A method for the synthesis of composite gels based on in situ mineralized bone-like hydroxyapatite, products and uses thereof

Abstract

The application discloses a synthesis method of a composite gel based on in-situ mineralized bone-like hydroxyapatite, which comprises the following steps: taking animal periosteum, and sequentially treating the animal periosteum with an immunostaining permeating liquid and a lysis liquid; sterilizing, detoxifying and washing the treated periosteum tissue, and then freeze-drying the periosteum tissue; grinding the periosteum tissue into powder, and then digesting the powder in a solution to obtain extracellular matrix; dissolving calcium chloride in ethanol, adding triethylamine under stirring, then adding an ethanol solution of phosphoric acid under stirring, centrifuging to discard the supernatant, and washing and resuspending the calcium phosphate mineralization precursor with ethanol to obtain the calcium phosphate mineralization precursor; uniformly mixing the calcium phosphate mineralization precursor with the extracellular matrix to obtain a gel, and in-situ biomimetic mineralizing the gel to obtain bone-like hydroxyapatite, which is used as the composite gel. The application further provides the composite gel prepared by the above synthesis method and application of the composite gel in preparation of a bone defect repair product. The composite gel has excellent biocompatibility, bone conduction, bone induction, blood vessel formation capacity and mechanical performance in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medical material synthesis, and particularly relates to a synthesis method of a composite gel based on in-situ mineralized bone-like hydroxyapatite, a product thereof and application. BACKGROUND

[0002] Bone, as the main hard tissue supporting the human body, is formed by hierarchical assembly of nanoscale hydroxyapatite (length 30-50 nm, width 20-25 nm, thickness 1.5-4 nm) and organic matrix. Due to the complexity of bone tissue composition and structure, the healing of bone defects needs to go through multiple physiological processes, including early inflammation and immune regulation, angiogenesis, osteogenic differentiation and biomineralization. Large area bone defects (such as maxillofacial trauma, tumor ablation, intervertebral disc injury or degeneration, etc.) are often difficult to heal by themselves, and are the most common and challenging clinical problems in orthopedics. At present, the intervention of transplantation surgery (including autologous transplantation, allogeneic transplantation or transplantation of exogenous biomaterials) is often needed to provide a suitable environment for effective healing of bone tissue. However, these transplantation materials have some limitations: (1) autografts may involve limited availability of tissue morbidity and donors; (2) allogeneic transplants have a risk of serious infection and immunogenic rejection; (3) the size and shape of solid biomaterials such as metal or ceramic implants are not easy to adapt to the defect tissue. Although the latest progress in three-dimensional printing technology provides the possibility of plasticity of materials, it is also not easy to apply in the shape of the damaged site in implantation surgery. Therefore, a new type of biomaterial that can completely adapt to the bone defect site is needed to guide cell growth and differentiation and promote the deposition of new bone tissue.

[0003] In recent years, multifunctional hydrogel materials have good cell compatibility, cell adhesion, and promotion of cell proliferation, migration and differentiation, and have been widely used in the field of biomedical materials. In the repair of bone defects, biomimetic composite hydrogel is an ideal material, its viscoelasticity is close to that of natural tissue, which can well simulate the osteogenic microenvironment, thereby promoting the repair and regeneration of bone tissue. And it can fully adapt to bone defects of different shapes and depths, and be used in minimally invasive implant surgery, and establish close contact with the host tissue to limit fibrosis and facilitate bone conduction. Such composite hydrogel materials select organic polymers with good biocompatibility (such as alginate, chitosan, hyaluronic acid, polyethylene glycol, gelatin, etc.) to form an organic matrix network. However, it lacks bone-related bioactive molecules, which has limitations in the application of bone tissue repair. In order to solve this defect, extracellular matrix (ECM) produced by tissue decellularization is developed and applied, which can relatively completely retain the original biological activity and functional complexity of the tissue. However, the mechanical properties of such ECM-based hydrogel materials are weak, and often need to be doped with inorganic particles to increase the mechanical support. Nano-hydroxyapatite, as an inorganic component of natural bone tissue, is often added to the organic matrix network to enhance the bone conduction, osteoinduction and angiogenic ability of the material. However, the morphology and structure of this artificially synthesized nano-hydroxyapatite are not similar to those of natural bone tissue, and it lacks comprehensive and accurate regulation of the physiological process of bone defect repair. Moreover, due to the lack of strong interaction between the inorganic and organic networks, the hydroxyapatite aggregates and disperses unevenly, which has limitations in clinical applications. The generation of hydroxyapatite in bone tissue often requires a complex biological mineralization process, in which inorganic ions or liquid-like precursors form sheet-like nano-hydroxyapatite under the regulation of related organic matrix and assemble with collagen. Due to the high water content in the hydrogel, the inorganic content is very low, and the interaction between the inorganic and organic matrices is weak, which leads to the separation of the two phases, so it has been a technical difficulty in the synthesis of bone-like hydroxyapatite in hydrogels.

[0004] In summary, a method for synthesizing bone-like hydroxyapatite in hydrogels is needed to prepare a biomimetic composite gel with biocompatibility, bone conduction, osteoinduction, angiogenic ability and mechanical support, which can promote inflammation, immune regulation, angiogenesis, osteogenic differentiation and biomineralization in the repair of bone defects. SUMMARY

[0005] The present application provides a method for synthesizing a composite gel based on in-situ mineralized bone-like hydroxyapatite, as well as products and applications thereof. The synthesized composite gel has excellent biocompatibility, bone conduction, osteoinduction, angiogenic ability and mechanical properties in vitro and in vivo, providing a suitable application material for bone defect repair.

[0006] The present application provides the following technical solutions:

[0007] A method for synthesizing a composite gel based on in-situ mineralized bone-like hydroxyapatite, comprising the following steps:

[0008] (1) Taking animal periosteum tissue, treating it with immunostaining permeation liquid and lysis liquid in sequence, and destroying the cells inside the periosteum tissue;

[0009] (2) Sterilizing, detoxifying and washing the periosteum tissue treated in step (1), and then freeze-drying it;

[0010] (3) Grinding the periosteum tissue in step (2) into powder, digesting it in a solution, centrifuging to discard the precipitate, and adjusting the pH to neutral, to obtain the acellular extracellular matrix of the periosteum tissue;

[0011] (4) Dissolving calcium chloride in ethanol, adding triethylamine under stirring, and then adding phosphoric acid ethanol solution under stirring, centrifuging to discard the supernatant, and resuspending and washing with ethanol, to obtain calcium phosphate mineralization precursor;

[0012] (5) Mixing the calcium phosphate mineralization precursor prepared in step (4) and the acellular extracellular matrix of the periosteum tissue prepared in step (3) uniformly to obtain a gel, and in-situ biomimetic mineralizing the calcium phosphate mineralization precursor in the gel to obtain bone-like hydroxyapatite, as a composite gel.

[0013] In the present application, for steps (1) and (2), in order to ensure that the acellular extracellular matrix of the periosteum tissue has good functional integrity and biocompatibility, the concentration and action time of the immunostaining permeation liquid and the lysis liquid need to be adjusted to a suitable range, and the concentration and action time of peroxoacetic acid, Tris-HCl buffer, glacial acetic acid and pepsin also need to be adjusted.

[0014] In step (1), the immunostaining permeation liquid is polyethylene glycol octylphenyl ether, and the lysis liquid is SDS lysis liquid, NP-40 lysis liquid or RIPA lysis liquid.

[0015] Preferably, in step (1), the immunostaining permeation liquid is polyethylene glycol octylphenyl ether, with a concentration of 0.3-1% and a treatment time of 12-24h; and the lysis liquid is SDS lysis liquid, with a concentration of 0.5-1% and a treatment time of 3-6h.

[0016] Further preferably, in step (1), the concentration of the immunostaining permeation liquid is 1%, and the treatment time is 12h; and the lysis liquid is SDS lysis liquid, with a concentration of 1% and a treatment time of 3h.

[0017] The immunostaining permeation solution is selected as polyethylene glycol octylphenyl ether with a concentration of 1% and a treatment time of 12 h. This non-ionic surfactant can help the cell membrane permeation in the original periosteum tissue. The lysis solution is selected as SDS lysis solution with a concentration of 1% and a treatment time of 3 h. As an anionic detergent, it can promote the lysis of the cell membrane and is particularly suitable for the dissolution of the cytoskeleton protein in the periosteum tissue. The above two steps can further ensure that the cells in the periosteum tissue are completely removed, while maintaining good functional integrity.

[0018] Preferably, in step (2), peracetic acid sterilization and Tris-HCl buffer detoxification are used; the concentration of the peracetic acid is 1-3%, the treatment time is 1-4 h, the Tris-HCl buffer detoxification time is 24-48 h, and the freeze-drying time is 24-48 h.

[0019] Further preferably, in step (2), the concentration of the peracetic acid is 3%, the treatment time is 2 h, the Tris-HCl buffer detoxification time is 36 h, and the freeze-drying time is 24 h.

[0020] The concentration of peracetic acid used in the sterilization process is 3%, the treatment time is 2 h, and the Tris-HCl buffer detoxification time is 36 h. The peracetic acid at this concentration can completely kill the bacteria in the extracellular matrix of the decellularized periosteum tissue, and the long-time buffer soaking and regular liquid replacement during the detoxification process ensure the biocompatibility of the extracellular matrix.

[0021] Preferably, in step (3), glacial acetic acid and pepsin solution digestion is used, the concentration of the glacial acetic acid is 0.1-1 M, the concentration of the pepsin is 0.5-2 mg / mL, the digestion time is 36-72 h. The centrifugal speed is 500-800 g, the centrifugal time is 10-15 min, and the pH adjustment is 7.0-7.5.

[0022] Further preferably, in step (3), the concentration of the glacial acetic acid is 0.5 M, the concentration of the pepsin is 0.8 mg / mL, and the digestion time is 48 h. The centrifugal speed is 500 g, the centrifugal time is 10 min, and the pH adjustment is 7.4.

[0023] During the digestion of glacial acetic acid and pepsin, the basic units in the extracellular matrix of the decellularized periosteum tissue will undergo gelation crosslinking, and then the pH is adjusted to 7.4 to maintain good biocompatibility.

[0024] In order to regulate the size and structure of the calcium phosphate mineralization precursor and ensure biomimetic mineralization in the gel, the reaction concentration and reaction time of calcium phosphate, triethylamine and phosphoric acid in the synthesis method need to be optimized.

[0025] wherein, in step (3), the bone tissue extracellular matrix is stored at 4°C.

[0026] Preferably, in step (4), the concentration of calcium chloride is 20-40 mM, the concentration of triethylamine is 0.4-2.0 M, the concentration of phosphoric acid is 10-30 mM, the stirring speed is 800-1200 rpm, and the stirring time is 4-12 h.

[0027] Further preferably, in step (4), the concentration of calcium chloride is 30 mM, the concentration of triethylamine is 0.8 M, the concentration of phosphoric acid is 20 mM, the stirring speed is 800 rpm, and the stirring time is 12 h.

[0028] In the process of synthesizing the calcium phosphate mineralization precursor, the optimized concentrations of calcium chloride and phosphoric acid can regulate the product to be an amorphous calcium phosphate precursor, and the slight excess of the concentration of triethylamine and the constant stirring can maintain the size of the calcium phosphate mineralization precursor at the nanometer level.

[0029] In order to ensure the in-situ mineralization of the bone-like hydroxyapatite in the biomimetic composite gel, there is a suitable interaction force between the organic phase and the inorganic phase, which provides good bone conduction, bone induction and mechanical strength, and provides a good microenvironment for bone defect healing. In the synthesis method, the reaction concentration and reaction time of the extracellular matrix after decellularization of the periosteal tissue and the calcium phosphate mineralization precursor need to be optimized.

[0030] Preferably, in step (5), the concentration of the calcium phosphate mineralization precursor is 10-20 mg / mL, the centrifugal speed is 6000-8000 rpm, the centrifugal time is 5-10 min, the concentration of the extracellular matrix of the decellularized periosteal tissue is 4-8 mg / mL, the mass percentage of the calcium phosphate mineralization precursor in the gel is controlled to be 60-70%, and the in-situ mineralization reaction time is 12-48 h.

[0031] Further preferably, in step (5), the concentration of the calcium phosphate mineralization precursor is 10 mg / mL, the centrifugal speed is 6000 rpm, the centrifugal time is 5 min, the extracellular matrix of the decellularized periosteal tissue is 8 mg / mL, the mass percentage of the calcium phosphate mineralization precursor in the gel is controlled to be 70%, and the in-situ mineralization reaction time is 24 h.

[0032] In the process of synthesizing the bone-like hydroxyapatite, the calcium ions of the precursor and the carboxyl groups of the extracellular matrix form a strong interaction force to form an organic-inorganic double gel network, and the mass percentage of the precursor in the biomimetic composite gel is controlled to be 70%, so that the inorganic precursor can be fully biomimetic mineralized under the regulation of the extracellular matrix, and the bone-like hydroxyapatite is constructed in the biomimetic gel.

[0033] wherein, in step (5), the mixture is homogenized at 4 DEG C and mineralized in situ at 37 DEG C.

[0034] The application further provides a composite gel based on in-situ mineralized bone-like hydroxyapatite prepared according to the above synthesis method.

[0035] The application further provides application of the composite gel based on in-situ mineralized bone-like hydroxyapatite in preparation of a bone defect repair product.

[0036] The technical concept of the application is to select the bone membrane tissue decellularized extracellular matrix as an in-situ biomimetic mineralization organic matrix, to remove cells in the tissue completely by using an immunostaining permeation liquid and a lysis liquid, and to obtain the bone membrane tissue decellularized extracellular matrix with complete functions and good biocompatibility by sterilization, detoxification and digestion; to select the amorphous calcium phosphate precursor as a mineralization precursor of bone-like hydroxyapatite, to obtain the nano-sized amorphous calcium phosphate mineralization precursor under the capping effect of triethylamine. The amorphous calcium phosphate precursor and the natural organic matrix form a composite gel through strong interaction, and the bone-like hydroxyapatite is constructed through in-situ biomimetic mineralization under the regulation of the organic matrix, so as to provide a good osteogenic repair microenvironment for the healing of bone defects.

[0037] The application obtains the bone-like hydroxyapatite through the in-situ biomimetic mineralization of the calcium phosphate mineralization precursor regulated by the bone membrane tissue decellularized extracellular matrix, and forms the strong interaction between the organic and inorganic at the molecular scale to form the biomimetic composite gel. Through the excellent biological functionality and biocompatibility of the organic matrix and the good osteoconductivity, osteoinductivity and mechanical strength of the bone-like hydroxyapatite, the application promotes the inflammation immune regulation, angiogenesis, osteogenic differentiation and biological mineralization in the bone repair process, and provides a safe and effective and flexible application biological medical material for the healing of bone defects. The application is successfully applied to the repair of critical-sized bone defects, greatly improves the repair of non-self-repairable bone tissue, and provides an effective new type of biological medical composite material for bone tissue repair. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Fig. 1 is a diagram of HE, Masson, Col1 and DAPI staining of the bone membrane tissue before and after decellularization; Fig. 2 is a diagram of DNA content statistics of the bone membrane tissue before and after decellularization; Fig. 3 is a diagram of the transmission electron microscope and XRD spectrum of the calcium phosphate mineralization precursor.

[0039] Figure 2A and B are the transmission electron microscope image and the length, width and thickness statistics of the bone-like hydroxyapatite, respectively; C, D and E are the XRD pattern, scanning electron microscope image and X-ray energy spectrum element image analysis diagram of the biomimetic composite gel, respectively; F is the compressive modulus of the biomimetic composite gel (BOH), extracellular matrix after decellularization of periosteal tissue (PECM) and PECM to which synthetic hydroxyapatite (PECMH) is added.

[0040] Figure 3 A and B are the alkaline phosphatase (ALP) staining diagram and content quantitative statistics of bone marrow mesenchymal stem cells (BMSC) after incubation for 7 days in the blank control, PECM, PECMH and BOH groups, respectively; C and D are the alizarin red (ARS) staining diagram and quantitative statistics of BMSC after incubation for 14 days in the blank control, PECM, PECMH and BOH groups, respectively; E and F are the microscope diagram and branch quantitative statistics of human umbilical vein endothelial cells (HUVEC) after incubation for 6 hours in the blank control, PECM, PECMH and BOH groups.

[0041] Figure 4 (A) Micro CT imaging, (B) bone volume fraction and (C) bone density quantitative statistics of the blank control, PECM, PECMH and BOH groups at the defect site for 4 weeks and 8 weeks in the rat critical skull defect model. DETAILED DESCRIPTION

[0042] In order to make the objects, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the protection scope of the present application. Example 1 synthesis of bone-like hydroxyapatite in a biomimetic composite gel using amorphous calcium phosphate mineralization precursor

[0043] Periosteum was harvested from the femur of Large White pigs and rinsed three times with deionized water until no blood remained. The sample was treated with immunostaining permeabilization buffer (1% polyethylene glycol octylphenyl ether) for 12 hours, followed by rinsing with deionized water. It was then treated with 1% SDS lysis buffer for 3 hours and rinsed with deionized water. The sample was then sterilized with 3% peracetic acid for 2 hours, transferred to Tris-HCl buffer for detoxification for 36 hours, washed with PBS buffer, and freeze-dried for 24 hours. The freeze-dried sample was ground into powder and digested in a solution of 0.5M glacial acetic acid and 0.8 mg / mL pepsin for 48 hours. After centrifugation at 500g for 10 minutes, the precipitate was discarded, and the pH was adjusted to 7.4 with 10M sodium hydroxide solution. The sample was stored at 4℃ to obtain decellularized extracellular matrix (PECM) of the periosteum for later use. 11.76 g of calcium chloride dihydrate was dissolved in 1.60 L of ethanol. Under magnetic stirring at 800 rpm, 221.79 mL of triethylamine was added to the solution, followed by the dropwise addition of an ethanol solution of phosphoric acid (4.18 mL of phosphoric acid dissolved in 80 mL of ethanol). The mixture was stirred, centrifuged, and the supernatant was discarded. The solution was then washed and resuspended with ethanol to obtain the calcium phosphate mineralization precursor. The precursor was washed three times with deionized water and dispersed to a concentration of 10 mg / mL. 20 mL of this precursor was mixed with 10 mL of PECM (8 mg / mL) at 4 °C and stirred thoroughly. The mixture was then incubated at 37 °C for 24 h to obtain BOH. Similarly, 20 mL of artificially synthesized nano-hydroxyapatite of the same concentration was mixed with 10 mL of PECM (8 mg / mL) at 4 °C and incubated at 37 °C for 24 h to obtain PECMH.

[0044] like Figure 1 As shown, tissue sections of periosteal tissue before and after decellularization were compared using hematoxylin-eosin (HE) staining, Masson staining, type I collagen immunohistochemical staining (Col1), DAPI nuclear fluorescence staining, and statistical comparison of internal DNA content. After decellularization, the cells in the periosteal tissue were completely detached, the internal DNA content was significantly reduced to zero, and the original collagen fiber structure of the tissue remained unchanged. Transmission electron microscopy revealed that the calcium phosphate mineralization precursor synthesized using this method was uniformly dispersed with a particle size of approximately 2-5 nm. XRD analysis showed that the calcium phosphate mineralization precursor was amorphous and could be used for subsequent biomimetic mineralization.

[0045] like Figure 2As shown, the biomimetic composite hydrogel (BOH) was observed by transmission electron microscopy, and the amorphous calcium phosphate mineralization precursor inside it had been regulated by the extracellular matrix of the decellularized periosteum tissue into a crystalline morphology. The length of the crystal was 46.29±6.31 nm, the width was 20.52±3.33 nm, and the thickness was 4.00±1.16 nm, which were similar to the morphology and size of natural bone hydroxyapatite. The biomimetic composite hydrogel was observed by XRD, and characteristic peaks appeared at (002), (211), (310), (222), (213), and (004), which were similar to natural bone hydroxyapatite. It was observed by scanning electron microscopy that the BOH had micron-level pores, which was conducive to cell growth. And through X-ray energy spectrum element image analysis, it was found that the inside of the BOH was composed of C, O, Ca, and P elements, which also confirmed that the biomimetic composite hydrogel was composed of organic and inorganic phases. At the same time, it was observed that the compressive modulus of the biomimetic composite hydrogel was 51.71±6.90 kPa, which was significantly higher than that of PECM (15.37±1.97 kPa) and PECMH (26.27±2.42 kPa), which also confirmed that the interaction between simply mixed artificially synthesized nanometer hydroxyapatite and organic matrix was limited, and there was a strong interaction between nanometer-sized calcium phosphate mineralization precursor and organic matrix.

[0046] Example 2 Application of biomimetic composite hydrogel with bone-like hydroxyapatite in the healing of bone defects in vitro and in vivo

[0047] The extracellular matrix gel of decellularized periosteum tissue (PECM), the PECM with artificially synthesized hydroxyapatite (PECMH), and the biomimetic composite hydrogel (BOH) were added to 96-well plates, 75 μL of gel was added to each well, and the wells without added materials were used as blank controls. Bone marrow mesenchymal stem cells (BMSCs) were inoculated in each well at a density of 2×10 3The cells were cultured in high glucose DMEM with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. After 7 days of culture, the medium was removed and the cells were washed twice with PBS buffer. Then 400 μL of alkaline phosphatase (ALP) staining solution was added to each well, and the plate was incubated in the dark for 1 h on a shaker. The staining solution was removed and the cells were washed twice with PBS buffer. After 14 days of culture, the medium was removed and the cells were washed twice with PBS buffer. Then 4% paraformaldehyde solution was added to each well, and the plate was incubated at room temperature for 30 min. The fixing solution was removed and the cells were washed twice with PBS buffer. Then 4% alcian blue staining solution was added to each well, and the plate was incubated for 10 min. The staining solution was removed and the cells were washed twice with PBS buffer. The staining of the BMSC under different conditions was observed under an optical microscope. PECM, PECMH and BOH were added to 96-well plates, respectively, and the wells without the addition of materials were used as blank controls. 100 μL of human umbilical vein endothelial cells (HUVEC) were added to each well at a seeding density of 1 x 10 5 The growth state of the HUVEC under different conditions was observed under an optical microscope.

[0048] Forty-eight 8-week-old Sprague Dawley male rats (weighing 250 ± 50 g) were randomly and equally divided into a PECM group, a PECMH group and a BOH group, and the wells without the addition of materials were used as blank controls. After anesthesia, the scalp of the rat skull was surface sterilized, and a surgical knife was used to make an incision, which was washed with a large amount of sterile saline and then a surgical drill was used to make a bilateral full-thickness critical-size skull defect (5 mm in diameter). The PECM, PECMH or BOH hydrogel was injected into the skull defect, and then the periosteum and scalp were sutured with a 2-0 nylon thread. The wells without the injection of any material were used as blank controls. The rats were individually raised in cages and kept at a constant temperature of 23 ± 3°C. On the 3rd day, the 4th week and the 8th week after transplantation (4 rats were taken from each group at each time point), the animals were euthanized by CO2 asphyxiation and cervical dislocation. The skull samples were obtained and fixed in 4% paraformaldehyde for subsequent immunological examination.

[0049] As Figure 3As shown, the content of alkaline phosphatase of BMSCs cultured in BOH hydrogel for 7 days was significantly higher than that in PECM and PECMH. After 14 days of culture, the extracellular deposited calcium nodules of BMSCs cultured in BOH hydrogel were significantly more than that in PECM and PECMH, as shown by alizarin red staining. It was proved that the biomimetic composite hydrogel with bone-like hydroxyapatite had a significant function of promoting the osteogenic differentiation and biomineralization of BMSCs in vitro, and had a more promoting effect than simply mixing artificially synthesized hydroxyapatite. After HUVEC was cultured in BOH gel, the morphology of the formed tube was significantly more complete than that in PECM and PECMH, and the number of branches also significantly increased. It was proved that BOH had good function of promoting angiogenesis in vitro.

[0050] As Figure 4 shown, in the critical size skull defect model of rats, the blank control group without injecting any material could hardly repair itself, and the bone volume fraction was only 4.59±0.38% and the bone density was 0.039±0.007 g / cm 3 after 4 weeks, and the bone volume fraction was 5.77±1.52% and the bone density was 0.047±0.006 g / cm 3 after 8 weeks. After injecting PECM, the growth of the defect was slow, the bone volume fraction was 8.56±0.51% and the bone density was 0.066±0.018 g / cm 3 after 4 weeks, and the bone volume fraction slightly increased to 11.06±1.36% and the bone density slightly increased to 0.106±0.012 g / cm 3 after 8 weeks. After injecting PECMH, the growth of the defect was slightly improved, the bone volume fraction was 13.20±1.23% and the bone density was 0.143±0.016 g / cm 3 after 4 weeks, and the bone volume fraction increased to 20.91±2.26% and the bone density slightly increased to 0.218±0.016 g / cm 3 after 8 weeks. After injecting BOH, the repair speed and effect of the defect were significantly improved, the bone volume fraction increased to 20.85±4.97% and the bone density was 0.199±0.013 g / cm 3 after 4 weeks, and the bone volume fraction significantly increased to 36.01±2.06% and the bone density increased to 0.398±0.024 g / cm 3 after 8 weeks. It was proved that the biomimetic composite hydrogel with bone-like hydroxyapatite had a significant effect on the repair of critical size bone defects in vivo.

[0051] The above detailed description of the specific embodiments of the present application has described the technical solutions and beneficial effects of the present application, and it should be understood that the above description is only the most preferred embodiment of the present application and is not intended to limit the present application. Any modifications, supplements and equivalent replacements made within the principle range of the present application shall be included in the protection range of the present application.

Claims

1. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite for the preparation of a bone defect repair product, characterized in that, The synthesis method of the composite gel comprises the following steps: (1) Take animal periosteum tissue, and sequentially treat with an immunostaining permeation liquid and a lysis liquid to destroy cells inside the periosteum tissue; (2) After the treatment in step (1), the periosteum tissue is sterilized, detoxified, washed, and then freeze-dried; (3) The periosteum tissue in step (2) is ground into powder and then digested in a solution, the precipitate is discarded after centrifugation, and the pH is adjusted to neutral to obtain a decellularized extracellular matrix of the periosteum tissue; (4) Calcium chloride is dissolved in ethanol, triethylamine is added under stirring, and then an ethanol solution of phosphoric acid is added dropwise under stirring, the supernatant is discarded after centrifugation, and the precipitate is resuspended and washed with ethanol to obtain a calcium phosphate mineralization precursor; (5) The calcium phosphate mineralization precursor prepared in step (4) is uniformly mixed with the decellularized extracellular matrix of the periosteum tissue prepared in step (3) to obtain a gel, and the calcium phosphate mineralization precursor is in-situ biomimetic mineralized in the gel to obtain bone-like hydroxyapatite as a composite gel; In step (5), the concentration of the calcium phosphate mineralization precursor is 10-20 mg / mL, the centrifugation speed is 6000-8000 rpm, the centrifugation time is 5-10 min, the concentration of the decellularized extracellular matrix of the periosteum tissue is 4-8 mg / mL, the mass percentage of the calcium phosphate mineralization precursor in the gel is controlled to be 60-70%, and the in-situ mineralization reaction time is 12-48 h; The particle size of the calcium phosphate mineralization precursor is 2-5 nm, and the size of the bone-like hydroxyapatite obtained by in-situ biomimetic mineralization in the gel is: length 46.29±6.31 nm, width 20.52±3.33 nm, and thickness 4.00±1.16 nm.

2. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite according to claim 1, characterized in that, In step (1), the immunostaining permeation liquid is polyethylene glycol octylphenyl ether, and the lysis liquid is an SDS lysis liquid, an NP-40 lysis liquid, or an RIPA lysis liquid.

3. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite according to claim 2, characterized in that, In step (1), the concentration of the immunostaining permeation liquid is 0.3-1%, and the treatment time is 12-24 h; and the concentration of the lysis liquid is 0.5-1%, and the treatment time is 3-6 h.

4. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite according to claim 1, characterized in that, In step (2), peroxoacetic acid is used for sterilization, and a Tris-HCl buffer is used for detoxification; the concentration of the peroxoacetic acid is 1-3%, the treatment time is 1-4 h, the detoxification time of the Tris-HCl buffer is 24-48 h, and the freeze-drying time is 24-48 h.

5. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite according to claim 1, characterized in that, In step (3), glacial acetic acid and pepsin solution are used for digestion; the concentration of the glacial acetic acid is 0.1-1 M, the concentration of the pepsin is 0.5-2 mg / mL, and the digestion time is 36-72 h; the centrifugation speed is 500-800 g, and the centrifugation time is 10-15 min; and the pH is adjusted to 7.0-7.

5.

6. Use of a composite gel based on in situ mineralized bone-like hydroxyapatite according to claim 1, characterized in that, In step (4), the concentration of the calcium chloride is 20-40 mM, the concentration of the triethylamine is 0.4-2.0 M, the concentration of the phosphoric acid is 10-30 mM, the stirring speed is 800-1200 rpm, and the stirring time is 4-12 h.

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