monomer γ-PGA-ADH;

[0019] Preparation of S40, γ-polyglutamic acid cross-linked product:

[0020] S41, dissolving 1-adamantaneacetic acid in a water / DMSO solution at a ratio of 1:3;

[0021] S42, adding appropriate amounts of EDC and NHS to the solution obtained in step S41, and reacting at room temperature for 1 h;

[0022] S43, weighing an appropriate amount of γ-PGA-ADH, dissolving it in pure water, and slowly adding it dropwise to the solution obtained in step S42, reacting at room temperature for 40 hours. After the reaction is completed, centrifuging the reaction solution to remove the precipitate, dialyzing it at 3000 rpm, centrifuging it for 10 minutes to remove the precipitate, and freeze-drying it to obtain Ad-γ-PGA-ADH;

[0023] S50. Preparation of biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid:

[0024] S51, taking an appropriate amount of the first cross-linking monomer and dissolving it in a PBS solution;

[0025] S52, taking an appropriate amount of the second cross-linking monomer and dissolving it in the PBS solution;

[0026] S53. Take an appropriate amount of the solution obtained in step 5.1 and an appropriate amount of the solution obtained in step S32, mix them quickly and evenly, and wait for gel formation in a 37°C water bath.

[0027] Preferably, the ratio of the CM-β-CD, EDC, NHS and gelatin is: 1 g of CM-β-CD corresponds to 1.5 mmol NHS, 1.5 mmol EDC and 1 g of gelatin.

[0028] Preferably, the dialysis refers to dialysis using a dialysis bag with a molecular weight cut-off of 8000-14000.

[0029] Preferably, the ratio of the β-CD-Gelatin to EDC and NHS is: every 0.5 g of CM-β-CD corresponds to 16 mmol of NHS and 16 mmol of EDC.

[0030] Preferably, the ratio of the γ-PGA, EDC, NHS and ADH is: every 2g of γ-PGA corresponds to 7.5mmol of NHS, 7.5mmol of EDC and 2.5g of ADH.

[0031] Preferably, the freeze-drying is carried out at -80°C.

[0032] Preferably, the ratio of the γ-PGA-ADH, 1-Ad, EDC and NHS is: every 1g of γ-PGA-ADH corresponds to 2.5mmol of NHS, 2.5mmol of EDC and 0.24g of 1-Ad.

[0033] Preferably, the ratio of the first cross-linking monomer to the second cross-linking monomer is as follows: 25 wt% of β-CD-Gelatin-NHS and 4 wt% of Ad-γ-PGA-ADH are mixed to form the target hydrogel.

[0034] A biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid, with the structural formula

[0035] The hydrogel is prepared by the method according to any one of claims 1 to 8.

[0036] Preferably, the biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid is used to reshape the cartilage microenvironment and promote cartilage regeneration.

[0037] The beneficial effects of the present invention are:

[0038] 1. A biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid. The biomimetic hydrogel, which is double-crosslinked by host-guest interaction and amide bonds, has excellent biocompatibility, biodegradability, good mechanical properties and self-healing properties. For example, when experiencing pressure, the first layer of reversible physical crosslinking network can effectively de-crosslink to dissipate energy and reduce carrier pressure. When relaxed, it can quickly recover through reversible crosslinking bonds. Another layer of strong chemical crosslinking network ensures that the hydrogel structure does not disintegrate while providing space for loading therapeutic substances. It can provide lubrication and support effects when used in cartilage areas.

[0039] 2. The hydrogel system modified with the VPM-pm26 TGF-β1 peptide drug has the advantages of responding to matrix metalloproteinase-3 (MMP-3) in the microenvironment to inhibit cartilage matrix degradation and inflammation, while simultaneously inducing the directed differentiation of stem cells and promoting cartilage regeneration. In addition to excellent biocompatibility, biodegradability, adjustable mechanical properties, self-healing properties, high water retention, stem cell adhesion, and the ability to buffer cartilage tissue pressure, this hydrogel system can be loaded with peptide drugs to promote inflammation resolution and directed cartilage differentiation of BMSCs, and has broad potential clinical application prospects.

[0040] 3. The N-amino group of the VPM-pm26TGF-β1 (VPMSMRGG-ACESPLK(Ac)RQCGGGS) polypeptide sequence was amidated with the NHS on the β-CD-Gelatin-NHS chain to obtain the final VPM-pm26TGF-β1-modified hydrogel system, which was used to reshape the damaged cartilage microenvironment and promote the directional cartilage differentiation of BMSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The preparation route for β-CD-Gelatin-NHS and Ad-γ-PGA-ADH;

[0042] Figure 2 For CM-β-CD, Gelatin, β-CD-Gelatin and β-CD-Gelatin-NHS 1 H NMR spectroscopy

[0043] Figure 3 for β-CD-G-NHS, pm26TGF-β1 and pm26TGFβ1-βCD-G-NHS 1 HNMR spectroscopy;

[0044] Figure 4 for β-CD-G-NHS, VPM-TGF-β1 and VPM-TGF-β1-βCD-G-NHS 1 HNMR spectroscopy;

[0045] Figure 5 MALDI-TOF spectra and HPLC analysis of pm26TGF-β1;

[0046] Figure 6 MALDI-TOF spectra and HPLC analysis of VPM-pmTGFβ1;

[0047] Figure 7 For ADH, γ-PGA, γ-PGA-ADH and Ad-γ-ADH 1 H NMR spectroscopy;

[0048] Figure 8 The infrared spectra of Gelatin, β-CD-Gelatin and β-CD-G-NHS;

[0049] Figure 9 The infrared spectra of γ-PGA, γ-PGA-ADH and Ad-γ-ADH;

[0050] Figure 10 Representative SEM images of hydrogel samples with three different ratios;

[0051] Figure 11 To study the in vitro degradation of hydrogel samples with different ratios;

[0052] Figure 12 To study the in vitro swelling properties of hydrogel samples with different proportions;

[0053] Figure 13 for β-CD-Gelatin-NHS and Ad-γ-PGA-ADH 1 H NMR and NOSEY two-dimensional spectra of the host-guest interaction between β-CD and 1-Ad in the hydrogel;

[0054] Figure 14 The mechanical properties and self-healing properties of β-CD-Gelatin-NHS / VPM-pm26TGF-β1 hydrogel;

[0055] Figure 15 The temporal and rheological behaviors of β-CD-Gelatin-NHS / VPM-pm26TGF-β1 hydrogel;

[0056] Figure 16 To study the biocompatibility and peptide release properties of hydrogels;

[0057] Figure 17 To investigate the effect of VPM-pm26TGF-β1 on inflammation-induced chondrocytes in vitro. DETAILED DESCRIPTION

[0058] The embodiments of the present invention are described in detail below. The following embodiments are implemented based on the technical solutions of the present invention, and provide detailed implementation methods and specific operating procedures. However, the protection scope of the present invention is not limited to the following embodiments.

[0059] The invention relates to a method for preparing a biomimetic double cross-linked hydrogel (β-CD-Gelatin-NHS / Ad-γ-PGA-ADH) system based on gelatin and polyglutamic acid (γ-PGA) and its application in cartilage regeneration, and relates to the technical field of biomimetic hydrogels for cartilage regeneration. The structural formula of the biomimetic double cross-linked hydrogel is:

[0060]

[0061] This hydrogel system has excellent mechanical and self-healing properties. For example, when subjected to pressure, the first layer of reversible physical cross-linking network can effectively de-crosslink to dissipate energy and reduce carrier pressure, and can quickly recover through reversible cross-linking bonds when relaxed; while another layer of strong chemical cross-linking network ensures that the hydrogel structure does not collapse while providing space for loading therapeutic substances, which can provide lubrication and support effects when used in cartilage areas.

[0062] The hydrogel system modified with the VPM-pm26 TGF-β1 peptide drug has the advantages of responding to matrix metalloproteinase-3 (MMP-3) in the microenvironment to inhibit cartilage matrix degradation and inflammation, while also inducing the directional differentiation of stem cells and promoting cartilage regeneration. In addition to its excellent biocompatibility, biodegradability, adjustable mechanical properties, self-healing properties, high water retention, stem cell adhesion, and ability to buffer cartilage tissue pressure, this hydrogel system can be loaded with peptide drugs to promote inflammation resolution and directional cartilage differentiation of BMSCs, showing broad potential for clinical application.

[0063] Specific preparation of the double-crosslinked hydrogel system:

[0064] 1. Preparation of the first cross-linked product.

[0065] 0.5 g of CM-β-CD was dissolved in 100 mL of ultrapure water and stirred thoroughly at 40°C. 1.5 mmol of NHS and 1.5 mmol of EDC were added to activate the carboxyl groups on the CM-β-CD for 1 hour. Subsequently, 1 g of gelatin was dissolved in 50 mL of ultrapure water and slowly added dropwise to the CM-β-CD solution. The reaction was continued at a pH of 4-5 for 24 hours. At the end of the reaction, the solution was dialyzed (8000-14000 MWCO) for 3 days and lyophilized at -80°C to harvest the β-CD-gelatin. For the synthesis of β-CD-g-NHS, 0.5 g of β-CD-Gelatin was dissolved in 16 mL of ultrapure water, stirred thoroughly, activated with 16 mmol of EDC for 1 h, and finally 20 mmol of NHS was added to the CM-β-Gelatin solution and allowed to react for 24 h. The solution was dialyzed (8000-14000 MWCO) for more than 3 days and freeze-dried at -80°C to obtain β-CD-Gelatin-NHS.

[0066] To chemically couple the peptides to the hydrogel chains, 10 mg of VPM-pm26TGF-β1 was added to 1 wt% β-CD-Gelatin-NHS and reacted at 4°C for 5 hours to deplete the NHS groups. The solution was then titrated to neutral pH with 5N NaOH, dialyzed against cold ultrapure water for at least 3 days, and lyophilized at -80°C to obtain the VPM-pm26TGF-β1 covalently coupled to β-CD-g-NHS.

[0067] 2. Preparation of the Second Cross-linked Product

[0068] 2g of γ-PGA was dissolved in 200mL of ultrapure water. 7.5mmol of NHS and 7.5mmol of EDC were then added to the solution and allowed to react for 1h. Finally, 2.5g of ADH was added to the reaction solution and allowed to react at a pH of approximately 5.5 for 24h. The reaction solution was dialyzed (8000MWCO) for at least 3 days, and the product was lyophilized at -80°C. To synthesize Ad-γ-PGA-ADH, 0.24g of 1-Ad was dissolved in 100mL of water / DMSO (1:3), 2.5mmol of EDC and 2.5mmol of NHS were added to the mixture, and stirred at room temperature for 2h. 1g of γ-PGA-NH2 dissolved in 100mL of ultrapure water was added dropwise to the 1-Ad mixture and allowed to react at room temperature for 40h. The reaction solution was centrifuged to remove the precipitate, dialyzed (8000MWCO) for at least 3 days, and lyophilized at -80°C to yield Ad-γ-PGA-ADH. Prepare the target hydrogel.

[0069] 25 wt% of β-CD-Gelatin-NHS and 4 wt% of Ad-γ-PGA-ADH were weighed and dissolved in PBS solution respectively to prepare a hydrogel with a specific ratio. The target hydrogel was formed by mixing them at 37°C.

[0070] The performance test results of the biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid are as follows:

[0071] 1. Testing of the mechanical properties of hydrogels. The storage modulus (G') and loss modulus (G") of the hydrogel samples were tested using a rotational rheometer (Malvern, UK). Time sweep tests were performed in the time range of 0-20 min, and frequency sweep tests were performed in the frequency range of 0.1-10 Hz to obtain the final G' and G". The stress-strain curve of each hydrogel sample was obtained by placing a cylindrical sample (diameter = 1 cm, height = 1 cm) in a universal materials testing machine (INSTRON3365) and compressing it at a speed of 0.5 mm / min to reach the maximum yield point.

[0072] 2. Degradation cycle of hydrogel. Briefly, cylindrical hydrogel samples (diameter: 1 cm, height: 1 cm) were prepared and freeze-dried, and their weights were recorded. The freeze-dried hydrogel samples were immersed in simulated body fluid (SBF) at 37°C, samples were taken out regularly and freeze-dried, and their weights were recorded. The degradation cycle of the hydrogel was ultimately obtained from the ratio of mass loss to total mass. The cylindrical hydrogel composed of 15% β-CD-g-NHS and 2% Ad-γ-ADH degraded almost 85% after 20 days in simulated body fluid (SBF), while the 25% β-CD-g-NHS / 4% Ad-γ-ADH hydrogel degraded about 30% within 20 days in SBF. n=3, *p<0.05, ***p<0.001, indicating that the 25% β-CD-g-NHS / 4% Ad-γ-ADH hydrogel has a stable structure and a longer degradation cycle in vivo.

[0073] 3. Swelling properties of hydrogels. To investigate the structural stability of hydrogels, different samples were immersed in SBF to simulate a physiological environment. Different hydrogel samples were placed in SBF at a concentration of 0.1 g / mL, cultured at 37°C for 5 days, and then weighed again. The swelling rate of the hydrogel was determined by the weight change before and after immersion. All hydrogel samples reached swelling equilibrium after immersion in SBF for 24 hours. The swelling rate of the 25% βCD-g-NHS / 4% Ad-γ-ADH hydrogel was lower than that of the other hydrogel samples.

[0074] 4. Testing of mechanical properties of hydrogel.

[0075] The storage modulus (G') and loss modulus (G") of β-CD-Gelatin-NHS / Ad-γ-PGA-ADH hydrogels with different ratios were measured within 20 minutes using a rotational rheometer in time scan mode. In order to evaluate the stability of the hydrogel, the changes in G' and G" were monitored at different frequencies from 0.1 to 10 Hz. At high frequencies, G' was significantly higher than the G" value, indicating that the hydrogel was in a solid-like state.

[0076] In addition, hydrogel samples with different ratios were prepared. Mechanical tests (tensile and compression) were carried out using a universal material testing machine. For the tensile test, a dumbbell-shaped hydrogel (total length of 15 mm, measuring area of ​​3×1 mm2, gripping area of ​​6×4 mm2, and thickness of 1 mm) was fixed on the testing machine holder and stretched at a rate of 2 mm / min until it broke. The stress was recorded as a function of strain, and the obtained stress / strain curve was used to extract the elastic modulus, ultimate tensile strength, elongation and fracture energy of each specimen (n=5). The double-crosslinked hydrogel can withstand more than 90% deformation and completely recover its original morphology when the pressure is removed, indicating that it has excellent elastic properties.

[0077] 5. Testing of self-healing properties of hydrogels

[0078] The self-healing property of the hydrogel is achieved by adhering several rhodamine-stained cut gels to each other, forming a stretchable hydrogel series structure within a dozen seconds. This double-crosslinked hydrogel combines the biological advantages of Gelatin and γ-PGA, and has excellent mechanical properties. For example, during pressure-stretching, the chemical amide network remains intact due to strong interaction forces, while the host-guest interaction between cyclodextrin and adamantane and the large number of hydrogen bond physical networks gradually unravel due to weak forces to effectively dissipate energy. When the stretching is removed, the reversible physical force restores the original network system, indicating that the hydrogel has strong mechanical properties and certain self-healing properties, which can provide lubrication and cushioning for cartilage tissue.

[0079] 6. Test the on-demand release performance of VPM-pm26TGF-β1 hydrogel.

[0080] The inhibition of MMP-3 by VPM-pm26TGF-β1 was assessed by detecting MMP-3 activity in the mixed supernatant using a rat MMP-3 ELISA kit. Briefly, a mixture containing equal amounts of MMP-3 (20 nM) VPM-modified β-CD-Gelatin-NHS or VPM-pm26TGF-β1-modified β-CD-Gelatin-NHS was dissolved in 100 μL of buffer and then added to a 96-well plate pre-coated with an MMP-3-specific monoclonal antibody. The mixture was incubated at 37°C in the dark for 90 minutes, and the fluorescence intensity of each sample at the endpoint was measured using UV light at a wavelength of 450 nm. FITC-labeled pm26TGF-β1 was used to assess its release profile from the VPM-pm26TGF-β1-loaded hydrogel. The peptide-loaded hydrogels were added to 96-well plates (100 μL per well). Then, 200 μL of PBS and / or rMMP-3 (2 nM) were added and incubated at 37°C for 15 days, with rMMP-3 replaced every two days to maintain enzyme activity. The collected solution was centrifuged at 15,000 rpm for 5 minutes at 4°C, and the absorbance of the supernatant, FITC-mpTGF-β1, was measured using a UV-visible spectrophotometer under 480 nm excitation. The cumulative percentage of peptide released from the grafted material was calculated by comparison with a pre-established standard curve.

[0081] 7. Cell biocompatibility testing of hydrogels

[0082] The survival rate of BMSCs cultured in hydrogels on days 1, 3, 5, and 7 was assessed using calcein (AM) / propidium iodide (PI). A live / dead dye solution was prepared by adding 1 μL of calcein AM and 2 μL of PI to 1 mL of PBS. The BMSCs-loaded hydrogels were stained with live / dead dye for 30 minutes at room temperature and recorded using a confocal laser scanning microscope with a 10x objective. In addition, the proliferation of BMSCs over time was determined using a CCK-8 kit. CCK-8 staining was performed using a 10% (total culture medium volume) CCK-8 solution and an incubation time of 1 hour.

[0083] 8. Directed chondrogenic differentiation of BMSCs in hydrogels:

[0084] BMSCs were cultured in chondrogenic medium with or without the addition of MMP-3 in BMSC-loaded hydrogel dilutions for 4 weeks. Total RNA was extracted from the hydrogels (n=3) using Trizol and reverse transcribed into cDNA using the MMLV cDNA synthesis kit. Real-time PCR was performed on cartilage-related genes (ColII, Sox9, Aggrecan, ColI) using a real-time PCR system with GAPDH as the reference gene. In addition, the BMSCs-loaded hydrogels were paraffin-embedded, sectioned, dewaxed in xylene, dehydrated in a graded series of ethanol solutions, incubated with primary antibodies against ColI and ColII at 4°C overnight, and incubated with a rabbit anti-mouse IgG biotin secondary antibody for 30 minutes. Immunohistochemical staining was performed, and the chondrogenic reaction of BMSCs was observed using Alcian blue and toluidine blue staining.

[0085] 9. Detection of peptide-loaded hydrogels inhibiting chondrocyte inflammation

[0086] To simulate the in vitro inflammatory state of cartilage, chondrocytes were seeded into 12-well plates and cultured with IL-1β (10 ng / mL) for 12 hours. After washing with PBS, the cells were replaced with normal growth medium and treated with TGF-β1 (10 ng / mL) or VPM-pmTGF-β1 hydrogel precursor solution (50 μL) for 24 hours. Immunofluorescence staining was performed to detect the nuclear translocation of NF-κB in the cells. Immunofluorescence and Western Blot results showed that VPM-pmTGF-β1 hydrogel inhibited NF-κB nuclear translocation, indicating that VPM-pmTGF-β1 hydrogel has the effect of inhibiting IL-1β-induced inflammation compared with the blank hydrogel group.

[0087] Based on the above analysis and measurement results, it can be seen that in the above embodiment, the present invention uses gelatin and γ-PGA as carriers, firstly modifies cyclodextrin (β-CD) and NHS to the gelatin molecular chain to obtain β-CD-Gelatin-NHS, and then cross-links the N-amino group of the VPM-pm26TGF-β1 polypeptide sequence with the NHS-amide on the β-CD-Gelatin-NHS chain to obtain a VPM-pm26TGF-β1-modified β-CD-Gelatin-NHS molecular chain. In addition, γ-PGA is first modified with ADH, and then 1-Ad is modified on γ-PGA to obtain Ad-γ-PGA-ADH. Finally, the two are mixed to construct a new type of biomimetic double-cross-linked hydrogel, in which the VPM-pm26TGF-β1 integrated peptide has an MMP-3 cleavage site, which can release pm26TGF-β1 on demand in response to MMP-3 to inhibit inflammation and promote cartilage directional differentiation. Based on the above reasons, the above hydrogel system has excellent biocompatibility, biodegradability, adjustable mechanical properties, self-healing properties, high water retention, stem cell adhesion, and the ability to buffer cartilage tissue pressure. It can also be loaded with polypeptide drugs to promote inflammation resolution and BMSCs directional cartilage differentiation, and has broad potential clinical application prospects.

[0088] It can be seen that compared with other natural hydrogel products, the polypeptide double-crosslinked hydrogel not only has the excellent biocompatibility and biodegradability of natural hydrogels, but also has the characteristics of optimized mechanical strength and easy modification of synthetic hydrogels. The hydrogel has cell adhesion sites that can load stem cells, improve cell activity and promote subsequent repair; the polypeptide components that make up the hydrogel are derived from the human body, giving it more excellent biocompatibility and biodegradability, and the degradation products can be reused by the body to provide energy units that can be used by the human body. The large number of hydrogen bonds, amino groups, etc. in the hydrogel can provide self-repair and strong adhesion, which is also not available in other hydrogel products. It is tailor-made for cartilage tissue and has the effects of buffering tissue mechanical pressure and providing lubrication without being easily disintegrated.

[0089] The above describes in detail the preferred embodiments of the present invention. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible by those skilled in the art without inventive effort. Therefore, any technical solution that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid, characterized in that: The method comprises the following preparation steps: S10. Preparation of gelatin intermediate: S11, dissolving carboxymethyl-β-cyclodextrin (CM-β-CD) in pure water at room temperature and stirring to obtain a carboxymethyl-β-cyclodextrin solution; S12, add EDC·HCl and NHS to the solution obtained in S11, and stir at pH 4.75-5 for 1 h; S13. Dissolve gelatin in pure water and stir thoroughly at 40°C; S14, slowly adding the gelatin solution obtained in S13 dropwise to the solution obtained in S12 to react for 24 hours. After the reaction is completed, the reaction solution is dialyzed and freeze-dried to obtain the first cross-linked monomer intermediate of β-CD-Gelatin; S20. Preparation of gelatin cross-linked product: S21, dissolve β-CD-Gelatin in pure water, stir evenly and add EDC·HCl to activate for 1h. S22, adding an appropriate amount of NHS to the solution obtained in S21 and reacting for 24 hours. The solution was dialyzed for 3 days, centrifuged to remove the precipitate, and lyophilized to obtain the β-CD-Gelatin-NHS product; S23, adding the VPM-pm26 TGF-β1 polypeptide to 1 wt% β-CD-Gelatin-NHS, reacting at 4°C for 5 h, then titrating the solution to a neutral pH with NaOH, dialyzing the solution against pre-chilled ultrapure water for 3 days, and then lyophilizing to obtain the polypeptide covalently coupled to β-CD-Gelatin-NHS; S30, Preparation of γ-polyglutamic acid intermediate: S31, dissolve γ-polyglutamic acid (γ-PGA) in pure water and stir thoroughly to mix; S32, adding appropriate amounts of EDC and NHS to the solution obtained in step S31, and reacting at room temperature for 2 h; S33, adding an appropriate amount of adipic acid dihydrazide (ADH) to the solution obtained in step S32, reacting at a pH of 5.5 for 24 hours, dialyzing the reaction solution after the reaction, and freeze-drying to obtain the second cross-linking intermediate monomer γ-PGA-ADH; Preparation of S40, γ-polyglutamic acid cross-linked product: S41, dissolve 1-adamantaneacetic acid (1-Ad) in a water / DMSO solution at a ratio of 1:3; S42, adding appropriate amounts of EDC and NHS to the solution obtained in step S41, and reacting at room temperature for 1 h; S43, weighing an appropriate amount of γ-PGA-ADH, dissolving it in pure water, and slowly adding it dropwise to the solution obtained in step S42, reacting at room temperature for 40 hours. After the reaction is completed, centrifuging the reaction solution to remove the precipitate, dialyzing it at 3000 rpm, centrifuging it for 10 minutes to remove the precipitate, and freeze-drying it to obtain Ad-γ-PGA-ADH; S50. Preparation of biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid: S51, take an appropriate amount of peptide covalently coupled β-CD-Gelatin-NHS and dissolve it in PBS solution; S52, take an appropriate amount of Ad-γ-PGA-ADH and dissolve it in PBS solution; S53. Take an appropriate amount of the solution obtained in step S51 and an appropriate amount of the solution obtained in step S52, mix them quickly and evenly, and wait for gel formation in a 37°C water bath.

2. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The ratio of CM-β-CD, EDC, NHS and gelatin is as follows: 1 g of CM-β-CD corresponds to 1.5 mmol NHS, 1.5 mmol EDC and 1 g of gelatin.

3. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The dialysis is performed using a dialysis bag with a molecular weight cut-off of 8000-14000.

4. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The ratio of the γ-PGA, EDC, NHS and ADH is as follows: every 2 g of γ-PGA corresponds to 7.5 mmol of NHS, 7.5 mmol of EDC and 2.5 g of ADH.

5. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The freeze-drying is carried out at -80°C.

6. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The ratio of the γ-PGA-ADH, 1-Ad, EDC, and NHS is as follows: 1 g of γ-PGA-ADH corresponds to 2.5 mmol of NHS, 2.5 mmol of EDC, and 0.24 g of 1-Ad.

7. The method for preparing a biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 1, characterized in that: The ratio of the first cross-linking monomer to the second cross-linking monomer is as follows: 25 wt% of polypeptide covalently coupled β-CD-Gelatin-NHS and 4 wt% of Ad-γ-PGA-ADH are mixed to form the target hydrogel.

8. A biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid, characterized in that: The structural formula is The hydrogel can be prepared by the method according to any one of claims 1 to 7.

9. The biomimetic double-crosslinked hydrogel based on gelatin and γ-polyglutamic acid according to claim 8, characterized in that: Used to reshape the cartilage microenvironment and promote cartilage regeneration.

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