A hydrogel scaffold and methods of making and using the same

Abstract

The present application relates to a kind of hydrogel scaffolds and its preparation method and application, the hydrogel is the short fiber reinforced hydrogel scaffold of bifunctional peptide loading.The hydrogel scaffold has good compression fatigue resistance, structural stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials, and particularly relates to a hydrogel scaffold and a preparation method and application thereof. BACKGROUND

[0002] Bone is a kind of hard and elastic hard tissue, which can provide mechanical support for the body and provide muscle attachment points for body movement. In addition, bone tissue also serves as a repository for calcium and phosphorus storage and bears the function of hematopoiesis of the body. However, bone tissue is easily damaged by external mechanical force, such as fracture, bone defect, or necrosis of bone tissue caused by tumors in the body, and thus the treatment of the damage of bone tissue is needed. At present, for fracture or non-critical bone tissue defect, steel nails, steel plates and other methods are usually used for auxiliary treatment, and for critical bone defect or bone tumor, bone scaffolds are usually used for auxiliary treatment. The commonly used bone scaffolds include titanium-nickel alloy scaffolds, autologous bone tissue scaffolds, allogeneic bone tissue scaffolds and the like. However, the titanium-nickel alloy scaffold lacks biological activity and is not degradable, and cannot realize in-situ regeneration of tissue; the autologous bone tissue scaffold has the disadvantages of limited donor source and secondary damage to the body caused by bone tissue sampling, thus limiting the application of autologous bone tissue.

[0003] With the development of tissue engineering scaffolds in the past few decades, they have gradually shown the advantages of personalized customization, good biocompatibility, and bone inductive activity in the field of bone tissue regeneration, and thus have attracted extensive attention and in-depth research of researchers. There are various technologies for preparing bone tissue engineering scaffolds, such as phase separation technology, short fiber-based freeze-drying technology, three-dimensional printing technology, gas foaming technology, and injectable hydrogel technology. The phase separation technology is limited to some high polymers such as PU (polyurethane) and PLA (polylactic acid); the three-dimensional bone scaffold prepared by the short fiber-based freeze-drying technology has poor mechanical properties; the bone scaffold prepared by the three-dimensional printing technology has a relatively complex process flow, and most three-dimensional printing technologies are based on high-temperature melting molding, which further limits the loading of bioactive substances; and the gas foaming technology can prepare three-dimensional scaffolds, but the process flow introduces toxic chemicals and relatively expensive equipment, which further limits its application in bone tissue engineering; and the hydrogel scaffold can be based on in-situ injection molding technology to introduce photosensitive groups into materials with good biocompatibility to prepare photosensitive hydrogels such as methacrylated gelatin (GM), methacrylated silk fibroin (SM), and methacrylated hyaluronic acid (HM), which are widely used in tissue damage repair.

[0004] Although the in-situ injectable hydrogel GM is widely used in wound repair, cartilage regeneration, and three-dimensional printing technology for bone regeneration, the mechanical properties of the GM after gelation are poor and the GM hydrogel is easy to break under stress, and the GM hydrogel itself does not have bone induction regeneration performance. SUMMARY

[0005] In view of the defects of the prior art, the technical problem to be solved by the present application is to provide a short fiber reinforced hydrogel scaffold loaded with bifunctional peptides and a preparation method and application thereof.

[0006] The preparation method of the hydrogel material of the present application comprises:

[0007] (1) AG@CP short nanofiber preparation: mixing polylactic acid PLA, gelatin Gel, peptide CP and solvent to obtain AG@CP spinning solution, electrospinning to obtain AG@CP nanofiber membrane, homogenizing treatment to obtain AG@CP short nanofiber suspension, and freeze-drying to obtain short AG@CP nanofiber;

[0008] (2) F127 solution and GM solution are respectively configured, and then the F-127 solution and the GM solution are mixed to obtain MF solution;

[0009] (3) mixing and pouring the MF solution, the peptide BP and the nanofiber of step (1), and crosslinking to obtain the hydrogel material.

[0010] The preferred mode of the above preparation method is as follows:

[0011] The preparation of the BP and CP functional peptides: based on the peptide synthesis reaction of amino acids, a short peptide BP for recruiting endogenous VEGF is synthesized, and a functional peptide CP with Ca 2+ performance is prepared.

[0012] The BP and CP are prepared by the peptide synthesis reaction of amino acids, wherein the sequence of the 12-amino-acid BP short peptide is DRVQRQTTTVVA, as shown in SEQ ID NO. 1, and the amino acid sequence of the 12-amino-acid CP short peptide is E-(Pip)-aH-(Acp)-(Acp)-(Acp)-Fv-(D-γ-E)-vt, as shown in SEQ ID NO. 2; but the functional peptide sequence can increase or decrease amino acids at both ends at present.

[0013] The amino acid sequence of the peptide CP in the step (1) is E-(Pip)-aH-(Acp)-(Acp)-(Acp)-Fv-(D-γ-E)-vt; the solvent is at least one of hexafluoroisopropanol HFIP and tetrahydrofuran, but is not limited thereto, and can also be other reagents that can dissolve high molecular compounds; the mass ratio of the polylactic acid PLA to the gelatin Gel is 3 / 7-5 / 5, and the concentration of the peptide CP in the spinning solution is 0.5-1.5 mg / mL.

[0014] The solid content of the AG@CP electrospinning solution in the step (1) can be 8-10 wt% (g / mL), and the CP loading content is 0.1-1 wt% (mg / mL).

[0015] The electrospinning process parameters in the step (1) are as follows: the voltage is 8-12 kV, the spinning solution propelling speed is 1.0-2.0 mL / h, the distance between the syringe needle and the receiving device during spinning is 8-15 cm, the receiving is performed by using a roller with a speed of 100-120 rpm, and the preparation time is 60-120 min; the homogenization treatment is as follows: the prepared nanofiber membrane is cut into pieces and placed in tert-butyl alcohol, the rotation speed is 8000-13000 rpm, and the homogenization time is 8-10 min, wherein the ratio of the nanofiber membrane to the tert-butyl alcohol is (1-2) g:(100-150) mL; the freeze-drying is as follows: the mixed liquid of the broken fibers is poured into a mold, after pouring, the mold is treated at (-75)~(-80) ℃ for 12-24 h, and then freeze-dried at (-20)~(-40) ℃ for 24-48 h.

[0016] The methacrylated gelatin GM solution in the step (2) further contains a photo initiator; the concentration of the photo initiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate salt LAP is 10-20 mg / mL; and the volume ratio of the F-127 solution to the GM solution is 1 / 9-2 / 8.

[0017] The polymer in the step (2) is F-127, the F-127 solution is obtained by dissolving F-127 in water at 2-7 ℃, wherein the solid content of the F-127 solution is 3-5 wt% (g / mL); and the GM solution is obtained by dissolving GM and a photo initiator in water at 35-37 ℃, wherein the solid content of the GM solution is 10-15 wt% (g / mL).

[0018] The mixing in the step (2) is performed by heating the F-127 aqueous solution to 18-20 ℃ and cooling the GM-containing aqueous solution to 25-27 ℃, and the volume ratio of the F-127 aqueous solution to the GM-containing aqueous solution is 1 / 9-2 / 8.

[0019] The difference in dissolution temperatures between GM and F-127 is because GM dissolves easily at high temperatures, while F-127 dissolves at low temperatures; using a temperature of 25℃ helps to ensure that the two components are mixed without phase separation.

[0020] The amino acid sequence of peptide BP in step (3) is DRVQRQTTTVVA; after mixing MF solution, peptide BP and nanofibers, the content of peptide BP is 0.1-1 wt% (mg / mL) and the content of nanofibers is 1-10 wt% (mg / mL).

[0021] The casting in step (3) is either mold casting or in-situ casting; the crosslinking is 405nm laser crosslinking for 0.5-1.5 min.

[0022] Hydrogel scaffolds with specific shapes can be prepared by casting using molds, or irregularly filled hydrogel scaffolds can be prepared by in-situ casting of tissue defects.

[0023] Furthermore, the hydrogel scaffold can be prepared by casting with a mold and then cross-linked to form a hydrogel scaffold with a specific shape. Alternatively, it can be prepared by in-situ casting of tissue defects to form an irregularly filled hydrogel scaffold (in-situ casting followed by 405nm laser cross-linking to form an in-situ filled irregularly filled tissue engineering scaffold).

[0024] A hydrogel material prepared by the method of the present invention.

[0025] The present invention relates to the application of the hydrogel material in the preparation of bone tissue damage modification materials.

[0026] This invention loads CP into nanofibers and prepares a hydrogel with MF, which helps to build a long-lasting sustained-release mechanism, delaying the action of CP. It can also be combined with BP to build a time-series sustained-release system, thereby combining the characteristics of bone tissue regeneration.

[0027] In this invention, GM and F-127 are compounded to form a copolymer, which can prepare a mechanical matrix scaffold with strong resistance to compressive fatigue. Combining electrospinning technology and short fiber preparation technology based on nanofiber membranes, CP is loaded onto AG (PLA / Gel) short nanofibers via electrospinning, and then prepared into AG@CP short nanofiber powder using homogenization and freeze-drying techniques. Figure 1 By loading CP short nanofibers and BP blends onto an MF mechanical matrix scaffold, a dual loading effect of BP and CP components can be achieved. Figure 2). Since CP is loaded in AG short nanofiber, its release needs to be released from short fiber to hydrogel, and then from hydrogel to the microenvironment of the tissue to be repaired, which helps to build a double time-release system with BP. This is because in the regeneration of bone tissue defects, the bone tissue preferentially builds a vascular network structure, which in turn promotes the mineralization of the bone tissue scaffold. Therefore, GM and F-127 are compounded to prepare a hydrogel solution, and short fibers loaded with CP are mixed with BP in the hydrogel solution, and then prepared into MF-AG@CBP bone tissue engineering scaffolds by photo-crosslinking agent.

[0028] Beneficial effects

[0029] (1) Based on the relationship between amino acid sequence and biological function, the BP with promoting blood vessel regeneration and the Ca 2+ performance of CP short peptide;

[0030] (2) Compared with short calcium peptide (E-(Pip)-aH) and (Fv-(D-γ-E)-vt), the 12-amino acid calcium peptide CP (E-(Pip)-aH-(Acp)-(Acp)-(Acp)-Fv-(D-γ-E)-vt) has stronger Ca 2+ recruitment performance due to the increase of Ca 2+ binding sites, and the molecular structure is also more stable due to the lengthening of the sequence;

[0031] (3) The GM hydrogel alone has poor compression cycle resistance, and after compounding with F-127 components, it has good compression fatigue resistance Figure 5 );

[0032] (4) CP is loaded in AG short nanofiber, which is further dispersed in a functional peptide BP-containing hydrogel solution to prepare a hydrogel by photo-crosslinking. The BP can play a role in the early stage of the tissue engineering scaffold, and since the CP slow release needs to go through two processes of fiber-hydrogel and hydrogel-tissue microenvironment, it can form a time-release system to meet the characteristics of blood vessel regeneration and tissue mineralization in bone tissue regeneration Figure 6 );

[0033] (5) The introduction of AG@CP short nanofiber in the hydrogel can further enhance the mechanical properties of the hydrogel, and also help to weaken the swelling rate of the hydrogel, thereby increasing the structural stability of the hydrogel scaffold. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 AG@CP short fiber preparation schematic diagram;

[0035] Figure 2 MF-AG@BCP hydrogel preparation process schematic diagram;

[0036] Figure 3 Hydrogel solution of Examples 1-4;

[0037] Figure 4 Hydrogel of Examples 1-4;

[0038] Figure 5 Compression mechanical stress-strain properties of hydrogels of Examples 1-4;

[0039] Figure 6 CT images of hydrogels of Examples 1-4 repairing skull for 6 weeks, with simple skull defects as a control group. DETAILED DESCRIPTION

[0040] The application will be further described below in connection with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content taught by the application, and these equivalent forms also fall within the scope of the appended claims.

[0041] Test: Place a cylindrical hydrogel with a diameter of 8 mm and a height of 10 mm on the test bench, and compress it downward at a speed of 5 mm / min until the hydrogel cylinder breaks. Analyze the stress-strain curve by software, and the maximum stress corresponds to the highest point on the curve.

[0042] Example 1

[0043] Preparation of GM hydrogel

[0044] (1) Preparation of GM solution: Dissolve 0.05 mg of LAP (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., 85073-19-4, 250 mg) photoinducer in 2 mL of deionized water to prepare a 0.025 mg / mL inducer stock solution. Dissolve 1 g of GM (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-GM-RF90, 1 g) and 1 mL of LAP photoinducer stock solution in 9 mL of deionized water, and stir at 37°C for 0.5 h to prepare a GM solution containing LAP inducer (as shown in Figure 3 ).

[0045] (2) Preparation of hydrogel: Pour the GM solution prepared in step (1) into a mold, or inject it in situ at the site of tissue defect, and then use a 405 nm laser to treat it for 1 min to obtain a GM hydrogel Figure 4 ).

[0046] (3) GM hydrogel: The hydrogel of this example has high transparency (as shown in Figure 4The hydrogel swelling rate is 25% at 24h in vitro swelling test, but the compression resistance is poor and the hydrogel is easy to break under uneven stress (as shown in Figure 5 The skull repair effect is general compared with the control group (as shown in Figure 6

[0047] Example 2

[0048] Preparation of MF hydrogel

[0049] (1) Preparation of GM solution: 0.05 mg of LAP (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., 85073-19-4, 250 mg photoinduced agent) was dissolved in 2 mL of deionized water to prepare a 0.025 mg / mL photoinduced agent stock solution. 1 g of GM (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-GM-RF90, 1 g) and 1 mL of LAP photoinduced agent stock solution were dispersed in 9 mL of deionized water, and the mixture was stirred at 37°C for 0.5 h to prepare a GM solution containing LAP photoinduced agent for standby;

[0050] (2) Preparation of F-127 solution: 3 g of F-127 (National Pharmaceutical Group Chemical Reagent Co., Ltd., 9003-11-6, 50 g) was dissolved in deionized water at 4°C to prepare a 3 wt% F-127 aqueous solution for standby;

[0051] (3) Preparation of MF solution: The prepared F-127 aqueous solution was heated to 20°C, and the GM aqueous solution was cooled to 25°C. Then, the F-127 aqueous solution and the GM aqueous solution were mixed by stirring according to the volume ratio (F-127 / GM, 1 / 9), and the mixture was stored at 25°C for standby (as shown in Figure 3

[0052] (4) Preparation of hydrogel: The MF solution prepared in step (3) was poured into a mold or injected in situ at the site of tissue defect, and then treated with 405 nm laser for 1.5 min to obtain MF hydrogel (as shown in Figure 4

[0053] (5) MF hydrogel: The prepared MF hydrogel has a certain degree of decrease in transparency (as shown in Figure 4 The mechanical compression resistance of the hydrogel itself is significantly improved, which is helpful for constructing a tissue engineering scaffold with optimized mechanical properties. The maximum compression stress is 405 KPa (as shown in Figure 5 The hydrogel swelling rate is 25% at 24h in vitro swelling test, but the compression resistance is poor and the hydrogel is easy to break under uneven stress (as shown in Figure 6

[0054] Example 3​​​​

[0055] MF-AG hydrogel preparation

[0056] (1) GM solution preparation: 0.05 mg of LAP (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., 85073-19-4, 250 mg photoinducer) was dissolved in 2 mL of deionized water to prepare a photoinducer stock solution with a concentration of 0.025 mg / mL. 1 g of GM (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-GM-RF90, 1 g) and 1 mL of LAP photoinducer stock solution were dispersed in 9 mL of deionized water, and the mixture was stirred at 37°C for 0.5 h to prepare a GM solution containing LAP photoinducer for standby;

[0057] (2) F-127 solution preparation: 3 g of F-127 (National Pharmaceutical Group Chemical Reagent Co., Ltd., 9003-11-6, 50 g) was dissolved in deionized water at 4°C to prepare a F-127 aqueous solution with a solid content of 3 wt% for standby;

[0058] (3) MF solution preparation: The prepared F-127 aqueous solution was heated to 20°C, and the GM aqueous solution was cooled to 25°C. Then, the F-127 aqueous solution and the GM aqueous solution were mixed by stirring according to the volume ratio (F-127 / GM, 1 / 9), and the mixture was stored at 25°C for standby;

[0059] (4) AG short nanofiber preparation: 0.6 g of polylactic acid PLA (molecular weight 300000, Guangzhou Maipu Regenerative Medicine Co., Ltd.) and 0.4 g of gelatin Gel (Beijing Innochem, B76801) were dispersed in 10 mL of HFIP and stirred for 24 h to obtain an electrospinning solution. Then, the electrospinning solution was prepared into a nanofiber membrane by electrospinning, with a voltage of 10 kV, a spinning solution pushing speed of 1.0 mL / h, a distance between the syringe needle and the receiving device of 10 cm during spinning, and a receiving speed of 120 rpm. The collection time was 120 min. The collected AG nanofiber membrane was prepared into an AG short nanofiber suspension by homogenization, with a rotation speed of 10000 rpm and a homogenization time of 8 min. The AG short nanofiber suspension was prepared into AG short nanofiber powder by freeze-drying, with the fiber mixture poured into a mold, and then placed in a refrigerator at -80°C for 12 h. Then, the mixture was placed in a freeze-drying machine at -20°C for 48 h to completely freeze-dry to obtain AG short nanofiber;

[0060] (5) MF-AG solution preparation: 5 mg of AG short nanofiber was added to 10 mL of MF solution, and the mixture was mixed well to prepare a MF-AG solution for standby (as shown in Figure 3 ;

[0061] (6) Hydrogel preparation: The MF-AG solution prepared in step (5) was poured into a mold or injected in situ at the site of tissue defect, and then a MF-AG hydrogel was obtained after 405 nm laser treatment for 1.5 min (as shown in Figure 4 );

[0062] (7) MF-AG hydrogel: The prepared MF-AG hydrogel had further decreased transparency compared to the MF hydrogel ( Figure 4 ), but the mechanical compression resistance of the hydrogel itself was further improved, and the mechanical properties could be further optimized, with a maximum compression stress of 472 KPa (as shown in Figure 5 ); the swelling rate of the hydrogel was 15% at 24 h in the in vitro swelling test, and the introduction of short fibers helped to weaken the swelling rate; compared with the control group, the skull repair effect was general (as shown in Figure 6 ).

[0063] Example 4

[0064] MF-AG@CBP hydrogel preparation

[0065] (1) GM solution preparation: 0.05 mg of LAP (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., 85073-19-4, 250 mg of light inducer was dissolved in 2 mL of deionized water to prepare a 0.025 mg / mL inducer stock solution, 1 g of GM (Suzhou Yongqinquan Intelligent Equipment Co., Ltd., EFL-GM-RF90, 1 g) and 1 mL of LAP light inducer stock solution were dispersed in 9 mL of deionized water, and the mixture was stirred at 37°C for 0.5 h to prepare a GM solution containing LAP inducer for use;

[0066] (2) F-127 solution preparation: 3 g of F-127 (National Pharmaceutical Group Chemical Reagent Co., Ltd., 9003-11-6, 50 g) was dissolved in deionized water at 4°C to prepare a 3 wt% F-127 aqueous solution for use;

[0067] (3) MF solution preparation: The prepared F-127 aqueous solution was heated to 20°C, and the GM aqueous solution was cooled to 25°C. Then, the F-127 aqueous solution and the GM aqueous solution were mixed by stirring according to the volume ratio (F-127 / GM, 1 / 9), and the mixture was stored at 25°C for use;

[0068] (4) AG@CP short nanofiber preparation: 0.6 polylactic acid PLA (molecular weight 300000, Guangzhou Maipu Regenerative Medicine Co., Ltd.), 0.4 g gelatin Gel (Beijing innochem, B76801) and 1 mg CP peptide (provided by Qiangyao Biotechnology Co., Ltd.) were dispersed in 10 mL HFIP and stirred for 24 h to obtain an electrospinning solution; then the electrospinning solution was prepared into a nanofiber membrane by electrospinning, wherein the voltage was 10 kV, the spinning solution pushing speed was 1.0 mL / h, the distance between the syringe needle and the receiving device during spinning was 10 cm, the receiving was carried out by a roller with a speed of 120 rpm, and the preparation time was 120 min; the collected AG@CP nanofiber membrane was prepared into AG@CP short nanofiber suspension by homogenization, wherein the rotation speed was 10000 rpm and the homogenization time was 8 min; the AG@CP short nanofiber suspension was prepared into AG@CP short nanofiber powder by freeze-drying, wherein the crushed fiber mixture was poured into a mold, and after pouring, it was placed in a refrigerator at -80°C for 12 h, then placed in a freeze-drying machine at -20°C for 48 h to completely freeze-dry to obtain AG@CP short nanofiber;

[0069] (5) MF-AG@CBP solution preparation: 5 mg of AG@CP short nanofiber and 0.1 mg of functional peptide BP (provided by Qiangyao Biotechnology Co., Ltd.) were added to 10 mL of MF solution, and the MF-AG@CBP solution was prepared by thoroughly mixing (as shown in Figure 3 );

[0070] (6) Hydrogel preparation: the MF-AG@CBP solution prepared in step (5) was poured into a mold or injected in situ at the site of tissue defect, and then MF-AG@CBP hydrogel was obtained after 405 nm laser treatment for 1.5 min (as shown in Figure 4 );

[0071] (7) MF-AG@CBP hydrogel: the prepared MF-AG@CBP hydrogel had further decreased transparency compared to MF hydrogel (as shown in Figure 4 ); however, the mechanical compression resistance of the hydrogel itself was further improved, and the maximum compression stress was 500 KPa (as shown in Figure 5 ); in the in vitro swelling test for 24 h, the swelling rate of the hydrogel was 16%, and the introduction of short fibers helped to weaken the swelling rate; in addition, the BP loaded in the MF-AG@CBP hydrogel scaffold could efficiently promote blood vessel regeneration, and the CP calcium peptide had the performance of significantly recruiting Ca 2+ , and the skull repair effect was significantly improved compared with the control group (as shown in Figure 6 ), thus showing good application potential in bone tissue regeneration.

Claims

1. A method for preparing a hydrogel material, comprising: (1) Polylactic acid (PLA), gelatin gel, peptide CP, and solvent are mixed to obtain a spinning solution. Electrospinning is performed to obtain a nanofiber membrane. The membrane is homogenized and freeze-dried to obtain nanofibers. The amino acid sequence of peptide CP is E-(Pip)-aH-(Acp)-(Acp)-(Acp)-Fv-(D-γ-E)-vt. The solvent is at least one of hexafluoroisopropanol (HFIP) and tetrahydrofuran. The mass ratio of PLA to gelatin gel is 3 / 7-5 / 5, and the concentration of peptide CP in the spinning solution is 0.5-1.5 mg / mL. (2) Prepare F127 solution and methacrylated gelatin solution separately, and then mix F127 solution and methacrylated gelatin solution to obtain MF solution; wherein the solid content of methacrylated gelatin solution is 10-15 wt%; (3) Mix the MF solution, peptide BP and nanofibers from step (1) and cast them together to crosslink them and obtain a hydrogel material; the amino acid sequence of the peptide BP is DRVQRQTTTVVA; after mixing the MF solution, peptide BP and nanofibers, the peptide BP content is 0.1-1wt% and the nanofiber content is 1-10wt%.

2. The preparation method according to claim 1, characterized in that, The electrospinning process parameters in step (1) are as follows: voltage 8-12kV, spinning solution propulsion speed 1.0-2.0mL / h, distance between syringe needle and receiving device during spinning 8-15cm, receiving with roller at speed of 100-120rpm, preparation time 60-120min; the homogenization treatment is as follows: the prepared nanofiber membrane is cut into fragments and placed in tert-butanol, rotation speed 8000-13000rpm, homogenization time 8-10min, wherein the ratio of nanofiber membrane to tert-butanol is (1-2)g:(100-150)mL; the freeze-drying is as follows: treatment at (-75)~(-80)℃ for 12-24h, and then freeze-drying at (-20)~(-40)℃ for 24-48h.

3. The preparation method according to claim 1, characterized in that, The methacrylated gelatin solution in step (2) also contains a photoinducer with a concentration of 10-20 mg / mL; the photoinducer is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate LAP; the volume ratio of the F-127 solution and the methacrylated gelatin solution is 1 / 9-2 / 8.

4. The preparation method according to claim 1, characterized in that, In step (2), the F-127 solution is obtained by dissolving F-127 in water at 2-7℃, wherein the solid content of the F-127 solution is 3-5wt%; the methacrylated gelatin solution is obtained by dissolving methacrylated gelatin and a photoinducer in water at 35-37℃.

5. The preparation method according to claim 3, characterized in that, In step (2), the mixing process involves heating the F-127 aqueous solution to 18-20°C and cooling the methacrylated gelatin aqueous solution to 25-27°C before mixing. The volume ratio of the F-127 aqueous solution to the methacrylated gelatin aqueous solution is 1 / 9 to 2 / 8.

6. The preparation method according to claim 1, characterized in that, The casting in step (3) is either mold casting or in-situ casting; the crosslinking is 405 nm laser crosslinking for 0.5-1.5 min.

7. A hydrogel material prepared by the method of claim 1.

8. The use of the hydrogel material of claim 7 in the preparation of bone tissue damage repair materials.

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