Preparation method of photo-crosslinking injectable hydrogel for promoting bone repair

Abstract

The invention discloses a preparation method of a photo-crosslinking injectable hydrogel for promoting bone repair, by optimizing the composition of a photo-crosslinking system and a preparation process, the prepared hydrogel has injectability, gradient adaptive adhesion strength and excellent adhesion stability, and can be precisely regulated and cured through light control to avoid secondary operation stripping injury. The hydrogel is stable in structural performance and not prone to excessive swelling in a simulated bone physiological environment, the mechanical properties of the hydrogel can be matched with cortical bones and cancellous bones respectively, and the hydrogel has remarkable bone regeneration promoting activity and can provide a proper microenvironment for bone tissue regeneration. When cancellous bone is repaired, the hydrogel has excellent toughness and elastic recovery rate, can bear dynamic load and avoids stress shielding effect; due to the injectable characteristic, irregular bone defect parts can be accurately filled with the material, and the adaptability of the material and a defect area is improved. The hydrogel prepared by the method can provide a better and safer material for a bone repair operation, efficiently assists bone tissue regeneration, improves the operation effect and solves the limitation of the existing material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a method for preparing a photocrosslinked injectable hydrogel that promotes bone repair. Background Technology

[0002] Photocrosslinked injectable hydrogels used for defect repair and tissue fixation need to be adapted to the physiological and mechanical needs of human tissues. The core requirement is gradient adhesion strength: it must be able to strongly bond to the tissue surface to stabilize and fix the damaged site, while avoiding excessive adhesion that could lead to secondary surgical damage.

[0003] Traditional adhesives (such as PMMA) suffer from poor biocompatibility and exothermic curing that can damage tissues, limiting their applications. Photocrosslinked injectable hydrogels, on the other hand, allow for precise control of curing through light regulation and are injectable to fit irregular defects. They are primarily composed of natural polysaccharide derivatives or synthetic peptides, exhibiting excellent biocompatibility. However, in bodily fluid environments, most photocrosslinked hydrogels are prone to over-swelling, leading to performance degradation and interfacial delamination. Therefore, these hydrogels need to maintain stable adhesion under simulated human physiological conditions (pH 7.3-7.4, 37℃, dynamic mechanical stimulation), which can be achieved by introducing dynamic covalent bonds or nano-reinforcing phases. For the repair of load-bearing tissues, these hydrogels need to possess gradient mechanical properties: those adapted for dense tissues need to have increased tensile and compressive strength to 10-30 MPa, while those adapted for loose tissues need optimized toughness and elastic recovery rate to withstand dynamic loads, avoid stress shielding, and ensure a suitable microenvironment for tissue repair.

[0004] In summary, there is an urgent need for a photocrosslinked hydrogel that can solve the problems of weak adhesion at the wetting interface and poor environmental stability of traditional hydrogels, while also exhibiting excellent injectability. Summary of the Invention

[0005] In view of the above shortcomings, the present invention aims to provide a method for preparing a photocrosslinked injectable hydrogel for promoting bone repair. By introducing SH-Asp6 (thiol-modified aspartic acid hexapeptide) as a dedicated bone repair component, a hydrogel system with both strong interfacial bonding and structural stability is constructed to obtain a high-performance bone repair material that meets the clinical needs of bone defect repair.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0007] This invention first discloses a method for preparing a photocrosslinked, bone-repairing injectable hydrogel, comprising the following steps:

[0008] (1) Weigh 0.5g of phenylboronic acid and thiol aspartic acid hexapeptide co-modified methacrylamide hyaluronic acid and dissolve it in 5ml of deionized water. Stir magnetically until fully dissolved to obtain hydrogel precursor.

[0009] (2) Weigh out the natural organic compound, dissolve it in an appropriate amount of ethanol, and then add it to the hydrogel precursor and mix thoroughly to obtain the first mixed solution;

[0010] (3) Add LAP photoinitiator to the first mixed solution, then add it to the mold and irradiate it with UV light to obtain a photocrosslinked bone repair injectable hydrogel.

[0011] Further, the methacrylamide hyaluronic acid co-modified with phenylboronic acid and hexameric aspartic acid in step (1) is prepared by the following method:

[0012] (1.1) Weigh 100 mg of phenylboronic acid-grafted methacrylamide hyaluronic acid solid powder and dissolve it in 10 mL of phosphate buffer solution. Stir until completely dissolved to obtain HAMA solution for later use.

[0013] (1.2) Under magnetic stirring, slowly add 0.1 M NaOH solution to the HAMA solution and adjust the pH value to 8.0~8.5 to obtain HAMA-PBA solution;

[0014] (1.3) Add mercapto-hexameric aspartic acid powder to HAMA-PBA solution according to a molar ratio of mercapto to methacrylate groups of 1:10. Immediately isolate oxygen and stir continuously in the dark at 25 °C for 24 hours to obtain a reaction mixture.

[0015] (1.4) Transfer the reaction mixture to a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze it with deionized water for 3 days, changing the dialysis solution 3-4 times a day;

[0016] (1.5) Collect the purified solution in the dialysis bag and freeze-dry for 48 hours to obtain methacrylated hyaluronic acid co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide. The product is sealed and stored at -20℃ in a dry environment for later use.

[0017] Further, the phenylboronic acid grafted with methacrylamide hyaluronic acid in step (1.1) is prepared by the following method:

[0018] ① Dissolve 1 g of hyaluronic acid in 100 ml of deionized water, add 1 mL of methacrylic anhydride, adjust the pH to 8.0~8.5 with 1 mol / L NaOH solution, react for 2 h, then stir at 4℃ for 24 h, centrifuge, transfer the solution to a dialysis bag and dialyze with deionized water for 4 days, then freeze dry to obtain methacrylamide hyaluronic acid for later use.

[0019] ② Dissolve 1g of methacrylamide hyaluronic acid in 100ml of deionized water, add 1.4g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, then add 0.11g of 3-aminophenylboronic acid, react in the dark for 24 hours, dialyze the reaction solution with deionized water for at least 3 days, and then freeze-dry to obtain phenylboronic acid grafted with methacrylamide hyaluronic acid.

[0020] Furthermore, the thiol-modified aspartic hexapeptide is prepared by the following method:

[0021] (1) Weigh 0.18 g of 2-chlorotriphenylmethyl chloride resin, soak it in N,N-dimethylformamide for 30 min and then filter it; add 0.37 g of fluorenemethoxycarbonyl-aspartic acid-tert-butyl ester, 0.13 mL of N,N-diisopropylethylamine and 10 mL of N,N-dimethylformamide to the resin, stir and react at room temperature for 2-3 h, filter it and wash it three times each with N,N-dimethylformamide and dichloromethane to complete the first step of connecting amino acids with the resin;

[0022] (2) Repeat the following steps 5 times to complete peptide chain elongation:

[0023] (2.1) Add a 20% (v / v) piperidine / N,N-dimethylformamide solution and deprotect at room temperature for 10 min;

[0024] (2.2) Wash 5 times with N,N-dimethylformamide and 3 times with dichloromethane;

[0025] (2.3) Fmoc-Asp(OtBu)-OH (fluorenylmethoxycarbonyl-aspartic acid-tert-butyl ester) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide and pre-activated for 5 min;

[0026] (2.4) Add the activation solution to the resin and couple the reaction at room temperature for 1-2 hours to complete the peptide chain elongation;

[0027] (3) Coupling with Fmoc-Asp(OtBu)-OH (fluorenylmethoxycarbonyl-cysteine-triphenylmethyl instead of fluorenylmethoxycarbonyl-aspartic acid-tert-butyl ester), and removing the fluorenylmethoxycarbonyl protecting group according to the conditions in step (2.1) after coupling is completed.

[0028] (4) Add a mixture of trifluoroacetic acid, thioanisole, water and triisopropylsilane in a volume ratio of 90:5:3:2 to the resin and stir at room temperature for 2-3 h; filter and collect the filtrate, and slowly drop the filtrate into 10 times the volume of pre-cooled ether, and let it stand to precipitate; centrifuge at 4000 rpm for 10 min, collect the precipitate, wash it 3 times with cold ether and then vacuum dry to obtain crude mercapto-modified aspartic acid hexapeptide;

[0029] (5) The crude thiol-modified aspartic acid hexapeptide was dissolved in a 0.1% trifluoroacetic acid acetonitrile / water mixed solvent, purified by HPLC using a C18 column, the target elution peak was collected and freeze-dried to obtain the thiol-modified aspartic acid hexapeptide.

[0030] Furthermore, the natural organic compound mentioned in step (2) is selected from:

[0031] 50 mg dihydromyricetin or 10 mg gallic acid.

[0032] Further, step (3) involves irradiating with UV light for 30 seconds.

[0033] The present invention also discloses a photocrosslinked bone-repairing injectable hydrogel prepared according to any of the above preparation methods.

[0034] The beneficial effects of this invention are as follows:

[0035] 1. The hydrogel prepared by the present invention is based on methacryloyl hyaluronic acid-phenylboronic acid (HAMA-PBA). The hydrogel has both injectability and photocurability, and can seamlessly fill irregular tissue defects in liquid form. It can rapidly gel in situ after light exposure, providing good mechanical support and preventing drug loss.

[0036] 2. This invention utilizes the dynamic borate ester bond formed by the PBA group and the ortho-dihydroxyl groups in dihydromyricetin and gallic acid molecules. This not only solves the problem of the poor water solubility of dihydromyricetin and gallic acid, significantly improving the drug loading capacity, but also endows the material with intelligent response capabilities to the inflammatory microenvironment (low pH or high ROS), realizing on-demand drug release and long-term sustained release.

[0037] 3. The system of this invention integrates the excellent biocompatibility of HAMA with the potent anti-inflammatory, antioxidant and antibacterial activities of dihydromyricetin and gallic acid, which can effectively remove excess reactive oxygen species (ROS) at the lesion site, regulate the immune microenvironment, and thus significantly promote tissue regeneration and repair.

[0038] 4. The adhesion enhancement mechanism of SH-Asp6 (thiol-modified aspartic hexapeptide) in this invention stems from its molecular structure characteristics. Its carboxyl groups can form multiple hydrogen bonds and electrostatic interactions with the hydroxyl groups on the tissue surface. At the same time, its own thiol groups can undergo thiol-alkene addition reactions with HAMA-PBA, which not only strengthens the interfacial adhesion effect but also enhances the internal cohesion of the hydrogel. The preparation process is simple and controllable and has good application prospects. Attached Figure Description

[0039] Figure 1 The 1H NMR spectrum of phenylboronic acid grafted with methacrylamide hyaluronic acid (HAMA-PBA) in Example 1 of this invention;

[0040] Figure 2 The 1H NMR spectrum of SH-Asp6 (thiol-modified aspartic acid hexapeptide) in Example 2 of this invention;

[0041] Figure 3 The image shows a scanning electron microscope image of the hydrogel in Example 4 of this invention, where A is HAMA-PBA-DMY and B is HAMA-PBA-DMY-Asp6.

[0042] Figure 4 This is a diagram showing the injectability of the hydrogel in Example 4 of the present invention, where A represents the state before injection and B represents the state after injection.

[0043] Figure 5 This is a rheological characterization diagram of the hydrogel in Example 4 of the present invention;

[0044] Figure 6 This is a drug release diagram of the hydrogel in Example 4 of the present invention, where A is the drug standard curve and B is the release curve;

[0045] Figure 7 The stress-strain curve of the hydrogel in Example 4 of this invention;

[0046] Figure 8 These are antibacterial coating images of the hydrogels in Examples 4 and 5 of this invention;

[0047] Figure 9 The figures show the cytotoxicity of the hydrogels in Examples 4 and 5 of this invention. Detailed Implementation

[0048] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above content.

[0049] Example 1

[0050] Preparation of phenylboronic acid grafted with methacrylyl hyaluronic acid

[0051] Methacrylic anhydride and phenylboronic acid-modified hyaluronic acid was prepared via a two-step method. Specifically, 1 g of HA was dissolved in 100 mL of deionized water, and then excess methacrylic anhydride (1 mL) was added. The pH was adjusted with 1 mol / L NaOH solution to maintain the pH between 8.0 and 8.5, and the reaction was carried out for 2 h, followed by stirring at 4 °C for 24 h. After centrifugation, the solution was transferred to a dialysis bag (molecular weight cutoff 10000-14000 Da). The solution was dialyzed against deionized water for 4 days, and then freeze-dried to obtain a white, spongy solid product, methacrylamide hyaluronic acid.

[0052] Dissolve 1 g of methacrylamide hyaluronic acid in 100 ml of deionized water, add 1.4 g of DMTMM to activate the carboxyl group on the hyaluronic acid, and then add 0.11 g of 3-aminophenylboronic acid and react in the dark for 24 hours. Dialyze the reaction solution with deionized water (molecular weight cutoff 10000-14000 Da) for at least 3 days, and then freeze-dry to obtain a white, spongy solid product, phenylboronic acid grafted with methacrylamide hyaluronic acid.

[0053] Example 2

[0054] Preparation of thiol-modified aspartic acid hexapeptide

[0055] (1) Weigh 0.18 g of 2-chlorotriphenylmethyl chloride resin (resin loading 1.4 mmol / g), soak and swell with DMF (N,N-dimethylformamide) for 30 min and then filter; add 0.37 g of Fmoc-Asp(OtBu)-OH (fluorenylmethoxycarbonyl-aspartic acid-tert-butyl ester), 0.13 mL of DIPEA (N,N-diisopropylethylamine) and 10 mL of DMF to the resin, stir and react at room temperature for 2-3 h, filter and wash with DMF and DCM (dichloromethane) 3 times each to complete the first step of amino acid connection with resin.

[0056] (2) Repeat the following steps 5 times to complete peptide chain elongation: ① Add 20% piperidine / DMF solution and deprotect at room temperature for 10 min; ② Wash 5 times with DMF and 3 times with DCM; ③ Dissolve Fmoc-Asp(OtBu)-OH, HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate), and DIPEA (N,N-diisopropylethylamine) in DMF and preactivate for 5 min; ④ Add the activation solution to the resin and couple at room temperature for 1-2 h.

[0057] (3) Replace Fmoc-Asp(OtBu)-OH with Fmoc-Cys(Trt)-OH (fluorenylmethoxycarbonyl-cysteine-triphenylmethyl) for coupling. After coupling, remove the Fmoc (fluorenylmethoxycarbonyl) protecting group according to the conditions in step 2 ①.

[0058] (4) Add a mixed lysis buffer of TFA (trifluoroacetic acid) / TIS (thioanisole) / H2O / TIPS (triisopropylsilane) with a volume ratio of 90:5:3:2 to the resin and stir at room temperature for 2-3 hours; filter and collect the filtrate, and slowly drop the filtrate into 10 times the volume of pre-cooled ether, and let it stand to precipitate; centrifuge at 4000 rpm for 10 minutes, collect the precipitate, wash it 3 times with cold ether and then vacuum dry it to obtain crude SH-Asp6 (thiol-modified aspartic acid hexapeptide).

[0059] (5) The crude product was dissolved in a mixed solvent of acetonitrile / water containing 0.1% TFA, purified by HPLC (high performance liquid chromatography) on a C18 column, the target elution peak was collected and freeze-dried to obtain a white powder product.

[0060] Example 3

[0061] Preparation of methacrylated hyaluronic acid co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide

[0062] (1) Weigh 100 mg of phenylboronic acid grafted methacrylamide hyaluronic acid (HAMA-PBA) solid powder, dissolve it in 10 mL of phosphate buffer (PBS, pH 7.4), place it on a magnetic stirrer and stir at room temperature until completely dissolved to prepare a 1% (w / v) HAMA solution.

[0063] (2) Under magnetic stirring, slowly add 0.1 M NaOH solution to the above HAMA solution, and use a pH meter to monitor in real time to adjust the pH value of the solution to the range of 8.0 to 8.5 in order to activate the thiol group and initiate the Michael addition reaction.

[0064] (3) Weigh a measured amount of thiol-aspartic acid hexapeptide (SH-Asp6) powder according to a molar ratio of thiol (-SH) to methacrylate groups (C=C) of 1:10. Add the SH-Asp6 powder directly to the HAMA-PBA solution with the pH adjusted in step S2. Immediately seal the reaction vessel with a sealing film to isolate oxygen and prevent thiol oxidation. Stir the reaction continuously at 25°C in the dark for 24 hours.

[0065] (4) After the reaction is complete, the reaction mixture is transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da. The dialysis bag is placed in a large amount of deionized water for dialysis purification. The dialysis time is 3 days, and the dialysis solution is changed 3-4 times a day to remove unreacted free peptides and salt ions.

[0066] (5) Collect the purified solution from the dialysis bag and freeze-dry it in a freeze dryer for 48 hours to obtain a white cotton-like solid product, which is a methacrylamide hyaluronic acid (Asp6-s-HAMA-PBA) conjugate co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide. Store the product in a dry environment at -20℃ in a sealed container for later use.

[0067] Example 4

[0068] Preparation of Asp6-s-HAMA-PBA-DMY (methacryloyl hyaluronic acid-loaded dihydromyricetin co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide) hydrogel

[0069] (1) Weigh 0.5g Asp6-s-HAMA-PBA and dissolve it in 5 ml of deionized water. Stir magnetically until fully dissolved to obtain the hydrogel precursor.

[0070] (2) Weigh 50 mg of dihydromyricetin, dissolve it in an appropriate amount of ethanol, and add it to the solution in (1) and mix thoroughly.

[0071] (3) After mixing the solution of (1) with (2) material evenly, add LAP photoinitiator, then add it into the mold, irradiate with UV light for 30 seconds, and then obtain hydrogel.

[0072] Example 5

[0073] Preparation of Asp6-s-HAMA-PBA-GA (methacryloyl hyaluronic acid-loaded gallic acid co-modified with phenylboronic acid and mercaptoaspartic hexapeptide) hydrogel

[0074] (1) Weigh 0.5g Asp6-s-HAMA-PBA and dissolve it in 5 ml of deionized water. Stir magnetically until fully dissolved to obtain the hydrogel precursor.

[0075] (2) Weigh 10 mg of gallic acid (GA) and add it to the solution in (1) and mix thoroughly;

[0076] (3) After mixing the solution of (1) with (2) material evenly, add LAP photoinitiator, then add it into the mold, irradiate with UV light for 30 seconds, and then obtain hydrogel.

[0077] Experimental Example 1

[0078] Nuclear magnetic resonance characterization experiments were performed on Examples 1 and 2.

[0079] In the ¹H NMR spectrum obtained from HAMA, characteristic doublets are observed at δ=5.72 and δ=6.12, corresponding to the two trans-olefin protons of the methacrylate double bond. Their splitting pattern is consistent with the cis-trans isomerism of the double bond. These characteristic peaks are not present in the original HA spectrum. The singlet at δ=1.95 is the absorption peak of the -CH3 proton in the methacrylate, and the multiplets in the range of δ=3.30 to 4.50 represent the proton signal of the sugar ring. The HAMA-PBA spectrum shows a newly added broad peak of phenylboronic acid aromatic ring protons in the range of δ=7.35 to 7.80, while the characteristic double bond peaks remain clearly visible, indicating that the functionalization modification did not damage the double bond structure. Figure 2 The peaks at δ=2.67 and δ=8.0 represent the methylene and amide groups of SH-Asp6, respectively.

[0080] Experimental Example 2

[0081] SEM characterization experiments were performed on the hydrogel prepared in Example 3.

[0082] The Asp6-s-HAMA-PBA-DMY hydrogel was freeze-dried and subjected to liquid nitrogen fracture. Scanning electron microscopy was used to observe its internal microporous structure. The results showed that the Asp6-s-HAMA-PBA-DMY hydrogel exhibited a denser network morphology with increased pore wall thickness. This porous structure facilitates the exchange of nutrients and metabolic waste, and the suitable pore size promotes cell adhesion, growth, and proliferation.

[0083] Experimental Example 3

[0084] Injectability performance characterization experiments were conducted on the hydrogel prepared in Example 3.

[0085] Injectable Asp6-s-HAMA-PBA-DMY hydrogel possesses excellent injection performance, allowing for smooth injection using a standard 7-gauge needle. Its ability to transform from a solution to a gel state during use effectively fills complex infection areas, reducing the risk of trauma during the procedure. Test data shows that even with a high concentration of functional components (up to 10%), the injectability remains stable due to the uniform dispersion of active ingredients at the nanoscale. This hydrogel, prepared using a controllable process, ensures sufficient anti-inflammatory and antibacterial loading while preventing clogging of the injection device due to sudden viscosity changes. It provides a novel carrier solution for local infection control, offering advantages such as precise drug delivery and minimally invasive procedures.

[0086] Test Example 4

[0087] Rheological characterization experiments were performed on the hydrogel prepared in Example 3.

[0088] The storage modulus, also known as the elastic modulus, can reflect the amount of energy stored by the material due to elastic deformation, embodying the elasticity of the material; the loss modulus, also called the viscous modulus, reflects the amount of energy lost by the material due to viscous deformation, thus embodying the viscosity of the material. When G′ > G″, the energy stored by the sample due to elastic deformation is more than the energy lost due to viscous deformation, and the elastic performance is more prominent. The sample has a sufficient crosslinking network to support the gel state; when G′ < G″, the viscous performance is greater. The amplitude sweep curve determines the limit point of the linear viscoelastic region of the gel. Within the range of 0.1% - 10% strain, G′ > G″, presenting a gel state. As the angular frequency continuously increases, both G′ and G″ rapidly decrease until G′ = G″ reaches the gel transition point. When the angular frequency is further increased, G′ < G″, and at this time the gel structure is destroyed.

[0089] Experimental Example 5

[0090] Perform a drug release performance characterization experiment on the hydrogel prepared in Example 3

[0091] By collecting the supernatant of Asp6-s-HAMA-PBA-DMY at 1, 2, 4, 7, and 12 h, the corresponding absorbance was measured at a wavelength of 568 nm using an enzyme-labeled instrument. Combining with the standard curve, the cumulative release amount of DMY can be obtained. Observing the curve, it can be seen that the cumulative rate in the first 2 h is relatively fast, the release rate starts to slow down at the 4th h, and basically reaches a plateau at the 12th h. In the Asp6-s-HAMA-PBA-DMY hydrogel system, DMY is not in a free state but forms crosslinking bonds by reacting with other components in the system, thus being firmly fixed within the network structure of the hydrogel. In the initial stage of application, there is a small amount of DMY that has not fully participated in the reaction and is temporarily free, resulting in a faster release rate in the initial stage than in the later stage. After the free DMY has been released, the remaining DMY fixed in the crosslinking bonds will gradually break with the crosslinking bonds and be released from the hydrogel in a slow release form.

[0092] Experimental Example 6

[0093] Perform a mechanical property characterization experiment on the hydrogel prepared in Example 3

[0094] The compressibility of the hydrogels was tested using a texture analyzer, and stress-strain curves were plotted based on the results. During strain increase, the stress increase of HAMA was relatively slow. In contrast, the stress increase of HAMA-PBA and Asp6-s-HAMA-PBA-DMY was faster. Asp6-s-HAMA-PBA-DMY reached a stress of 0.6006 at a strain of 0.62 and continued to rise, indicating that its final compressive strength will exceed that of HAMA, becoming the highest. The stress of HAMA-PBA was higher than that of HAMA throughout the process. This is because the phenylboronic acid groups formed dynamically reversible phenylboronic acid ester bonds with the hydroxyl groups on the HAMA molecular chain, constructing a cross-linked network that dissipates external force energy, reducing stress concentration, and simultaneously improving intermolecular interactions, thereby enhancing compressive strength. The introduction of DMY provides additional covalent crosslinking sites or physical effects, such as hydrogen bonds and hydrophobic effects, which synergize with the dynamic crosslinking of PBA, increasing the network crosslinking density. This restricts the slippage of molecular chains, intensifies the stress response, and results in stronger load-bearing capacity under high strain conditions. Although no fracture points were observed within the test range, based on the stress growth trend, the highest compressive strength and fracture resistance are observed under continuous pressure.

[0095] Experimental Example 7

[0096] Antibacterial properties were tested on the hydrogel prepared in Example 3.

[0097] The basic HAMA group showed relatively weak antibacterial activity against both strains, with survival rates exceeding 80% for both strains. In contrast, the Asp6-s-HAMA-PBA-DMY and Asp6-s-HAMA-PBA-GA groups exhibited very prominent antibacterial effects, demonstrating that DMY and GA possess good antibacterial properties.

[0098] Experimental Example 8

[0099] Cytotoxicity test was performed on the hydrogel prepared in Example 3.

[0100] The biocompatibility of HAMA, Asp6-s-HAMA-PBA-DMY, and Asp6-s-HAMA-PBA-GA hydrogels was analyzed using CCK-8 assay. The HAMA group showed near 100% cell viability, indicating very low cytotoxicity and excellent biocompatibility. The Asp6-s-HAMA-PBA-DMY group showed slightly lower cell viability, but still above the 80% acceptable standard. The Asp6-s-HAMA-PBA-DMY group's cell viability fell between the two groups, suggesting that the GA-loaded hydrogel material exhibited better biocompatibility. The data demonstrate that the LAP, SH-Asp6, and phenylboronic acid bonds introduced during material preparation do not cause cytotoxicity, and all three materials exhibit good cell compatibility.

[0101] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A method for preparing a photocrosslinked injectable hydrogel for promoting bone repair, comprising the following steps: (1) Weigh 0.5 g of methacrylamide hyaluronic acid co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide and dissolve it in 5 ml of deionized water. Stir magnetically until fully dissolved to obtain hydrogel precursor. (2) Weigh out the natural organic compound, dissolve it in an appropriate amount of ethanol, and then add it to the hydrogel precursor and mix thoroughly to obtain the first mixed solution; (3) Add LAP photoinitiator to the first mixed solution, then add it to the mold and irradiate it with UV light to obtain a photocrosslinked bone repair injectable hydrogel.

2. The preparation method according to claim 1, wherein: The methacrylamide hyaluronic acid co-modified with phenylboronic acid and hexameric aspartic acid in step (1) is prepared by the following method: (1.1) Weigh 100 mg of phenylboronic acid-grafted methacrylamide hyaluronic acid solid powder and dissolve it in 10 mL of phosphate buffer solution. Stir until completely dissolved to obtain HAMA solution for later use. (1.2) Under magnetic stirring, slowly add 0.1 M NaOH solution to the HAMA solution and adjust the pH value to 8.0~8.5 to obtain HAMA-PBA solution; (1.3) Add mercapto-hexameric aspartic acid powder to HAMA-PBA solution according to a molar ratio of 1:10 of mercapto groups to methacrylate groups. Immediately isolate oxygen and stir continuously in the dark at 25°C for 24 hours to obtain a reaction mixture. (1.4) Transfer the reaction mixture to a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyze it with deionized water for 3 days, changing the dialysis solution 3-4 times a day; (1.5) Collect the purified solution in the dialysis bag and freeze-dry for 48 hours to obtain methacrylated hyaluronic acid co-modified with phenylboronic acid and mercaptoaspartic acid hexapeptide. The product is sealed and stored at -20℃ in a dry environment for later use.

3. The preparation method according to claim 2, wherein: The phenylboronic acid grafted with methacrylamide hyaluronic acid in step (1.1) is prepared by the following method: ① Dissolve 1 g of hyaluronic acid in 100 ml of deionized water, add 1 mL of methacrylic anhydride, adjust the pH to 8.0~8.5 with 1 mol / L NaOH solution, react for 2 h, then stir at 4 ℃ for 24 h, centrifuge, transfer the solution to a dialysis bag and dialyze with deionized water for 4 days, then freeze dry to obtain methacrylamide hyaluronic acid for later use. ② Dissolve 1g of methacrylamide hyaluronic acid in 100ml of deionized water, add 1.4g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, then add 0.11g of 3-aminophenylboronic acid, react in the dark for 24 hours, dialyze the reaction solution with deionized water for at least 3 days, and then freeze-dry to obtain phenylboronic acid grafted with methacrylamide hyaluronic acid.

4. The preparation method according to claim 2, wherein: The thiol-modified aspartic hexapeptide was prepared by the following method: (1) Weigh 0.18 g of 2-chlorotriphenylmethyl chloride resin, soak it in N,N-dimethylformamide for 30 min to swell, and then filter it. Add 0.37 g of fluorenemethoxycarbonyl-aspartic acid-tert-butyl ester, 0.13 mL of N,N-diisopropylethylamine and 10 mL of N,N-dimethylformamide to the resin, stir and react at room temperature for 2-3 h, filter it, and wash it three times each with N,N-dimethylformamide and dichloromethane to complete the first step of connecting the amino acid to the resin. (2) Repeat the following steps 5 times to complete peptide chain elongation: (2.1) Add a 20% (v / v) piperidine / N,N-dimethylformamide solution and deprotect at room temperature for 10 min; (2.2) Wash 5 times with N,N-dimethylformamide and 3 times with dichloromethane; (2.3) Fmoc-Asp(OtBu)-OH, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were dissolved in N,N-dimethylformamide and pre-activated for 5 min; (2.4) Add the activation solution to the resin and couple the reaction at room temperature for 1-2 h to complete the peptide chain elongation; (3) Replace Fmoc-Asp(OtBu)-OH with fluorenemethoxycarbonyl-cysteine-triphenylmethyl for coupling, and remove the fluorenemethoxycarbonyl protecting group according to the conditions in step (2.1) after coupling is completed; (4) Add a mixture of trifluoroacetic acid, thioanisole, water and triisopropylsilane in a volume ratio of 90:5:3:2 to the resin and stir at room temperature for 2-3 h; filter and collect the filtrate, and slowly drop the filtrate into 10 times the volume of pre-cooled ether, and let it stand to precipitate; centrifuge at 4000 rpm for 10 min, collect the precipitate, wash it 3 times with cold ether and then vacuum dry to obtain crude mercapto-modified aspartic acid hexapeptide; (5) The crude thiol-modified aspartic acid hexapeptide was dissolved in a 0.1% trifluoroacetic acid acetonitrile / water mixed solvent, purified by HPLC using a C18 column, the target elution peak was collected and freeze-dried to obtain the thiol-modified aspartic acid hexapeptide.

5. The preparation method according to claim 1, wherein: The natural organic compounds mentioned in step (2) are selected from: 50 mg dihydromyricetin or 10 mg gallic acid.

6. The preparation method according to claim 1, wherein: Step (3) involves irradiating the area with UV light for 30 seconds.

7. A photocrosslinked, bone-repairing injectable hydrogel prepared by the preparation method according to any one of claims 1 to 6.

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