Preparation method of bionic hydrogel for promoting epiphyseal plate repair by bidirectionally inducing BMSCs (bone mesenchymal stem cells) through regulation and control mechanics

Abstract

The invention relates to the technical field of biomedical materials, and particularly discloses a preparation method of bionic hydrogel capable of bidirectionally inducing BMSCs (bone marrow mesenchymal stem cells) to promote epiphyseal plate repair through regulation and control mechanics, which comprises the following steps: step 1, mixing 10wt% of methacrylated gelatin, 10wt% of methacrylated decellularized cartilage matrix and 0.5 wt% of phenyl (2, 4, 6-trimethyl-1, 3-pentanediol) according to a ratio of the methacrylated gelatin to the methacrylated The preparation method comprises the following steps: respectively preparing methacrylated gelatin and methacrylated acellular cartilage matrix pre-polymerization solutions according to the concentration of (1, 2, 6-trimethylbenzoyl) lithium phosphate, and carrying out 37 DEG C constant-temperature dark water bath until the methacrylated gelatin and methacrylated acellular cartilage matrix pre-polymerization solutions are completely dissolved for later use; and 2, mixing the 10 wt% methacrylated decellularized cartilage matrix prepolymer solution and the 10 wt% methacrylated gelatin prepolymer solution according to a ratio of 3: 7 to obtain a methacrylated gelatin / methacrylated decellularized cartilage matrix prepolymer solution, carrying out cross-linking under blue light with a wavelength of 365 nm for 5-20 s, and carrying out drying to obtain the methacrylated gelatin / methacrylated decellularized cartilage matrix. The methylacryloylated gelatin / methylacryloylated acellular cartilage matrix composite hydrogel is prepared from the methylacryloylated gelatin and the methylacryloylated acellular cartilage matrix.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and specifically to a method for preparing a bionic hydrogel for promoting epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs. Background Art

[0002] The epiphyseal plate (growth plate) is a cartilage area at the end of a child's long bone that controls the longitudinal growth of the bone. Its damage may be caused by fractures, infections, tumors or iatrogenic injuries, accounting for about 15%-30% of bone injuries in children. Once the epiphyseal plate is damaged, the epiphyseal cartilage tissue will gradually be replaced by bone tissue to form a "bone bridge", which often leads to unbalanced bone growth, such as growth retardation, angulation or rotational deformity, and even affects joint function, which has a huge impact on the physical and mental health of children. In clinical practice, bone bridge resection combined with material filling is often used to prevent the recurrence of bone bridges and allow the surrounding undamaged epiphyseal tissue to restore the longitudinal growth ability of bones, but the success rate is less than 30%. Research from the Mayo Clinic shows that up to 71% of patients require additional surgery after surgery, which adds a huge burden to families and society.

[0003] Therefore, the use of cartilage tissue engineering technology to construct bioactive epiphyseal cartilage has become a new research direction for the treatment of epiphyseal injuries in children. Its core is the ternary model of "seed cells + drugs or cytokines + scaffold materials", which uses chondrocytes or bone marrow mesenchymal stem cells (BMSCs) combined with scaffolds to achieve cartilage regeneration. However, the transplanted cells, drugs or growth factors used in current research mainly focus on how to promote chondrogenic differentiation and regenerate cartilage tissue, ignoring the prevention of local osteogenic differentiation and bone bridge formation after epiphyseal injury from the source in the early stage of injury. In addition, the sources of scaffold materials are mainly divided into two categories: natural materials and synthetic materials. Both have their own advantages and disadvantages, but it is difficult for them to fully meet the growth environment of natural epiphyseal tissues, and their microstructures are mostly inconsistent with the physiological structural characteristics of epiphyseal plates, resulting in disordered accumulation of regenerated chondrocytes, which cannot play their biological function of controlling the longitudinal growth of osteoblasts; in terms of scaffold components, cell transplantation therapy, such as mesenchymal stem cell (MSCs) transplantation, autologous cell transplantation and direct chondrocyte transplantation, is an effective method. MSCs have the ability to self-renew and differentiate, but their survival rate after transplantation is low and their cartilage differentiation is uncontrolled. The traditional view is that the cartilage differentiation of MSCs requires cytokine induction, such as TGF-β, but its non-specific effects are significant, and it is dose- and time-dependent, and the results are unstable.

[0004] Therefore, alternative strategies are needed to promote MSCs chondrogenesis and survival rate. In terms of scaffold materials, the ideal scaffold should have components similar to those of epiphyseal plate tissue, three-dimensional porous structure, mechanical strength and biocompatibility. The extracellular matrix (ECM) derived from the epiphyseal plate is rich in a variety of bioactive proteins and growth factors, providing cells with a suitable growth environment and guiding cell differentiation. Studies have shown that ECM scaffolds have been successfully used in muscle, bone and articular cartilage regeneration. ECM has advantages as a scaffold material for epiphyseal plate tissue engineering in epiphyseal plate structure reconstruction. Li WC et al. used a scaffold made of decellularized epiphyseal cartilage extracellular matrix (ACM) to treat epiphyseal plate injuries in white rabbits. The results showed that the scaffold formed a longitudinal columnar structure, cells grew well, and no bone bridges were formed. However, the mechanical properties of the pure ACM component are poor, the degradation-regeneration rate is not matched, and the regenerated tissue is prone to collapse or deformed growth. It cannot be used as the main body of the material to carry seed cells for epiphyseal plate repair, which limits its application. For this reason, a method for preparing a biomimetic hydrogel that promotes epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs is provided. Summary of the invention

[0005] The purpose of the present invention is to address the defects of the prior art and provide a method for preparing a bionic hydrogel that promotes epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs, so as to solve the problems raised by the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing a bionic hydrogel that promotes epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs, characterized in that: the specific steps are as follows:

[0007] Step 1: According to the concentrations of 10wt% methacryloyl gelatin (GelMA), 10wt% methacryloyl acellular cartilage matrix (ACMMA) and 0.5wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), respectively prepare (GelMA) and (ACMMA) prepolymer solutions, and place them in a constant temperature, light-proof water bath at 37°C until they are completely dissolved and then set aside;

[0008] Step 2: Mix 10wt% methacrylylated gelatin (GelMA) prepolymer solution with 10wt% methacrylylated decellularized cartilage matrix (ACMMA) prepolymer solution to obtain methacrylylated gelatin (GelMA) / methacrylylated decellularized cartilage matrix (ACMMA) prepolymer solution, and cross-link it under blue light of 365nm wavelength for 5-20s to prepare methacrylylated gelatin (GelMA) / methacrylylated decellularized cartilage matrix (ACMMA) composite hydrogel.

[0009] As a preferred technical solution of the present invention, the mixing ratio of the methacrylylated decellularized cartilage matrix and the methacrylylated gelatin in step 2 is 3:7.

[0010] An application of the bionic hydrogel prepared by the above-mentioned preparation method in the production of a material for promoting the regeneration and repair of epiphyseal cartilage.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention discloses an injectable bionic hydrogel and a preparation method thereof for promoting epiphyseal plate damage repair by regulating mechanical bidirectional induction of bone marrow mesenchymal stem cells (BMSCs). First, a methacrylated decellularized cartilage matrix prepared by the method disclosed in the present application is uniformly mixed with GelMA in a ratio of 3:7, and after cross-linking for 5-20 seconds under blue light of a wavelength of 365nm, a composite hydrogel is prepared, and BMSCs are mixed in the hydrogel; the hydrogel is then injected into the epiphyseal plate cartilage damage site, and the differentiation of BMSCs into chondrocytes is promoted by simulating the mechanics of the natural epiphyseal plate extracellular matrix and the natural epiphyseal plate, and then the regeneration and repair of the epiphyseal plate cartilage is promoted, thereby providing a new cartilage tissue engineering strategy for the clinical treatment of epiphyseal plate damage repair;

[0013] The overall implementation process of this method is relatively simple, with a wide range of sources, strong operability, great market application potential, and is conducive to promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the preparation and characterization results of ACMMA.

[0015] Figure 1 A is a schematic diagram of the preparation of ACMMA of the present invention, Figure 1 B is ACMMA 1 H NMR, Figure 1 C is the FTIR spectrum of ACMMA, Figure 1 D is the schematic diagram of 10% ACMMA gelation. Figure 1 E is a schematic diagram of the injectability of ACMMA;

[0016] Figure 2 These are the material characterization results of the ACMMA-GelMA composite hydrogels with different proportions in the present invention.

[0017] Figure 2 A- Figure 2 B is the stress-strain curves and elastic modulus quantitative diagrams of 11 groups of ACMMA-GelMA composite hydrogels with different ratios. Figure 2 C is the scanning electron micrograph of the four groups of ACMMA-GelMA composite hydrogels with different ratios screened for the first time. Figure 2 D- Figure 2 E are the degradation and swelling results of four groups of ACMMA-GelMA composite hydrogels with different ratios. Figure 2 F- Figure 2G are the shear thinning and energy storage loss results of four groups of ACMMA-GelMA composite hydrogels with different ratios;

[0018] Figure 3 These are the material characterization results of the G7A3 composite hydrogel with different photocrosslinking times of the present invention.

[0019] Figure 3 A- Figure 3 B is the stress-strain curve and elastic modulus quantitative diagram of G7A3 composite hydrogel with different photocrosslinking times. Figure 3 C is the energy storage loss result diagram of the G7A3 composite hydrogel with different photocrosslinking times that were finally screened out. Figure 3 D is the swelling result of the G7A3 composite hydrogel with different photocrosslinking times that were finally screened out. Figure 3 E is the scanning electron microscopy image of the G7A3 composite hydrogel with different photocrosslinking times that were finally screened;

[0020] Figure 4 The biocompatibility results of the composite hydrogels with photocrosslinking times of 5S and 20S of the present invention are shown.

[0021] Figure 4 A is the live-dead staining image of the composite hydrogel with photocrosslinking time of 5S and 20S. Figure 4 B is the quantitative graph of CCK-8 detection of composite hydrogel with photocrosslinking time of 5S and 20S;

[0022] Figure 5 To investigate the effects of the composite hydrogels with photocrosslinking times of 5S and 20S on the cell behavior and chondrogenic differentiation of RBMSCs, RT-qPCR was performed on genes related to chondrogenesis (SOX9, COL2A1, PRG4), osteogenesis (COL1A1, RUNX2, OCN) and hypertrophic cartilage (COLX). DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0024] Example:

[0025] 1: Preparation of methacrylic anhydride modified acellular cartilage matrix;

[0026] ① Obtain pig ears from the local vegetable market, peel the pig ears, and clean the outer skin as much as possible. Then cut the remaining ear cartilage tissue into small pieces and rinse them with deionized water. Then, freeze-dry these cartilage pieces and grind them into powder using an automatic sample freeze grinder;

[0027] ② First, dissolve 0.5% pancreatin and 0.02% Na2EDTA in 100mL PBS to prepare pancreatin solution. Place cartilage powder in an appropriate amount of pancreatin solution and shake on a 37°C constant temperature shaker for 24 hours. Change the digestion solution every 4 hours. After digestion, centrifuge at 4000rpm for 10 minutes, remove the supernatant and take the precipitate for subsequent experiments;

[0028] ③Secondly, add 50U / mL of deoxyribonuclease, 1U / mL of ribonuclease and 1mL of 1MTris-HCl buffer into PBS, make up to 100mL, add appropriate amount to the precipitate in step ②, shake and digest on a constant temperature shaker at 37℃ for 4 hours, and then centrifuge to obtain the precipitate for subsequent experiments;

[0029] ④ Next, take 1 mL of 1M Tris-HCl buffer and add it to 99 mL of PBS to obtain 10 mM Tris-HCl, then take 39 mL of it and add 1 mg of aprotinin to prepare a 100 KIU / mL aprotinin solution. Transfer the above precipitate to the aprotinin solution, resuspend it on a constant temperature shaker, shake and elute for 20 hours, and then centrifuge to obtain the precipitate;

[0030] ⑤ After eluting the precipitate from step ④ with 1% TritonX-100 in PBS for 24 hours, centrifuge and take out the precipitate, mix and wash it 3-4 times with ultrapure water, freeze-dry and weigh it;

[0031] ⑥ After adding pepsin at 3 mg / mL to 100 mL PBS, adjust the solution pH to 2-3 with glacial acetic acid. Add the freeze-dried decellularized precipitate to the pepsin solution at 25 mg / mL, stir and enzymolyze at 37°C for 24 hours, centrifuge and discard the precipitate, and take the viscous solution; adjust the solution pH to neutral with 5M NaOH solution, transfer to a 3500D dialysis bag, dialyze in ultrapure water for 3 days, freeze and freeze-dry the dialyzed solution to obtain decellularized cartilage matrix (ACM);

[0032] ⑦ Take 1g ACM and dissolve it with 10mL PBS solution, then pour it into a three-necked flask and stir it in a 37℃ water bath. At the same time, use a disposable syringe to slowly drip 1-1.5mL methacrylic anhydride (MA). During the reaction, use 5M NaOH solution to keep the pH of the solution between 8-10; after about 3 hours of reaction, add PBS with a pH of 8.5 to mix and terminate the reaction, and transfer the emulsion to a centrifuge tube, collect the supernatant solution by centrifugation; after adjusting the pH of the supernatant to neutral, transfer it to a 3500D dialysis bag and dialyze for at least 5 days, changing the dialysis water every day to remove unreacted methacrylic anhydride. Freeze-dry the dialyzed solution to constant weight, which is methacrylated decellularized cartilage matrix (ACMMA);

[0033] ⑧ The molecular structure of ACMMA was tested using Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The results are as follows Figure 1 As shown;

[0034] ⑨ In order to evaluate the photo-crosslinking performance of ACMMA bio-ink, 0.5%wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) was prepared with PBS and ACMMA was dissolved therein to make the concentration of ACMMA prepolymer solution 10%wt%. The prepolymer solution was transferred to a round-bottom glass bottle, which was tilted and irradiated with blue light (365nm) for 5 seconds to observe the flow of the hydrogel and preliminarily evaluate the photo-crosslinking performance of the ACMMA. Figure 1 B shows that after MA modification, the target group appears on the ACMMA molecular structure, representing the increase of the signal peaks of the double bond protons (=CH2) of methacrylic anhydride at 5.4 and 5.6 ppm; Figure 1 C shows that the stretching vibration of the NH bond is mainly located at 3318 cm -1 、3075cm -1 ; 1654cm -1 The peak at 1542 cm is mainly caused by the C=O vibration in the conjugated amide group; -1 The peak at 1029 cm-1 corresponds to the coupling of NH bending vibration and CN stretching vibration, indicating the formation of amide bond; the peak at 1029 cm-1 corresponds to the COC stretching vibration of methacrylic acid group. Figure 1 D shows that the ACMMA hydrogel prepolymer solution is a flowing translucent liquid before irradiation, and turns into a translucent gel after being irradiated with blue light of wavelength 365nm for 5 seconds. Figure 1 E shows that the hydrogel can be delivered by syringe before light curing, and the morphology is controllable. The above results all prove that the ACMMA prepolymer solution can be injected into the epiphyseal defect with complex geometry through a syringe and can be cured into gel in situ.

[0035] 2. Preparation of ACMMA / GelMA injectable biomimetic composite hydrogel;

[0036] ① Prepare GelMA and ACMMA prepolymer solutions according to the concentrations of 10wt% GelMA, 10wt% ACMMA and 0.5wt% LAP, respectively, and place in a constant temperature, light-proof water bath at 37°C until completely dissolved and set aside.

[0037] ② 10 wt % GelMA prepolymer solution and 10 wt % ACMMA prepolymer solution were first mixed in different proportions to obtain 11 groups of GelMA / ACMMA prepolymer solutions, which were then cross-linked under 365 nm wavelength blue light to prepare 11 groups of GelMA / ACMMA composite hydrogels with different proportions.

[0038] ③ First, the elastic modulus of these 11 groups of GelMA / ACMMA hydrogels was characterized by stress-strain testing. After the elastic modulus range that was suitable for cartilage growth was preliminarily screened out, scanning electron microscopy was used to observe the cross-sectional morphology, swelling, degradation, shear thinning, and storage modulus / loss modulus of the freeze-dried hydrogels. Figure 2 .

[0039] Depend on Figure 2 A- Figure 2 B shows that the elastic modulus of these 11 groups of GelMA / ACMMA hydrogels increases with the increase of GelMA ratio. Combined with the literature (①Biodegradable Dual-Cross-Linked Hydrogels with Stem CellDifferentiation Regulatory Properties Promote Growth Plate Injury Repair viaControllable Three-Dimensional Mechanics and a Cartilage-like ExtracellularMatrix.②Harnessing traction-mediated manipulation of the cell / matrixinterface to control stem-cell fate.), 4 groups of GelMA / ACMMA hydrogels with elastic modulus between 5-15kPa were preliminarily screened out to be in the elastic modulus range suitable for cartilage growth, and they were named G4A6, G5A5, G6A4, and G7A3. Figure 2 C- Figure 2It can be seen from G that the four groups of G4A6, G5A5, G6A4, and G7A3 all have porous structures. They swell in PBS for 24 hours, reaching 9.2±0.5, 9.25±0.22, 9.64±0.41, and 10.49±0.44 times of their dry weight, respectively. After being immersed in PBS at a constant temperature for 28 days, they lose 57%±2%, 61%±3%, 75%±3%, and 82%±2% of their dry weight, respectively. The kinematic viscosity of the four groups of prepolymer solutions is moderate and can be smoothly extruded. They all have relatively stable storage modulus (G') and loss modulus (G'). Regardless of the angular frequency, the curves of G' and G' are There is no tendency to converge and G' is much larger than the loss modulus G". The above results show that the four groups of hydrogels are internally stable gel-state solids with high hydrophilicity and interconnected porous structure. The in vitro degradation rate and elastic modulus are consistent with the rate of epiphyseal cartilage regeneration and differentiation. In order to further explore the effect of hydrogel matrix viscoelasticity on the repair of growth plate injury, the G7A3 group of composite hydrogels was finally selected to encapsulate RBMSCs. By changing the photo-cross-linking time, composite hydrogels with different viscoelasticity were obtained. The effect of composite hydrogels with different viscoelasticity on the differentiation of RBMSCs was studied to explore the effect of growth plate repair.

[0040] ④ 10 wt % GelMA prepolymer solution and 10 wt % ACMMA prepolymer solution were mixed in a ratio of 7:3 to form a prepolymer solution, and then cross-linked for 5S, 10S, 20S, and 30S under a blue light of 365 nm wavelength to prepare 4 groups of G7A3 (GelMA: ACMMA = 7:3) composite hydrogels with different photocrosslinking times. Figure 3 .Depend on Figure 3 A- Figure 3 B shows that as the photocrosslinking time increases, the elastic modulus of the hydrogel gradually increases. There is no significant difference between 5S and 10S, and there is no significant difference between 20S and 30S. Therefore, the elastic modulus of 5S is 5.62±0.94kPa and the elastic modulus of 20S is 10.21±1.34kPa as the final groups for testing. Figure 3 C shows that both the 5S and 20S groups have relatively stable storage modulus (G') and loss modulus (G"). Regardless of the angular frequency, the curves of G' and G" do not tend to converge and G' is much larger than the loss modulus G". Figure 3 D. After swelling in PBS for 24 hours, the weights reached 10.7±0.13 and 10.22±0.21 times of the dry weight, respectively. Figure 3 E As can be seen from the scanning electron microscope, both groups have porous structures, which are conducive to the adhesion of RBMSCs in the later stage.

[0041] 3. Biocompatibility evaluation;

[0042] ① Absorb 200μL of G7A3 prepolymerization solution, resuspend and mix the primary RBMSCs cells, and the cell density is 1×107 / ml / group; take 100ul of the mixed prepolymerization solution containing cells and evenly cover the bottom of the confocal dish, cross-link and solidify for 5S seconds and 20S respectively under 365nm wavelength blue light, add α-MEM complete culture medium, and culture in a cell culture incubator. After three days of culture, remove the culture medium in each group of confocal dishes and gently wash with PBS. Add an appropriate amount of Calcein-AM / PI staining solution (purchased from Yisheng) and incubate at 37℃ in the dark for 20 minutes. Aspirate the staining solution, wash with PBS, and observe under a confocal laser confocal microscope. Use 488nm excitation light to detect living cells stained with Calcein-AM (green), and use 555nm excitation light to observe dead cells stained with PI (red); the results are as follows Figure 4 shown.

[0043] Depend on Figure 4 As shown in A, the RBMSCs cultured in each group of hydrogels were round, with living cells dominating and only a few cells dying, showing good cell viability, indicating that each group of hydrogels had excellent biocompatibility.

[0044] ②Pipette 200 μL of G7A3 prepolymer solution and resuspend the primary RBMSCs cells to a cell density of 1×10 7 / ml / group. Take 100ul of the mixed cell-containing pre-polymer solution and drop it evenly on the curing ring (purchased from EFL). After cross-linking and curing for 5S seconds and 20S respectively under blue light of 365nm wavelength, put it into a 12-well plate, add α-MEM complete medium and culture it in a cell culture incubator, and replace α-MEM complete medium every other day. On the 1st, 3rd and 5th days, take out the RBMSCs curing ring to be tested and put it in a new 12-well plate, add CCK-8 detection reagent to the wells of each group, and incubate at 37℃ in the dark for 6 hours. Pipette 100μL CCK-8 detection reagent from each group of six-well plates and add it to a 96-well plate. Use an enzyme reader to measure the absorbance (OD value) of each well at 450nm, collect data and further analyze. The results are as follows Figure 4 shown.

[0045] like Figure 4 As shown in B, CCK-8 results showed that the cell activity of RBMSCs increased with the increase of culture time, which indicated that the composite hydrogel of 5S and 20S had good biological activity in promoting the proliferation of RBMSCs.

[0046] IV: In vitro verification of the effects of 5S and 20S composite hydrogels on cell chondrogenic differentiation;

[0047] ① Pipette 200 μL of G7A3 prepolymer solution and resuspend the primary RBMSCs cells to a cell density of 1×10 7 / ml / group. Take 100ul of the mixed prepolymer solution containing cells and drop it evenly in a 6-well plate. Cross-link and solidify it under 365nm wavelength blue light for 5S seconds and 20S respectively. Add α-MEM complete medium and culture it in a cell culture incubator. Replace the chondrogenic induction medium every other day (α-MEM complete medium contains 40ng / ml dexamethasone, 1nM sodium pyruvate, 50μg / ml ascorbic acid, 46μg / mL proline, 10ng / mL TGF-β1 and 1% ITS-G), and change it every two days. After 14 days of chondrogenic induction, the expression of chondrogenic-related genes (SOX9, COLⅡ, PRG4), hypertrophic cartilage-related genes (COL X) and osteogenesis-related genes (OCN, RUNX2, COLⅠ) was detected using the Bio-Rad real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) system. β-actin was used as the internal reference gene during the detection process, and 2 -ΔΔCT The relative expression data were obtained by the method. Figure 5 shown.

[0048] Depend on Figure 5 It can be seen that RT-qPCR experiments show that the composite hydrogel with a photocrosslinking time of 5S can specifically increase stem cell chondrogenesis-related genes but has no effect on hypertrophic cartilage-related genes, and can reduce osteogenesis-related genes. Therefore, it is proved that under the photocrosslinking time of 5S, the viscoelasticity of the composite hydrogel is conducive to the generation and repair of hyaline cartilage in the growth plate; and the composite hydrogel with a crosslinking time of 20S can also specifically increase stem cell chondrogenesis-related genes, but at the same time, it can also significantly increase hypertrophic cartilage-related genes and osteogenesis-related genes. Therefore, under the photocrosslinking time of 20S, the viscoelasticity of the composite hydrogel is more conducive to repairing the growth plate and the surrounding bone defect area. While repairing the growth plate cartilage, it can also repair the surrounding bone defects, achieving the effect of "killing two birds with one stone".

[0049] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic hydrogel that promotes epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs, characterized in that: The specific steps are as follows: Step 1: prepare methacrylated gelatin and methacrylated decellularized cartilage matrix prepolymer solutions according to the concentrations of 10wt% methacrylated gelatin, 10wt% methacrylated decellularized cartilage matrix and 0.5wt% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, respectively, and place them in a constant temperature, light-proof water bath at 37° C. until they are completely dissolved and then set aside; Step 2: Mix 10wt% methacrylylated gelatin prepolymer solution and 10wt% methacrylylated decellular cartilage matrix prepolymer solution to obtain methacrylylated gelatin / methacrylylated decellular cartilage matrix prepolymer solution, and cross-link it under blue light of 365nm wavelength for 5-20s to prepare methacrylylated gelatin / methacrylylated decellular cartilage matrix composite hydrogel.

2. The method for preparing a biomimetic hydrogel for promoting epiphyseal plate repair by regulating mechanical bidirectional induction of BMSCs according to claim 1, characterized in that: The mixing ratio of the methacrylylated decellularized cartilage matrix and the methacrylylated gelatin in step 2 is 3:

7.

3. Use of a bionic hydrogel prepared by the preparation method according to any one of claims 1 to 2 in the production of a material for promoting the regeneration and repair of epiphyseal cartilage.

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