Bone tissue repair material for promoting vascularization and new bone formation and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a bone tissue repair material for promoting vascularization and new bone generation and a preparation method thereof. The bone tissue repair material is composed of a decalcified bone matrix and a degradable biological source gel material. The repair material has a loose porous three-dimensional structure similar to that of natural cancellous bone, and the temperature-sensitive gel material filled in the pore structure of the repair material can form gel in situ in vivo and is attached to pores of a decalcified bone matrix, so that cell migration, adhesion and proliferation can be promoted, the degradation rate of gel in vivo can be reduced, and the repair material has a good application prospect. The continuous release of the pro-angiogenesis factor is ensured. Good biocompatibility is achieved, and cytotoxicity and immunogenicity are avoided; meanwhile, in the slow degradation process, angiogenesis promoting factors are continuously released for a long time, and angiogenesis and new bone generation are promoted. Therefore, the implanted biological scaffold prepared from the bone tissue repair material has excellent bone repair and regeneration promotion capability and wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a bone tissue repair material for promoting vascularization and new bone formation and a preparation method thereof. BACKGROUND

[0002] Bone defect is a common clinical problem caused by trauma, tumor resection, infection and congenital malformation. With the increasing number of orthopedic disease and trauma patients, the demand for bone repair materials continues to grow. The current clinical materials have significant limitations. In the face of the choice of bone repair materials, autologous bone, although being the gold standard, is limited in source and causes secondary damage, and cannot be applied to large-area bone defect surgery; allogeneic bone and xenogeneic bone have relatively extensive sources and have similar physical structure and biomechanical properties to autologous bone, but have potential immunogenicity, high viral risk and need to be decellularized. Traditional artificial bone materials mainly include inert metals, biologically inert ceramics, bioactive ceramics and high molecular polymers. Metals have high strength but the elastic modulus is much higher than that of cortical bone, and long-term implantation can easily lead to stress shielding bone resorption; inert ceramics are brittle and cannot match the micro-motion requirements of cancellous bone; bioactive ceramics can form chemical bonds with host bone, but the degradation rate does not match the new bone formation, often resulting in "collapse" or "excessive absorption". High molecular materials have strong processability, but lack bone inductivity, and acidic degradation products can easily cause local inflammation. More importantly, the above-mentioned materials generally lack a three-dimensional interpenetrating microvascular network, and the essence of bone regeneration is a highly vascular-dependent process: osteogenic precursor cells more than 200 μm away from blood supply will undergo apoptosis due to hypoxia, leading to "central necrosis", so that the scaffold can only form a thin layer of bone callus on the surface, making it difficult to achieve through repair of large defects.

[0003] Decalcified bone matrix is a composite natural bone graft material composed of collagen, non-collagen and a low concentration of growth factors, and is mainly derived from allogeneic bone and xenogeneic bone. It has relatively extensive sources, good biocompatibility, bone inductivity and bone conductivity. BMPs and various osteogenic factors in decalcified bone matrix are closely combined with collagen in bone matrix, which can induce mesenchymal cells to differentiate into cartilage tissue and osteoblasts, and finally form cartilage tissue and bone tissue. It is an ideal bone tissue engineering scaffold material. However, using decalcified bone matrix alone as a bone repair material will have low porosity, long bone repair time and no vascularization function, and therefore cannot achieve good repair effect in the face of large-area bone defect repair surgery. SUMMARY

[0004] In view of the above technical problems, the purpose of the present application is to provide a bone tissue repair material for promoting vascularization and new bone formation, which is prepared by combining a degradable biological gel with a porous antigen-removed freeze-dried decalcified bone matrix. After being implanted into the body, the temperature-sensitive biological gel can form a gel in situ in the porous antigen-removed freeze-dried decalcified bone matrix, forming nanometer-micron level connecting channels to meet the requirements of vascular invasion. The gel flexible network structure is combined with the rough surface and internal of the decalcified bone matrix, increasing the specific surface area to provide more sites for protein adsorption and cell adhesion, while also reducing the degradation rate of the gel in the body to ensure the sustained release of the vascularization factors in the gel.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: A bone tissue repair material for promoting vascularization and new bone formation, which is prepared by mixing a porous antigen-removed freeze-dried decalcified bone matrix and a degradable biological gel material at a mass ratio of (80-90):(10-20). The volume of the porous antigen-removed freeze-dried decalcified bone matrix is 0.1 cm 3 ~10 cm 3 , and the pore size is 0.1 mm-1 mm.

[0006] Preferably, the volume of the porous antigen-removed freeze-dried decalcified bone matrix is 1 cm 3 ~4 cm 3 , and the pore size is 0.3 mm-0.5 mm.

[0007] The purpose of selecting a decalcified bone matrix block with a size of 1 cm 3 ~4 cm 3 is to ensure the structural stability, biological activity and clinical practicability of the decalcified bone matrix during the preparation of the material. If the decalcified bone matrix is too small, it will lead to excessive decalcification, destroying the three-dimensional structure and mechanical properties of the bone matrix. Secondly, if the decalcified bone matrix is too large, it will lead to a prolonged decalcification time and a reduced content of bone formation-related factors. The present technical solution selects a tibial bone material, which can be precisely cut to prepare a decalcified bone matrix with a volume of 1 cm 3 ~4 cm 3 .

[0008] Secondly, the pore size of the porous antigen-removed decalcified bone matrix material is selected to be 0.3 mm-0.5 mm. The purpose is that a pore size greater than 0.3 mm can promote the growth of blood vessels, forming a vascular network in the bone repair material to ensure the circulation of nutrients. A bone scaffold material with a pore size of 0.5 mm can promote cell adhesion, proliferation and differentiation, and promote bone mineralization.

[0009] Preferably, the basic raw material of the porous antigen-removed freeze-dried decalcified bone matrix is a calcium bone matrix. The calcium bone matrix is derived from one or more of humans, non-human primates, fish, avian, bovine, porcine, equine, ovine, canine, mule, chicken, duck, and goose.

[0010] Preferably, the porous antigen-extracted freeze-dried decalcified bone matrix is derived from allogeneic cortical bone. The porous antigen-extracted freeze-dried decalcified bone matrix and the degradable biological gel material are derived from the same source.

[0011] The bone tissue repair material has the porous antigen-extracted freeze-dried decalcified bone matrix as the main body, the calcium residue in the material is less than 8%, and the bone matrix is a fully decalcified bone matrix, which can ensure the release of osteogenic factors in the bone matrix and the efficient bone induction and bone conduction functions. The porous characteristics of the decalcified bone matrix and the unique processing method enable the gel material to be fully filled in the pores and to be in situ gelled after being implanted in the body, thereby providing conditions for cell adhesion and proliferation during bone repair. The pro-angiogenic factors in the gel can induce the growth and infiltration of blood vessels, ensure the transmission of nutrients, and further promote bone repair. The porous antigen-extracted freeze-dried decalcified bone matrix and the degradable biological gel material are derived from the same source, which further improves the biocompatibility of the vascularized bone repair material.

[0012] Preferably, the degradable biological gel material is selected from one or more of homologous heart decellularized extracellular matrix hydrogel material, spleen decellularized extracellular matrix hydrogel material, bladder decellularized extracellular matrix hydrogel material, lung decellularized extracellular matrix hydrogel material, and liver decellularized extracellular matrix hydrogel material.

[0013] A preparation method of a bone tissue repair material for promoting vascularization and new bone formation, comprising the following steps: S1. Preparation of a porous antigen-extracted freeze-dried decalcified bone matrix: Fresh natural bone is cut into bone blocks with a size of 0.1 cm 3 ~10 cm 3 The bone blocks are subjected to decalcification and decellularization antigen extraction treatment, and then array drilling treatment is performed on the bone matrix with a pore size of 0.1-1 mm. After freeze-drying, a porous antigen-extracted freeze-dried decalcified bone matrix block is obtained. S2. Preparation of a degradable biological gel material: Fresh natural organs are cut into tissue blocks with a size of 0.5 mm 3 ~5 mm 3 The tissue blocks are subjected to decellularization antigen extraction treatment, and then freeze-drying is performed to obtain a decellularized tissue material. Then, an "acid + enzyme" method is used to prepare a gel. After adjusting the pH value, freeze-drying and crushing are performed to obtain a degradable biological gel material. S3. Preparation of a bone tissue repair material: The degradable biological source gel material in S2 is placed in a PBS solution and dissolved for 20-24 h under stirring at 4°C to obtain a degradable biological source gel solution; The mass concentration of the degradable biological source gel solution is 15-40 mg / mL; The porous deantigenic freeze-dried decalcified bone matrix in S1 is placed in a vacuum impregnation tank, and a vacuum impregnation method is used to vacuum the impregnation tank to 0.09-0.20 MPa and maintain for 30-90 min. In this state, the degradable biological source gel solution is slowly injected into the impregnation tank through an injection device, and then the tank pressure of the impregnation tank is restored to normal atmospheric pressure within 10-15 min to obtain a mixed material; The ratio of the porous deantigenic freeze-dried decalcified bone matrix to the degradable biological source gel solution is (0.5-10 g):(2-40 ml); The mixed material is subjected to freeze-drying and irradiation sterilization.

[0014] As preferred, in S2, the fresh natural organs are first cleaned of blood water on the surface with physiological hydrochloric acid, then soaked in hypertonic sodium chloride saline for 10-24 h, then cut into tissue blocks, and cleaned with purified water until no blood water is present. The fresh natural organs include one or more of heart, spleen, bladder, lung, and liver. The specific process of the decellularized deantigenic treatment is as follows: The tissue blocks are treated with 0.1-2% sodium dodecyl sulfate at 37°C for 24-48 h, and then cleaned with purified water until no residue is present. The freeze-drying time is 24-42 h.

[0015] As preferred, in S2, the preparation process of the gel is as follows: The decellularized tissue material, hydrochloric acid, and pepsin are mixed at a ratio of 150 mg:(7-20 ml):(9-20 mg) for 24-48 h to obtain a mixed solution treated with pepsin; The mixed solution treated with pepsin is subjected to high-speed centrifugation, and the supernatant is taken and the precipitate is discarded. The pH of the supernatant is adjusted to 7.2-7.6 with 0.8-1.2 M sodium hydroxide, then 1 / 9 volume of 10×PBS solution is added, and the mixture is uniformly mixed, then freeze-dried, and crushed to 0.05 mm-1 mm to obtain a degradable biological source gel material; The molar concentration of the hydrochloric acid is 0.008-0.012 M.

[0016] As preferred, in S3, the mass concentration of the degradable biological source gel solution is 25-35 mg / mL. The ratio of the porous deantigen freeze-dried decalcified bone matrix to the degradable biological gel solution is (1-4g):(5-20ml).

[0017] The application of a bone tissue repair material promoting vascularization and new bone formation in the preparation of an implant biological scaffold for orthopedic repair.

[0018] Compared with the prior art, the application has at least the following technical effects: The application provides a bone tissue repair material promoting vascularization and new bone formation, which is combined with a degradable biological gel and a porous deantigen freeze-dried decalcified bone matrix; after being implanted into the body, the temperature-sensitive biological gel can be in-situ gelled in the porous deantigen freeze-dried decalcified bone matrix, forming nanometer-micron level connecting channels to meet the requirement of vascular invasion. The gel flexible network structure is combined with the rough surface and the interior of the decalcified bone matrix, increasing the specific surface area, providing more sites for protein adsorption and cell adhesion, and also reducing the degradation speed of the gel in the body, so as to ensure the sustained release of the vascularization factor in the gel.

[0019] The preparation method of the bone tissue repair material promoting vascularization and new bone formation is that the porous deantigen freeze-dried decalcified bone matrix and the degradable biological gel material are prepared respectively, and then the two components are combined by physical adsorption and cross-linking through vacuum impregnation, so that the bone repair material has both osteogenic efficacy and vascularization promoting function.

[0020] In the specific preparation process, 1) the decalcified bone matrix is punched in an array drilling manner, compared with the conventional block-shaped decalcified bone matrix material, the artificial hole can promote the growth of blood vessels and provide a basis for subsequent bone repair; compared with the granular decalcified bone matrix material, the bone repair material has certain mechanical properties. 2) The vacuum impregnation method is used, compared with the conventional stirring and mixing, the gel can be better filled into the pores of the decalcified bone matrix, promote cell adhesion and proliferation, and by reducing the exposure area of the gel in the body, the degradation speed of the gel is reduced, so that the vascularization growth factor can be continuously and slowly released, and long-term and efficient bone repair effect is achieved.

[0021] The bone tissue repair material promoting vascularization and new bone formation is a porous biological scaffold providing osteogenic function and a bone tissue repair material promoting vascularization and cell adhesion.

[0022] The bone tissue repair material for promoting blood vesselization and new bone formation has good biocompatibility and bone induction repair function, both of which are subjected to decellularization treatment, so that the bone repair material has extremely low immunogenicity; drilling treatment is conducted by using a customized device, the surface area of the material is increased on the premise of retaining the mechanical properties of the bone repair material to a large extent, and a special impregnation process is used to ensure that the degradable biological gel material is fully filled into the bone matrix; the bone repair material is convenient to use and can be shaped, and can be customized to the required size according to the clinical use requirements, and after being implanted into the body, the temperature-sensitive gel material in the bone repair material can be gelled in situ, which can accelerate the bone repair process to a certain extent and provide a better bone repair material on the spot. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Cell toxicity test chart and cell morphology schematic diagram of the finished product prepared for Example 1; Figure 2 CT scan chart and new bone volume / tissue volume result data chart of the finished product material prepared for Example 1 implanted in the skull defect for 4, 8 and 12 weeks; Figure 3 HE staining chart of the finished product prepared for Example 1 implanted in the skull defect for 4, 8 and 12 weeks. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the present application will be briefly introduced below in combination with the drawings and the description of the embodiments or the prior art. Obviously, the following description of the drawings structure is only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. It should be noted that the description of these embodiment modes is used to help understand the present application, but does not constitute a limitation on the present application.

[0025] The technical solution of one specific embodiment of the present application is: A preparation method of a bone tissue repair material for promoting blood vesselization and new bone formation, comprising the following steps: S1, preparation of porous antigen-free freeze-dried decalcified bone matrix: S1-1: Select allogeneic cortical bone as raw material, use bone sawing machine to saw the bone into 0.1 cm 3 ~10 cm 3 blocky bone, then wash the blood stains and grease on the surface of the bone block with a high-pressure water gun; Preferably, the volume of the blocky bone: 1 cm 3 ~4 cm 3 .

[0026] S1-2: The bone pieces are added with isopropyl alcohol for degreasing treatment according to a ratio of 1 g: 4-8 mL, and are cleaned by alternately using ultrasonic and oscillation for 6 h, and the isopropyl alcohol is replaced every 2 h, then purified water is added, and the cleaning is performed for no less than 6 times, to obtain the degreased bone pieces; Preferably, the ratio of the bone pieces to isopropyl alcohol is 1 g: 5 mL.

[0027] S1-3: The degreased bone pieces are placed into a chemical sterilization solution, oscillated at 4°C and 100-300 rpm / min for 2 h for virus inactivation, and the purified water is cleaned for no less than 6 times, to obtain the virus-inactivated bone pieces; the chemical sterilization solution contains 6%-10% ethanol and 0.1%-0.4% peroxyacetic acid; S1-4: The virus-inactivated bone pieces are subjected to decalcification treatment in hydrochloric acid containing sucrose, the residual calcium content of the decalcified bone pieces is less than 8%, then the purified water is cleaned, and the cleaning solution is replaced every 1 h until the pH of the cleaning solution is 5.5-7.5.

[0028] Preferably, the concentration of the hydrochloric acid is 0.6 mol / L, and the content of the sucrose is 8%.

[0029] S1-5: The decalcified bone pieces are placed into a 1% Triton X-100 solution for stirring and decellularization treatment, and then the purified water is cleaned until there is no chemical reagent residue; Preferably, the ratio of the bone pieces to Trion X-100 is 1 g: 10 mL; Preferably, the stirring speed is 300 r / min, and the temperature is 37°C; S1-6: A customized device is used to perform array hole transfer on the decalcified bone matrix, the hole diameter is 0.3-0.5 mm, and the distance between adjacent holes is 0.5-1 mm; S1-7: The above decalcified bone matrix is placed in a vacuum freeze dryer for freeze drying for 48 h.

[0030] S2, Preparation of a degradable biological gel material: S2-1: An allogeneic organ is selected as a raw material, the surface blood is cleaned with physiological saline, then the allogeneic organ is soaked in a high-osmotic sodium chloride solution at 4°C for more than 16 hours, and then is cut into tissue blocks with a size of 0.5 mm 3 -5 mm 3 ; S2-2: 1% SDS solution is added according to a ratio of 1 g: 10 mL, stirring and decellularization are performed at 37°C and 300 r / min for 48 h, and the purified water is cleaned for at least 2 d; S2-3: The decellularized tissue blocks are pre-frozen at -80°C for 3 h, and then are vacuum freeze-dried for 48 h, to obtain a decellularized tissue material; S2-4: A certain amount of decellularized tissue material is weighed, and a solution containing 1 mg / ml of pepsin hydrochloride is added at a concentration of 15 mg / ml. The mixture is stirred at 25°C for 24 hours, then centrifuged to retain the supernatant. The pH is adjusted to 7.5 using 1M sodium hydroxide solution, and 1 / 9 volume of 10x PBS solution is added. After mixing, a degradable biological gel is obtained. S2-5: The degradable biological gel is freeze-dried and crushed to a size of 50-400 μm to obtain a degradable biological gel material. S3, Preparation of bone tissue repair material for promoting vascularization and new bone formation: S3-1: The porous antigen-free freeze-dried decalcified bone matrix is placed in a vacuum impregnation tank and kept in a vacuum state for 3 hours. S3-2: The degradable biological gel material is dissolved in PBS solution in advance to prepare a gel solution with a concentration of 28-35 mg / ml. The gel solution is then slowly added through an injection device, and the vacuum impregnation is maintained for 2 hours. The ratio of decalcified bone matrix to gel solution is 1 g:4 mL. S3-3: The mixed material obtained in S3-2 is freeze-dried and sterilized by irradiation to obtain a bone tissue repair material for promoting vascularization and new bone formation. The dose of irradiation sterilization is 15-25 kGy.

[0031] A specific embodiment: porous antigen-free freeze-dried decalcified bone matrix: natural bone tissue is sawn into bone blocks, and after defatting, virus inactivation, decalcification, decellularization, and array drilling, the bone blocks are washed with purified water until there is no chemical reagent residue. Finally, vacuum freeze-drying is performed to obtain a porous antigen-free freeze-dried decalcified bone matrix.

[0032] One implementable process for the porous antigen-free freeze-dried decalcified bone matrix is as follows: cortical bone of the same species or different species is cut into blocks using a bone sawing machine, and the blood stains and grease on the surface of the bone blocks are washed off using a high-pressure water gun. The bone blocks are defatted in isopropanol for 8 hours, washed clean with purified water, and then treated with a chemical sterilization solution composed of peroxoacetic acid and alcohol at 4°C for 2 hours. The bone blocks are then decalcified in 0.6M hydrochloric acid at 4°C for 7 days, washed with PBS and purified water alternately until neutral, and then decellularized with 1% Triton X-100 at 37°C for 48 hours. After washing with purified water, the decalcified bone matrix is array drilled using a customized device, with a hole diameter of 0.5 mm and a distance of 0.5 mm between two holes. Finally, the bone matrix is freeze-dried at -60°C for 48 hours using a freeze-drying machine to obtain a porous antigen-free freeze-dried decalcified bone matrix, which mainly contains collagen, non-collagen proteins, and a low concentration of growth factors.

[0033] A specific embodiment: degradable biological gel material: natural organ tissue is cut into blocks and washed with purified water until there is no blood water. After decellularization, freeze-drying, digestion into gel, and re-freeze-drying and crushing, the degradable biological gel material is obtained.

[0034] One implementable process for preparing the degradable biological gel material is as follows: a natural organ tissue is cut into 1 mm 3 size, then purified water is used for washing until no blood water is left, a 1% SDS solution is used for decellularization treatment at 37°C, a completely decellularized tissue is obtained, purified water is used for washing until no reagent residue is left, and then vacuum freeze-drying is performed for 48 h. A certain amount of freeze-dried tissue is taken, an "acid + enzyme" method is used to digest the tissue, NaOH and 10×PBS solution are added, a tissue gel is obtained, the gel is freeze-dried, and then is crushed to prepare the degradable biological gel material.

[0035] It is pointed out that, through experimental research, when the concentration of the degradable biological gel material is greater than 28 mg / ml, the adhesion can reach a good plasticity ability, and the anti-scattering ability is also strong. It is also pointed out that the bone repair material prepared by the above method needs to be packaged and sealed with two layers of bacteria isolation plastic bags after freeze-drying, and then is used for irradiation sterilization.

[0036] Example 1: A preparation method of a bone tissue repair material for promoting vascularization and new bone formation, comprising the following steps: S1: bone block processing: cortical bone is processed into 1 cm 3 size, then a high-pressure water gun is used for washing for 10 min. Then, isopropanol is added at 1 g:5 mL, and ultrasonic and oscillation are alternately used for defatting treatment for 6 h, and then purified water is used for washing until no odor is left. Chemical sterilization liquid (containing 8% ethanol and 0.2% peracetic acid) is added at 1 g:5 mL, oscillation is performed at 4°C for 2 h, and then purified water is used for washing until no peracetic acid residue is left. Decalcification liquid (0.6 mol / L hydrochloric acid containing 8% sucrose) is added at 1 g:10 mL, stirring is performed at 4°C for 8 d, and the decalcification liquid is replaced every 2 d, and then the bone block is washed to be neutral with purified water. 1% Triton X-100 is added at 1 g:10 mL, stirring is performed at 37°C for 48 h, the decellularization liquid is replaced once in the middle, and then purified water is washed for 2 d. A customized device is used to uniformly drill holes on the decalcified bone matrix, the hole diameter is 0.5 mm, and the distance between adjacent holes is 1 mm. Vacuum freeze-drying is performed at -60°C and 0.04 mbar for 48 h to obtain a porous antigen-free freeze-dried decalcified bone matrix.

[0037] S2: the organ tissue is washed with physiological saline to remove surface blood water, then is soaked in a 10% sodium chloride solution for 16 h, and then is cut into 5 mm 3Size of the tissue block. Washed with purified water until the tissue block was no blood color, according to 1 g: 10 mL, 1% SDS solution, 37 ℃ stirring decellularization 48 h, during the replacement of decellularization fluid, then purified water washing 2d. Freeze-drying at -60 ℃, 0.04 mbar for 48 h. A certain amount of freeze-dried tissue material was weighed, and 0.01M hydrochloric acid (containing 0.1% pepsin) was added at 15 mg / ml, and the solution was stirred at 25℃ for 24h, centrifuged at 10000rpm / min, and the supernatant was adjusted to pH 7.0 with 1M sodium hydroxide. A 1 / 9 volume of 10×PBS solution was added, and the mixture was stirred to obtain a degradable biological gel. Secondary freeze-drying: The degradable biological gel was freeze-dried again to obtain a degradable biological gel material; S3: 24h before material mixing, dissolve the degradable biological gel material in purified water to prepare a gel solution with a concentration of 30mg / ml, stir at 4℃ for 24h, and exclude gas from the solution.

[0038] S4: Put 1g porous antigen-free freeze-dried decalcified bone matrix into a vacuum impregnation tank, maintain vacuum state for 3h, and remove gas in the pores. Slowly inject 10mL of gel solution into the vacuum impregnation tank through the injection device, and mix under vacuum for 3h; S5: Freeze-dry the mixed material at -60℃, 0.04 mbar for 48h. Package and sterilize by irradiation: package the material in two layers of sterile plastic bags, seal, and sterilize by irradiation at a dose of 15kGy.

[0039] Example 2: A method for preparing a bone tissue repair material for promoting vascularization and new bone formation, comprising the following steps: According to the preparation method of Example 1, the difference is S4: The mass-volume ratio of porous antigen-free freeze-dried decalcified bone matrix to gel solution is 1g:5mL, and the other preparation methods remain the same.

[0040] Example 3: According to the preparation method of Example 1, the difference is S4: The mass-volume ratio of porous antigen-free freeze-dried decalcified bone matrix to gel solution is 1g:20mL, and the other preparation methods remain the same.

[0041] Example 4: According to the preparation method of Example 1, the difference is S3: The concentration of the gel solution is 25mg / ml.

[0042] Example 5: According to the preparation method of Example 1, the difference is S1: The material and Triton X-100 were mixed at a mass-volume ratio of 1 g:5 mL, and the other preparation methods were consistent.

[0043] Detection test Test 1: DNA residue detection Test method: 10 mg of the bone tissue repair material for promoting vascularization and new bone formation was accurately weighed, and was subjected to enzymolysis with proteinase K and RNase in a 56℃ water bath, and then genomic DNA was extracted (operation was performed according to the instructions of the DNA extraction kit), and then the DNA content of the sample was detected according to YY / T 1876-2023 Animal-derived Biomaterial DNA Residue Determination Method for Tissue Engineering Medical Products.

[0044] The materials prepared in Examples 1-5 were subjected to DNA residue detection.

[0045] Table 1

[0046] According to the results in Table 1, the DNA content of the materials prepared in Examples 1-5 was all <50 ng / mg, meeting the requirements of decellularized materials.

[0047] Test 2: In vitro cytotoxicity detection Preparation of extraction solution: the bone tissue repair material for promoting vascularization and new bone formation prepared in Example 1 was added into DMEM culture medium at 0.2 g / ml, and was subjected to extraction at 37℃ and 120 rpm for 72 h, and then the supernatant was collected by centrifugation.

[0048] Cell preparation: the mouse fibroblasts were resuscitated using DMEM culture medium containing 10% FBS, and were cultured at 37℃, 5% CO2 and saturated humidity, and were subcultured for 2-3 times to logarithmic growth phase, and then the cells were digested with trypsin, and the cell concentration was adjusted to 1×10 5 cells / mL for the following experiments.

[0049] Preparation of negative samples: DMEM culture medium containing 10% FBS Preparation of positive samples: DMEM culture medium containing 10% DMSO was prepared Experimental method: the cell suspension was added into a 96-well plate, 100 μL per well, and a total of 1×10 4Cells were cultured in 96-well plates at 37°C, 5% CO2 for 24 hours. After the end of the culture, the medium in the plate was discarded, and each sample was added to the negative sample and the positive sample, 6 wells in each group, and placed in a 37°C, 5% CO2 saturated humidity incubator. The blank sample group was only added with 10% FBS DMEM medium without cells. After 24 hours of culture, the photographs were taken under a microscope and recorded, the medium in the plate was discarded, 10% CCK-8 reagent was added to the DMEM medium, and it was placed in a 37°C, 5% CO2 saturated humidity incubator for 1 hour. After the end of the culture, it was mixed for 30 minutes in the dark, and then it was placed in an enzyme marker at 450 nm for absorbance detection.

[0050] As shown in Figure 1 FIG. 1 is a schematic diagram of the cell morphology of the cytotoxicity test of the finished product of Example 1, and the scale is 100 μm.

[0051] Table 2: Cytotoxicity test table

[0052] The results combined Figure 1 with Table 2 show that the cell morphology and quantity of the Example 1 (material group) group did not change significantly compared with the control group, and it has good biocompatibility.

[0053] Test 3: Rat skull defect test Forty-five male SD rats (250-300 g) were randomly divided into a control group, a decalcified bone matrix group (ZL2023104481813), and a bone tissue repair material group for promoting vascularization and new bone formation, 15 rats in each group. The animals were anesthetized by intraperitoneal injection of 3% sodium pentobarbital at a dose of 50 mg / kg, and after the anesthetic took effect, the skull area was prepared for skin, disinfected, and the rats were placed in a prone position. A 2-3 cm longitudinal incision was made on the rat skull, the skin, subcutaneous tissue, and muscle-membrane layer were cut open and flapped, and the skull bone plate was exposed by blunt dissection. A 5 mm outer diameter bone drill was used to make a circular full-thickness periosteal bone defect with a diameter of 5 mm in the midline of the skull. The control group was not treated; the decalcified bone matrix group was only filled with decalcified bone matrix; and the bone tissue repair material group for promoting vascularization and new bone formation was implanted with the bone repair material prepared in Example 1.

[0054] Penicillin was injected immediately after the operation. The animals were sacrificed at 4, 8, and 12 weeks after the operation, and the skulls at the defect site were taken together with the periosteum, placed in 10% neutral formalin solution for preservation, and then subjected to CT scanning and HE staining after decalcification.

[0055] Figure 2 The CT scan images and new bone volume / tissue volume data graphs of the finished product material prepared in Example 1 implanted in the skull defect at 4, 8, and 12 weeks. Figure 3 The HE staining images of the tissue sections implanted with the finished product prepared in Example 1 at 4, 8, 12 weeks, the scale of the picture is 200 μm.

[0056] Results Figure 2 The results of CT scanning (in order left) and the statistical chart of the new bone volume (BV) / tissue volume (TV) (in order right) show that the new bone volume of Example 1 group > decalcified bone matrix group > control group at 4 weeks, 8 weeks, 12 weeks, which indicates that Example 1 group effectively accelerates bone regeneration.

[0057] Results Figure 3 The results of HE staining show that Example 1 group has the whole cycle of angiogenesis effect, and the number of new vessels in the new bone tissue is significantly increased.

[0058] Although the decalcified bone matrix group also has new vessels, the number is significantly lower than that of the bone tissue repair material group with angiogenesis and new bone formation.

[0059] The above two experimental results show that the bone tissue repair material with angiogenesis and new bone formation prepared in the application has good osteogenesis effect.

[0060] Finally, it should be noted that: the above only for the preferred embodiments of the application, and not for limiting the protection scope of the application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A bone tissue repair material that promotes angiogenesis and new bone formation, characterized in that, The bone tissue repair material is prepared by mixing porous deantigenated lyophilized bone matrix and biodegradable bio-derived gel material in a mass ratio of (80~90):(10~20); The volume of the porous deantigenated lyophilized bone matrix is ​​0.1 cm³. 3 ~10cm 3 The aperture is 0.1mm~1mm.

2. The bone tissue repair material for promoting angiogenesis and new bone formation according to claim 1, characterized in that, The volume of the porous deantigenated lyophilized bone matrix is ​​1 cm³. 3 ~4cm 3 The aperture is 0.3mm~0.5mm.

3. The bone tissue repair material for promoting angiogenesis and new bone formation according to claim 1, characterized in that, The base material of the porous antigen-removed freeze-dried decalcified bone matrix is ​​calcium bone matrix. The calcium-bone matrix is ​​derived from one or more of the following: humans, non-human primates, fish, birds, cattle, pigs, horses, sheep, dogs, mules, chickens, ducks, and geese.

4. The bone tissue repair material for promoting angiogenesis and new bone formation according to claim 1, characterized in that, The porous deantigen-free lyophilized decalcified bone matrix is ​​derived from allogeneic cortical bone. The porous deantigenated lyophilized bone matrix and the biodegradable bio-derived gel material are from the same source.

5. The bone tissue repair material for promoting angiogenesis and new bone formation according to claim 1, characterized in that, The biodegradable bio-derived gel material is selected from one or more of the following: cardiac decellularized extracellular matrix hydrogel materials, spleen decellularized extracellular matrix hydrogel materials, bladder decellularized extracellular matrix hydrogel materials, lung decellularized extracellular matrix hydrogel materials, and liver decellularized extracellular matrix hydrogel materials.

6. A method for preparing a bone tissue repair material that promotes angiogenesis and new bone formation as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Preparation of porous deantigenated lyophilized bone matrix: Fresh natural bone is cut into 0.1 cm pieces using a bone saw. 3 ~10 cm 3 Bone blocks of various sizes were prepared and decalcified and decellularized to remove antigens. Then, the bone matrix was drilled with an array of holes with a pore size of 0.1~1mm. After freeze-drying, porous deantigenated freeze-dried decalcified bone matrix blocks were obtained. S2. Preparation of biodegradable bio-based gel materials: Cut fresh, natural organs into 0.5mm pieces. 3 ~5 mm 3 The tissue blocks were divided into several sizes, and the tissue blocks were decellularized and deantigenated, and then freeze-dried to obtain decellularized tissue material. Then, the gel was prepared using the "acid + enzyme" method. After adjusting the pH, the gel was freeze-dried and pulverized to obtain a biodegradable bio-derived gel material. S3. Preparation of bone tissue repair materials: The biodegradable bio-derived gel material in S2 was placed in PBS solution and dissolved at 4°C for 20-24 hours with stirring to obtain a biodegradable bio-derived gel solution. The mass concentration of the biodegradable bio-derived gel solution is 15~40 mg / mL; The porous deantigenated lyophilized bone matrix in S1 was placed in a vacuum impregnation tank. The vacuum impregnation method was used. The impregnation tank was evacuated to 0.09~0.20MPa and maintained for 30~90min. Under this condition, the biodegradable bio-derived gel solution was slowly injected into the impregnation tank through an injection device. Then, the tank pressure was restored to normal atmospheric pressure within 10~15min to obtain the mixed material. The ratio of the porous deantigenated lyophilized bone matrix to the biodegradable bio-derived gel solution is (0.5~10g):(2~40ml). The mixture is freeze-dried and sterilized by irradiation to obtain the final product.

7. The method for preparing a bone tissue repair material that promotes angiogenesis and new bone formation according to claim 6, characterized in that, In step S2, physiological hydrochloric acid is first used to wash away the blood on the surface of the fresh natural organ. Then, the organ is soaked in hypertonic sodium chloride saline for 10-24 hours and cut into tissue blocks. The tissue blocks are then washed with purified water until there is no blood. The fresh, natural organs include one or more of the heart, spleen, bladder, lungs, and liver; The specific procedure for the decellularization and antigen removal process is as follows: Use sodium dodecyl sulfate with a mass fraction of 0.1-2% to rapidly process the tissue at 37°C for 24-48 hours, then wash with purified water until no residue remains. The freeze-drying time is 24~42h.

8. The method for preparing a bone tissue repair material that promotes angiogenesis and new bone formation according to claim 6, characterized in that, In step S2, the specific process for preparing the gel is as follows: A cell-free tissue material, hydrochloric acid, and pepsin were selected and mixed in a ratio of 150 mg: (7~20 ml): (9~20 mg) for 24~48 h to obtain the pepsin-treated mixture. The mixture treated with pepsin was centrifuged at high speed, the supernatant was collected, the precipitate was discarded, the pH of the supernatant was adjusted to 7.2-7.6 with 0.8-1.2M sodium hydroxide, then 1 / 9 volume of 10×PBS solution was added, mixed evenly, and then freeze-dried and pulverized into 0.05mm-1mm particles to obtain a biodegradable bio-derived gel material. The molar concentration of the hydrochloric acid is 0.008~0.012M.

9. The method for preparing a bone tissue repair material that promotes angiogenesis and new bone formation according to claim 6, characterized in that, In step S3, the mass concentration of the biodegradable bio-derived gel solution is 25~35 mg / mL; The ratio of the porous deantigenated lyophilized bone matrix to the biodegradable bio-derived gel solution is (1~4g): (5~20ml).

10. The use of a bone tissue repair material that promotes angiogenesis and new bone formation as described in any one of claims 1 to 5 in the fabrication of implantable biological scaffolds for orthopedic repair.

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