Injectable bone repair material and preparation method thereof

Abstract

The invention belongs to the technical field of bone repair, and particularly relates to an injectable bone repair material and a preparation method thereof. Comprising the following steps: carrying out chemical crosslinking on methacrylated gelatin of EFL, methacrylated gelatin containing amino functional groups and polyacrylic acid containing carboxyl functional groups, and then carrying out physical crosslinking with silk fibroin to finally form a double-network structure with physical-chemical crosslinking. The mechanical property of the material is enhanced through physical and chemical bi-crosslinking, osteogenesis in cartilage is induced in vivo in a pure material mode, and finally bone repair is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair, and particularly relates to an injectable bone repair material and a preparation method thereof. Background Art

[0002] The description of the background art of the present invention belongs to the related art related to the present invention, and is only used to illustrate and facilitate the understanding of the content of the present invention, and should not be construed as the applicant clearly believing or presuming that the applicant believes it is the prior art on the filing date of the first application of the present invention.

[0003] Bone is an important part of the human body. It provides structural support for the body, ensures the body's ability to move, and protects internal organs. At the same time, bone is also an important endocrine organ, which secretes osteocalcin and other substances to regulate surrounding cells and organs, produces various blood cells for the body, and stores some non-renewable minerals such as calcium, phosphorus and essential ions. Bone defects are usually caused by trauma, osteoarthritis or bone tumors, and are a serious problem in public health. Problems such as prolonged hospital stay, postoperative complications and increased treatment costs brought by it have imposed a considerable burden on patients. It is statistically shown that more than 2 million bone transplantation surgeries are performed globally every year, which is the second largest tissue transplantation after blood transfusion. Clinically, autologous or allogeneic bone grafts are usually used to repair critical-sized bone defects. However, these methods are often limited by problems such as bone source, bone defect shape, and immune rejection. In addition, alternative scaffolds for repairing large-segment bone defects also have many limitations, such as low osteogenic efficiency, insufficient angiogenesis and invasion, and slow bone remodeling. Therefore, there is an urgent need for an effective and sustainable treatment strategy for bone defect repair. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide an injectable bone repair material and a preparation method thereof. A composite material mainly composed of PAA is used, combined with GelMA and SF, and physical and chemical double crosslinking is used to enhance the mechanical properties of the material, and endochondral ossification is induced in vivo in a pure material manner, and finally bone repair is achieved.

[0005] A preparation method of an injectable bone repair material includes the following steps: Use methacrylated gelatin of EFL. After the methacrylated gelatin containing amino functional groups is chemically crosslinked with polyacrylic acid containing carboxyl functional groups, it is then physically crosslinked with silk fibroin, and finally a double-network structure with physical-chemical crosslinking is formed.

[0006] Further, it includes the following steps: Use methacrylated gelatin of EFL, dissolve it into a 10% mass fraction GelMA solution, add 1% mass of a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), then add polyacrylic acid (PAA) containing carboxyl functional groups, stir and react in a 40°C water bath, then add 5% mass fraction of silk fibroin, and photocure under ultraviolet light irradiation to finally form a double-network structure with physical-chemical crosslinking.

[0007] An injectable bone repair material is prepared by the above preparation method.

[0008] The embodiments of the present invention have the following beneficial effects:

[0009] In the present invention, GelMA and carboxylated polyacrylic acid (PAA) are chemically crosslinked through double bonds and then physically crosslinked with silk fibroin (SF) to form a double-network injectable hydrogel with chemical-physical crosslinking, which has a stable structure and excellent mechanical properties. The carboxyl functional groups provided by PAA can chelate iron ions to simulate a hypoxic environment, improve the survival environment of chondrocytes, form a corresponding cartilage mechanism, and ultimately promote the process of endochondral ossification. This method of inducing endochondral ossification by pure materials, combined with materials with good biocompatibility of GelMA and SF, realizes a method of inducing endochondral ossification without cells and growth factors. Description of the Drawings

[0010] Figure 1 It is the mechanical property diagram of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention; it shows that after adding SF and PAA, the mechanical properties of the hydrogel material are significantly improved.

[0011] Figure 2 It is the swelling rate diagram of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention; it shows that after adding SF and PAA, the swelling rate of the hydrogel material does not change significantly, indicating that the hydrogel material can maintain a relatively stable volume in vivo.

[0012] Figure 3 It is the in vitro degradation rate of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention; it shows that after adding SF and PAA, the hydrogel material will not degrade in a short time, can maintain for a relatively long time, and is beneficial to the continuation of osteogenesis.

[0013] Figure 4 It is the scanning electron microscope diagram of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention; it shows that after adding SF and PAA, the hydrogel has good porosity and pore size.

[0014] Figure 5 These are the co - culture live / dead staining and CCK - 8 experimental result graphs of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention, showing that the hydrogel materials have good biological activity.

[0015] Figure 6 These are the safranin O staining and alcian blue staining graphs of chondrospheres of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vitro chondrogenic induction ability.

[0016] Figure 7 These are the ACAN fluorescence staining of chondrogenesis of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vitro chondrogenic induction ability.

[0017] Figure 8 These are the chondrogenic RT - PCR of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vitro chondrogenic induction ability.

[0018] Figure 9 These are the in vitro angiogenesis performance graphs of three hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vitro angiogenesis induction ability.

[0019] Figure 10 These are the explorations of the in vitro osteogenic mechanism (volcano plot, GO enrichment analysis) of three hydrogels (GelMA and GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material promotes osteogenesis through the endochondral ossification mechanism.

[0020] Figure 11 These are the explorations of the in vitro osteogenic mechanism (gene heat map) of three hydrogels (GelMA and GelMA / SF / PAA) in the present invention, showing that after adding PAA, the GelMA / SF / PAA hydrogel material promotes osteogenesis through the endochondral ossification mechanism.

[0021] Figure 12Micro-CT analysis of bone regeneration in the femoral condyle defects of SD rats with three hydrogels (GelMA and GelMA / SF / PAA) in the present invention. It shows that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vivo osteogenic ability. Detailed implementation manners

[0022] The following further introduces the present application in combination with embodiments.

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, in the following description, different "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. Different embodiments can be replaced or combined. For those of ordinary skill in the art, other implementation manners can also be obtained based on these embodiments without creative efforts.

[0024] The applicant's research found that: Traditional bone tissue engineering focuses on simulating the intramembranous ossification pathway, which naturally occurs in the formation of flat bones in the human body such as the skull. However, a major drawback of this method for repairing large bone defects is the limited applicable size. Large tissue-engineered bone structures require rapid vascularization to provide sufficient oxygen and nutrients to the cells in the core of the structure. Intramembranous ossification is prone to ischemic necrosis and central area degradation. Endochondral ossification can overcome this limitation because chondrocytes can also grow well under hypoxic conditions. The mechanism of endochondral ossification has been increasingly emphasized and applied in tissue engineering. Its theoretical basis is the natural development pattern of endochondral ossification during the formation of long bones in embryonic development and the fracture healing process. The formation of long bones begins with the condensation of mesenchymal stromal cells (MSCs), which then differentiate into chondrocytes. These chondrocytes secrete cartilage-specific matrix, rich in type II collagen and glycosaminoglycans (GAGs). During the terminal hypertrophic differentiation process, chondrocytes then recruit mixed cells responsible for the ossification and vascularization of the cartilage template, ultimately promoting vascularization. In this context, many current biomaterials combine biomaterials with related progenitor cells to construct hypertrophic chondrocytes in vitro for implantation in vivo to induce endochondral ossification. However, the long pre-culture time before implantation, the high cost of growth factors, and potential complication problems have greatly hindered its further clinical application. In recent years, many cell-free and growth factor-free strategies have also been developed to promote endochondral ossification induced by pure materials. For example, relevant research has constructed channel-like pore structures and ordered extracellular matrices to directly recruit related progenitor cells and induce the occurrence of endochondral ossification.

[0025] In endochondral osteogenesis materials, the endochondral ossification process begins with the generation of a cartilage template, which highly depends on the formation, stability, and subsequent transformation of chondrocytes into hypertrophic chondrocytes. Then, the deposition and secretion of these cells can further provide a matrix for future bone formation. Currently, several signaling pathways have been identified to regulate the initial differentiation of chondrocytes from mesenchymal progenitor cells into mature hypertrophic chondrocytes. Among them, hypoxia-inducible factor 1α (HIF-1α) is a basic helix-loop-helix transcription factor expressed in a hypoxic environment, which plays an important role in cartilage development, promotes the expression of Sox9, and also switches metabolism from glucose to glycolysis to meet the energy requirements for chondrocyte proliferation and survival. HIF-1α plays an important role in both early cartilage development and later bone formation development, and its regulation in vivo mainly relies on low oxygen levels or the elimination of iron ions. Since it is difficult to regulate oxygen levels in vivo, most studies have focused on reducing iron ions to stabilize the HIF-1α pathway, thereby inducing endochondral ossification to promote bone repair. The most common iron chelator is deferoxamine (DFO), which has been widely used to stabilize the expression of HIF-1α and activate its downstream functions. However, the poor biocompatibility and short plasma half-life of DFO have severely hindered its further application. Therefore, it is very necessary to explore alternative methods for in-situ chelating iron ions with ideal biocompatibility to promote the effective expression of HIF-1α. However, to our knowledge, there is still a lack of a pure biomaterial method that can effectively chelate iron ions without introducing a hypoxia mimetic, let alone biomaterials mimicking hypoxia to reproduce and improve ECO to achieve effective bone regeneration.

[0026] In recent years, methacrylated gelatin (GelMA) has been widely used in the field of biomaterials due to its good biocompatibility, injectability, and photocuring properties. Silk fibroin (SF) is a natural high-purity protein secreted and synthesized by endothelial cells on the inner wall of the silk gland of silkworms. Its final degradation products can be absorbed by the body, and its molecular mass can also be adjusted by modifying the composition of silk proteins to meet the requirements of different in-vivo environments. Therefore, it has good biocompatibility.

[0027] Therefore, the present invention intends to chemically crosslink GelMA and carboxylated polyacrylic acid (PAA) through double bonds and then physically crosslink with silk fibroin (SF) to form an injectable hydrogel with chemical-physical crosslinked double network, which has a stable structure and excellent mechanical properties. The carboxyl functional groups provided by PAA can chelate iron ions to simulate a hypoxic environment, improve the survival environment of chondrocytes, form a corresponding cartilage mechanism, and ultimately promote the endochondral ossification process. This method of inducing endochondral ossification by pure materials, combined with materials with good biocompatibility such as GelMA and SF, realizes a method of inducing endochondral ossification without cells and growth factors.

[0028] Most bone defects caused by high-violence injuries are complex bone defects with different shapes, and achieving precise adaptation is the first key problem to be solved in the present invention. After bone defects caused by various reasons, the defect site is in an injury microenvironment such as oxidative stress, ischemia and hypoxia, and inflammatory cell infiltration. Whether the GelMA / SF / PAA bone repair material can adsorb Fe3+, induce a hypoxic environment, thereby inhibiting oxidative stress and accelerating the formation of neurovascularization is the second key problem to be solved in the present invention. Bone development includes two forms: intramembranous ossification and endochondral ossification, and the limb bones are endochondral ossification. At present, the research focus of bone tissue engineering at home and abroad has been on bone regeneration through intramembranous ossification. Therefore, this project will study the endochondral ossification ability of the GelMA / SF / PAA bone repair material and explore its mechanism, aiming to provide a new idea for the repair of bone defects. This is the third key problem to be solved in the present invention.

[0029] A preparation method of an injectable bone repair material includes the following steps: using methacrylated gelatin (GelMA) of EFL, dissolving it into a 10% mass fraction GelMA solution, adding 1% mass of a photoinitiator (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate), then adding polyacrylic acid (PAA) containing carboxyl functional groups, stirring and reacting in a 40°C water bath, then adding 5% mass fraction of silk fibroin (SF), and photocuring under ultraviolet light irradiation to finally form a double network structure with physical-chemical crosslinking.

[0030] An injectable bone repair material is prepared by the above preparation method.

[0031] Characterization of the GelMA-SF-PAA injectable bone repair material:

[0032] Figure 1 The mechanical properties of the material were tested by compression test.

[0033] Three identical hydrogels (GelMA, GelMA / SF, GelMA / SF / PAA) were prepared using a mold with a diameter of 1.5 cm and a height of about 8 cm, and then their compressive properties were tested under a compression testing machine.

[0034] Figure 2 The material stability was determined by measuring the swelling ratio.

[0035] Different samples were placed in PBS and soaked at 37 °C for 24 hours. After sufficient swelling, their weight (Ws) was measured. Then, the samples were freeze-dried to obtain their dry weight (W0). The swelling ratio was calculated using the formula

[0036]

[0037] Figure 3 The in vitro degradation rate of the material was measured.

[0038] For the UV-crosslinked injectable bone repair material, different samples were freeze-dried and their weight (W0) was measured. Then, the freeze-dried samples were placed in a PBS solution containing 2 μg / mL type II collagenase (C6885-500 MG, Sigma-Aldrich, Germany) and soaked at 37 °C. The solution was changed every two days, and the samples were taken out on the 1st, 2nd, 3rd, 5th, 7th, 9th, 14th, and 21st days. After rinsing twice with deionized water, the samples were freeze-dried and weighed (W1). The formula for the remaining weight was

[0039]

[0040] Figure 4 The microstructure of the material was observed by scanning electron microscopy.

[0041] The micro-morphology of the material was observed using a scanning electron microscope (SEM; ZEISS Sigma 300, Carl Zeiss AG, Jena, Germany). After fixing the samples with 2.5% glutaraldehyde solution for half an hour, they were rinsed with PBS and then dehydrated with a gradient of ethanol. At the critical point, they were dried, coated with gold, and observed with a scanning electron microscope.

[0042] The UV-crosslinked injectable bone repair material was reacted in 40 mL of FeCl3 solution for 6 h. After biosorption, the FeCl3 solution was filtered to remove the biosorbent, and the biosorption amount was calculated by ICP to test the biosorption ability of the injectable bone repair material for Fe3+.

[0043] Biological effects of BMSCs, HUVECs, RAW264.7, and SCs in vitro on the GelMA-SF-PAA injectable bone repair material:

[0044] (1) Cell source. HUVECs and RAW264.7 were purchased from Cyagen Biosciences Inc., and primary cells of SCs and BMSCs were extracted.

[0045] ① Primary extraction of BMSCs: Four-week-old SD rats were selected. After being sacrificed by cervical dislocation, they were soaked and disinfected in 75% alcohol for 10 minutes. The bilateral femurs and tibias were dissected under sterile conditions in a laminar flow hood and placed in pre-cooled sterile PBS. The two ends of the femoral shaft were cut off to fully expose the bone marrow cavity. The bone marrow cavity was flushed with 1 ml of syringe containing DMEM culture medium, and flushed 4 - 5 times until the marrow cavity turned white. The flushing fluid was collected, pipetted and mixed evenly, and filtered through a sterile cell sieve to remove excess impurities. The cells were centrifuged at 1200 rpm for 5 minutes, and the precipitated cells were collected. The cells were resuspended in complete DMEM medium and seeded in a 10-cm culture dish, and cultured routinely in a cell incubator overnight. Then the culture medium was changed to remove the non-adherent cells on the surface, and continued to culture. The cell culture medium was changed every three days; when the cells grew to 90% confluence, the culture medium was discarded, and the cells were rinsed 2 - 3 times with PBS, then trypsin was added for digestion, and then complete culture medium was added to neutralize. The cells were passaged at a ratio of 1:3 and continued to be cultured routinely. The cells of passages P3 - P5 were used in this experiment.

[0046] ② Primary extraction of SCs: 1 - 2-day-old SD neonatal rats were taken. After being sacrificed by decapitation, the bilateral sciatic nerves were isolated and extracted. After obtaining 10 sciatic nerves, they were carefully trimmed under a stereomicroscope to remove the perineurium, vascular tissue, etc. After gently washing with D-Hanks solution, they were minced with ophthalmic scissors. After digestion and centrifugation, the supernatant was discarded. An appropriate amount of complete culture medium was added to make a homogeneous cell suspension, which was inoculated into a 6-well plate pre-coated with PLL. After culturing for 24 hours, the culture medium was completely changed, and the replaced culture medium was DMEM / F12 medium containing 10% FBS and cytosine arabinoside at a final concentration of 2 μg / ml. After 3 days, half of the culture medium was changed, and after another 2 days, the culture medium was completely changed to DMEM / F12 medium containing bovine pituitary extract at a final concentration of 20 μg / ml and 10% FBS. When the density of SCs > 95%, the culture medium was discarded, and the cells were gently washed 3 times with D-Hanks solution, and trypsin at 0.25% was added for digestion and passage. The cells of passages P3 - P5 were used in this experiment.

[0047] (2) BMSCs were co-cultured with injectable bone repair materials. After the liquid injectable bone repair materials were filtered through a 0.22-μm sterile filter, BMSCs were resuspended and added to a 96-well plate at 100 μL per well for Live / Dead and CCK-8 assays to detect cell viability and proliferation. Immunofluorescence staining was used to observe cell morphology.

[0048] ①CCK-8: Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, add 100 μL to each well of a 96-well plate, and adjust the density of the cells to be tested to (1,000 - 10,000) / well. Culture the cells in 2.5% CO2 at 37 °C for 24 hours. Add 10 μL of CCK-8 solution to each well. Continue to incubate in the cell culture incubator for 4 hours. Using this as the time point, measure the absorbance at 450 nm at 12 h, 1 d, 3 d, 5 d, and 7 d later ( Figure 5 ).

[0049] ②Live / Dead: Perform cell culture on the material. Take out the cells on 1 d, 3 d, and 5 d, discard the culture medium, wash 3 times with PBS, add an appropriate amount of live / dead staining reagent, place it back in the cell culture incubator and incubate for 15 minutes, then take it out, discard the staining solution, add a little PBS, and then observe and take pictures under a laser confocal microscope.

[0050] (3) Co-culture BMSCs after chondrogenic induction with an injectable bone repair material, and detect the expression levels of chondrogenic differentiation-related proteins (SOX-9, GAG, COL-2) by chondrocyte pellet staining, immunofluorescence, and qRT-PCR;

[0051] ① As Figure 6 shown, Safranin O staining and Alcian blue staining: Culture cells under 2D conditions. First, culture them with a conventional cell culture medium. When the cells reach about 70% confluence, change to a chondrogenic induction culture medium. Digest the cells and centrifuge them into a 15 ml centrifuge tube. Replace the fresh culture medium every 3 days; On the 14th day of culture, take out the cells, gently wash 3 times with PBS, then fix them with 4% paraformaldehyde at room temperature for 15 minutes. Finally, use deionized water, section and stain them with a cryostat, and then stain them using a Safranin O staining / Alcian blue staining kit according to the staining steps. Then mount the slides and observe and take pictures under a Nikon microscope.

[0052] ② Immunofluorescence: Culture cells under 2D conditions. First, culture them with a conventional cell culture medium. When the cells reach about 70% confluence, change to a chondrogenic induction culture medium. Digest the cells and centrifuge them into a 15 ml centrifuge tube. Replace the fresh culture medium every 3 days; On the 14th day of culture, take out the cells, gently wash 3 times with PBS, then fix them with 4% paraformaldehyde at room temperature for 15 minutes. Discard the paraformaldehyde, wash 3 times with PBS, add Triton X-100, and incubate at room temperature for 15 minutes to perforate the cells; Discard Triton X-100, wash 3 times with PBS, add the target primary antibodies (SOX-9, GAG, COL-2), and incubate overnight at 4 °C; Wash 3 times with PBS, add an appropriate fluorescently labeled secondary antibody, and incubate at 37 °C for 1 hour; Wash 3 times with PBS, add DAPI staining solution; Wash 3 times with PBS, and observe and take pictures under a laser confocal microscope (as Figure 7 )

[0053] ③ Detect the expression levels of osteogenic differentiation-related proteins (ALP, COL-1, OCN, OPN, RUNX2) by qRT-PCR. After extracting RNA samples, measure the concentration and purity by ultraviolet absorption method, prepare the gel and perform electrophoresis, then observe and take pictures under ultraviolet transillumination, and then synthesize the cDNA of the samples. Perform qRT-PCR on the housekeeping gene (β-actin) of the standard product with gradient dilution and the sample to be tested, prepare the DNA template for drawing the standard curve of gradient dilution, and perform qRT-PCR on the gene to be tested of the sample to be tested.

[0054] (4) Co-culture the chondrogenically induced BMSCs with the injectable bone repair material, and detect the expression levels of chondrogenic differentiation-related proteins (N-Cadherin, SOX-9, GAG, COL-2, COL-X) by immunofluorescence, WB and qRT-PCR. The specific methods are as above, and the results are as Figure 8 shown.

[0055] (5) Co-culture HUVECs with the injectable bone repair material, observe the tubular structure by cytoskeleton staining, and detect the expression levels of HIF-1α and VEGF by immunofluorescence, WB and qRT-PCR. The specific methods are as above. The results are as Figure 9 shown.

[0056] (6) Co-culture SCs with the injectable bone repair material, and detect the expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (Ncam) secreted by SCs by Elisa and qRT-PCR. The specific methods are as above.

[0057] Investigation on the effectiveness and mechanism of GelMA-SF-PAA injectable bone repair material in repairing femoral condyle defects in rats:

[0058] Make femoral condyle bone defects in SD rats. The experiment is divided into four groups: A (blank group), B (GelMA-PAA group), C (GelMA-SF group) and D (GelMA-SF-PAA group), with 15 rats in each group.

[0059] (1) Animal model establishment: Male Sprague-Dawley (SD) rats at 7 weeks of age with an average body weight of about 300 g were selected. After anesthesia by intraperitoneal injection of sodium pentobarbital (3.5 mg / 100 g), the lower limbs of the rats were shaved and disinfected. In a sterile environment, a longitudinal incision was made on the lateral side of the distal femur, and the femoral condyle was exposed by blunt dissection; the center of the femoral condyle was located, and a drill was slowly started to drill a hole in the center, and the hole was gradually enlarged with drills of diameters 1 mm, 2 mm, and 3 mm to create a cylindrical defect that penetrated both cortical bones. During the drilling, the site was irrigated with ice-cold saline; the irradiated and disinfected scaffold material was implanted into the bone defect site; then the muscle, fascia, and skin were closed layer by layer, the incision was disinfected, and the rats were returned to the cage for continued feeding after waking up.

[0060] (2) Study on the bone defect repair mechanism. Samples were taken 7 days after surgery, and through high-throughput sequencing of whole-transcriptome RNA, GO, KEGG, etc. were analyzed to find the enriched regions of up-regulated genes and down-regulated genes, and molecular biology experiments such as dual-luciferase reporter gene and qRT-PCR were carried out to further analyze the genes related to endochondral ossification and perform signal pathway analysis. Figure 10 This is the exploration of the in vitro osteogenic mechanism of three hydrogels (GelMA and GelMA / SF / PAA) in the present invention (volcano plot, GO enrichment analysis); it shows that after adding PAA, the GelMA / SF / PAA hydrogel material promotes osteogenesis through the mechanism of endochondral ossification.

[0061] (3) Study on endochondral ossification

[0062] ① X-ray and Micro-CT: Rats were sacrificed at 2, 4, and 8 weeks after surgery respectively, the femoral shafts were isolated, wrapped with wet gauze soaked in saline, stored on ice, and immediately subjected to X-ray, Micro-CT scanning and three-dimensional reconstruction to analyze and evaluate bone regeneration, and BMD, BV / TV, Tb.Th, and Tb.Sp were quantitatively analyzed.

[0063] Figure 11 This is the exploration of the in vitro osteogenic mechanism of three hydrogels (GelMA and GelMA / SF / PAA) in the present invention (gene heat map); it shows that after adding PAA, the GelMA / SF / PAA hydrogel material promotes osteogenesis through the mechanism of endochondral ossification.

[0064] Figure 12 This is the mirco-ct analysis of bone regeneration in the femoral condyle defect of SD rats with three hydrogels (GelMA and GelMA / SF / PAA) in the present invention. It shows that after adding PAA, the GelMA / SF / PAA hydrogel material has good in vivo osteogenic ability.

[0065] ② Histology: Samples were taken at 2, 4, and 8 weeks after surgery, decalcified, and subjected to HE staining, Masson staining, and Safranin-O histological staining to observe bone, collagen fiber, and cartilage formation.

[0066] Preparation of paraffin sections: The excised femurs were immediately immersed in 4% paraformaldehyde for 48 hours after Micro-CT scanning; the fixed specimens were taken out and placed in a 10% EDTA decalcifying solution for decalcification, with the decalcifying solution changed every two days for one week; after decalcification, the specimens were rinsed under running water overnight to remove residual EDTA for subsequent embedding; the rinsed tissue specimens were successively dehydrated in 70% ethanol for 5 minutes, 80% ethanol for 2 - 4 hours, 95% ethanol for 2 - 4 hours, 95% ethanol for 2 - 4 hours, 100% ethanol for 2 - 4 hours, and 100% ethanol for 2 - 4 hours; the dehydrated specimens were then successively made transparent in an anhydrous ethanol / xylene (1:1) mixed solution for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes; the transparent tissue blocks were embedded after impregnation with paraffin wax, and the embedded wax blocks were stored at -20°C. HE staining: The paraffin sections were successively immersed in xylene I and xylene II for 30 minutes each; then the sections were successively immersed in absolute ethanol I for 2 minutes, absolute ethanol II for 2 minutes, 95% ethanol I for 2 minutes, 95% ethanol II for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and rinsed with distilled water; the sections were immersed in hematoxylin stain for 2 minutes, rinsed with running water, immersed in 1% hydrochloric acid alcohol solution for differentiation for 3 seconds, rinsed with running water for 5 minutes until fully blued, and examined under the microscope to check if the cell nuclei were appropriate; the sections were immersed in alcoholic eosin solution for staining for 15 seconds, and then successively rinsed in 85%, 95%, 95%, 100%, and 100% ethanol for 1 minute each; the sections were successively immersed in xylene I and xylene II for 1 minute each; the transparent sections were taken out, the xylene outside the specimens was wiped clean, and the sections were sealed with neutral gum.

[0067] The pre-culture of chondrocyte-derived osteogenic induction materials with growth factors for a long time before implantation, the high cost of growth factors, and potential complication problems have greatly hindered their further clinical application. While cell-free and growth factor-free pure materials such as deferoxamine have problems with poor biocompatibility, which greatly limits their clinical application.

[0068] Therefore, we plan to use a composite material based on PAA, combined with GelMA and SF, with physical and chemical double crosslinking to enhance the mechanical properties of the material, induce endochondral ossification in vivo in the form of a pure material, and ultimately achieve bone repair. The cell-free and growth factor-free material avoids the cost and safety problems related to growth factors and also greatly saves time costs. At the same time, this composite hydrogel has good biocompatibility and has broad clinical application prospects.

[0069] It should be noted that the above embodiments can be freely combined as needed. The above introduction is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an injectable bone repair material, characterized in that: The method comprises the following steps: using methacryloyl gelatin of EFL, chemically cross-linking the methacryloyl gelatin containing amino functional groups with polyacrylic acid containing carboxyl functional groups, and then physically cross-linking with silk fibroin to finally form a double network structure with physical-chemical cross-linking.

2. The method for preparing an injectable bone repair material according to claim 1, characterized in that: The method comprises the following steps: using methacrylated gelatin of EFL, dissolving it into a 10% by mass GelMA solution, adding a 1% by mass photoinitiator (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate), then adding polyacrylic acid (PAA) containing a carboxyl functional group, stirring and reacting in a 40°C water bath, then adding 5% by mass silk fibroin, and photocuring under ultraviolet light to finally form a double network structure with physical-chemical cross-linking.

3. An injectable bone repair material, characterized in that: Prepared by the preparation method described in claim 1 or 2.

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