A method for preparing decalcified bone matrix

Abstract

The present application provides a method for preparing a decalcified bone matrix, which is combined with specific pre-treatment, disinfection, defatting, decellularization, decalcification, freeze-drying and sterilization methods to better retain the integrity of the bioactive components and structure in the material, especially using the decalcification method of the present application, adding sugars and polyol protein protective agents to traditional decalcification reagents, which can achieve efficient removal of mineral components while reducing the damage to BMP-2 and various cytokines during decalcification. The decalcified bone matrix prepared by the present application retains the spatial structure of the bone intact, has good biocompatibility, biodegradability and bone repair ability, can be used for the repair and reconstruction of bone defects, and has broad application prospects.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a method for preparing a decalcified bone matrix. Background Art

[0002] Bone tissue defects caused by severe fracture trauma, orthopedic surgery, etc., lead to serious loss of motor function, a sharp decline in quality of life, and even threaten life. With the continuous improvement of living standards and the further aggravation of the aging trend, the current demand for bone tissue repair is increasing, which brings huge challenges to the clinical application of orthopedics and the research field of bone tissue reconstruction and repair.

[0003] Natural bone transplant materials represented by demineralized bone matrix (DBM) have become a hot topic in the current research field of bone transplant materials due to their good osteoinduction, osteoconduction and osteogenic abilities. Demineralized bone matrix is ​​a matrix material composed of collagen, non-collagenous proteins and low-concentration growth factors after removing some or all of the minerals in bone tissue. Its active role in promoting bone formation mainly depends on its rich bone morphogenetic proteins (BMPs) and various osteogenic factors. The BMPs and various osteogenic factors in the demineralized bone matrix are tightly bound to the collagen in the bone matrix, which can induce mesenchymal cells to differentiate into cartilage tissue and osteoblasts, and finally form cartilage tissue and bone tissue. However, in normal intact bones, BMPs are surrounded and isolated by dense mineral components, making it difficult to effectively play the function of inducing osteogenic function. After decalcification, on the one hand, the BMPs are freed from their encapsulation, allowing them to be released smoothly and thus play an osteogenesis-inducing role. On the other hand, the unique pore structure of decalcified bone ensures that BMPs can be released slowly and stably, allowing BMPs to play a long-term role. Therefore, decalcification is an important step that determines the effectiveness of decalcified bone matrix. The difficulty of the decalcification process lies in removing minerals while minimizing the damage to BMPs and various cytokines. Therefore, how to improve the retention of BMPs and various cytokines while removing minerals and improve the osteogenesis-inducing ability of decalcified bone matrix is ​​the current research focus of preparing decalcified bone matrix. Summary of the invention

[0004] The purpose of the present application is to provide a method for preparing a decalcified bone matrix, so as to achieve efficient removal of mineral components in the decalcified bone matrix and reduce the damage to BMP-2 and various cytokines.

[0005] The first aspect of the present application provides a method for preparing a decalcified bone matrix, which comprises the following steps:

[0006] Pretreatment: Take animal cortical bones, remove the surface soft tissue and periosteum, cut into bone blocks with a thickness of 3-20 mm, and crush into bone particles with a particle size of 100-3000 μm;

[0007] Disinfection: Mix the bone particles with a disinfectant according to a material-liquid mass volume ratio of 1 g: 3-10 mL, and shake for 60-150 min; wherein the disinfectant is selected from at least one of a 0.1-1 wt% peracetic acid solution and a 0.5-3 wt% hydrogen peroxide solution;

[0008] Degreasing: Mix the bone particles with a degreasing agent according to a material-liquid mass volume ratio of 1g:5-10mL, and shake for 3-6h; wherein the degreasing agent is selected from at least one of isopropanol, acetone and anhydrous ethanol;

[0009] Decellularization: mixing the bone particles with a decellularization reagent according to a material-liquid mass volume ratio of 1 g: 5-10 mL, stirring at 0-10° C. for 16-48 hours; wherein the decellularization reagent is selected from at least one of a 0.2-1.0 wt % sodium dodecyl ether sulfate solution, a 0.1-1 wt % Triton X-100 solution, and a 0.5-2 wt % sodium deoxycholate solution;

[0010] Decalcification: adding a protein protective agent to a 0.4-0.8 mol / L hydrochloric acid solution to obtain a decalcified solution; mixing the bone particles with the decalcified solution according to a mass volume ratio of 1 g: 3-15 mL, stirring at 0-10° C. for 2-8 hours, and changing the solution every hour; wherein the mass volume ratio of the protein protective agent to the hydrochloric acid solution is 1-20 g: 100 mL; wherein the protein protective agent is selected from at least one of a carbohydrate and a polyol; wherein the carbohydrate is selected from at least one of sucrose, glucose, mannose and trehalose, and the polyol is selected from at least one of polyethylene glycol and glycerol;

[0011] Freeze drying: freeze drying the bone particles;

[0012] Sterilization: freezing the bone particles at low temperature and sterilizing them by irradiation to obtain decalcified bone matrix; wherein the irradiation sterilization is cobalt 60 irradiation sterilization with an irradiation dose of 18-25 kGy.

[0013] The second aspect of the present application provides a decalcified bone matrix prepared by the preparation method of the first aspect of the present application, which comprises bone morphogenetic protein 2 and collagen, wherein the content of bone morphogenetic protein 2 based on the decalcified bone matrix is ​​17-35 ng / g.

[0014] The preparation method of decalcified bone matrix provided by the present application adopts a combination of specific pre-treatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods, which can effectively remove antigenic substances in animal tissues while better retaining the integrity of the biologically active components and structure in the material. In particular, the decalcification method of the present application is adopted, and sugars and polyol protein protective agents are added to the traditional decalcification reagents, which can achieve efficient removal of mineral components while reducing the damage to BMP-2 and various cytokines during the decalcification process. Furthermore, the preparation conditions of the present application are simple, the quality is controllable, and the cost is low.

[0015] The decalcified bone matrix prepared by the present application retains the spatial structure of the bone intactly, has good biocompatibility, biodegradability and bone repair ability, and can be used for the repair and reconstruction of bone defects. Furthermore, the decalcified bone matrix prepared by the present application is low in price and has good market application prospects.

[0016] Of course, implementing any product or method of the present application does not necessarily require achieving all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0018] Figure 1 This is a hematoxylin-eosin staining (HE staining) image of the decalcified bone matrix particles of Example 1;

[0019] Figure 2 This is a fluorescence image of the adhesion and growth of cells on the decalcified bone matrix particles of Example 2;

[0020] Figure 3 This is a Goldner staining image of the decalcified bone matrix particles in Example 3 during ectopic osteogenesis induction in vivo;

[0021] Figure 4 This is the Goldner staining image of the decalcified bone matrix particles of Comparative Example 1 during ectopic osteogenesis in vivo. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field based on the present application belong to the scope of protection of the present application.

[0023] The first aspect of the present application provides a method for preparing a decalcified bone matrix, which comprises the following steps:

[0024] Pretreatment: Take animal cortical bones, remove the surface soft tissue and periosteum, cut into bone blocks with a thickness of 3-20 mm, and crush into bone particles with a particle size of 100-3000 μm;

[0025] Disinfection: Mix the bone particles with a disinfectant according to a material-liquid mass volume ratio of 1 g: 3-10 mL, and shake for 60-150 min; wherein the disinfectant is selected from at least one of a 0.1-1 wt% peracetic acid solution and a 0.5-3 wt% hydrogen peroxide solution;

[0026] Degreasing: Mix the bone particles with a degreasing agent according to a material-liquid mass volume ratio of 1g:5-10mL, and shake for 3-6h; wherein the degreasing agent is selected from at least one of isopropanol, acetone and anhydrous ethanol;

[0027] Decellularization: mixing the bone particles with a decellularization reagent according to a material-liquid mass volume ratio of 1 g: 5-10 mL, stirring at 0-10° C. for 16-48 hours; wherein the decellularization reagent is selected from at least one of a 0.2-1.0 wt % sodium dodecyl ether sulfate solution, a 0.1-1 wt % Triton X-100 solution, and a 0.5-2 wt % sodium deoxycholate solution;

[0028] Decalcification: adding a protein protective agent to a 0.4-0.8 mol / L hydrochloric acid solution to obtain a decalcified solution; mixing the bone particles with the decalcified solution according to a mass volume ratio of 1 g: 3-15 mL, stirring at 0-10° C. for 2-8 hours, and changing the solution every hour; wherein the mass volume ratio of the protein protective agent to the hydrochloric acid solution is 1-20 g: 100 mL; wherein the protein protective agent is selected from at least one of a carbohydrate and a polyol; wherein the carbohydrate is selected from at least one of sucrose, glucose, mannose and trehalose, and the polyol is selected from at least one of polyethylene glycol and glycerol;

[0029] Freeze drying: freeze drying the bone particles;

[0030] Sterilization: freezing the bone particles at low temperature and sterilizing them by irradiation to obtain decalcified bone matrix; wherein the irradiation sterilization is cobalt 60 irradiation sterilization with an irradiation dose of 18-25 kGy.

[0031] The inventors found in their research that the decalcified bone matrix prepared by crushing bone blocks into particles with a size of 100-3000 μm has higher osteogenic induction activity.

[0032] The inventors found in their research that the fat content of the decalcified bone matrix prepared by the degreasing method and degreasing reagent of the present application is low, at 0.05-0.5wt%. It should be noted that the purity of the isopropanol and acetone in the degreasing reagent of the present application is above 99%.

[0033] The inventors found in their research that by adopting the disinfection method of the present application and selecting peracetic acid and hydrogen peroxide as disinfectants, the osteoinductive ability of the decalcified bone matrix can be retained while achieving the purpose of disinfection.

[0034] The inventors found in their research that the sterilization method of the present application, freezing the bone particles at low temperatures, and using irradiation sterilization treatment with an irradiation dose of 18-25 kGy, can not only stably and effectively sterilize, but also will not destroy the osteoinductive ability of the decalcified bone matrix. It should be noted that the present application does not limit the method of low-temperature freezing, as long as the purpose of the present application can be achieved, for example, dry ice can be spread around the sample to maintain the low temperature.

[0035] The inventors found in their research that the decalcified bone matrix prepared by the decellularization method and decellularization reagent of the present application basically retains the complete structure, and the matrix components are basically unaffected. Furthermore, the decellularization method of the present application reduces the damage to BMP-2 and various cytokines during the decalcification process.

[0036] The inventors found in their research that by using the decalcification method of the present application, adding sugars and polyol protein protectants to traditional decalcification reagents, and controlling the decalcification temperature and decalcification time within the range of the present application, the damage to BMP-2 and various cytokines during the decalcification process is reduced.

[0037] It should be noted that freeze-drying is a common operation in the art and is not limited in the present application. For example, the bone particles can be freeze-dried at -50 to -30°C for 48 to 72 hours.

[0038] In the present application, a combination of specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods can better preserve the integrity of the material structure and bioactive components, especially the retention of BMP-2 and various osteogenic factors that promote bone formation, thereby improving the bone-inducing ability of the decalcified bone matrix.

[0039] In some embodiments of the present application, the animal cortical bone is derived from cattle or pigs; preferably, the animal cortical bone is derived from cattle aged 20-30 months or pigs aged 4-6 months.

[0040] In some embodiments of the present application, the protein protective agent is selected from at least one of sucrose, glucose, trehalose and polyethylene glycol. The inventors found in their research that adding sucrose, glucose, trehalose or polyethylene glycol as a protein protective agent during the decalcification process can reduce the dissolution or denaturation of BMP-2 and various cytokines, and can provide stable protection for BMP-2 and various cytokines in solution or in a dry state.

[0041] In some embodiments of the present application, the temperature in the decalcification step is 2-8° C. and the time is 3-6 h.

[0042] In some embodiments of the present application, the temperature in the decellularization and decalcification steps is 2-8° C. The inventors found in their research that the decellularization and decalcification temperatures are controlled within the range of the present application, which reduces the damage to BMP-2 and various cytokines during the decellularization and decalcification processes.

[0043] It should be noted that the decellularization and decalcification steps of the present application are all carried out in a stirring manner. The inventors have found in their research that the decellularization and decalcification in a stirring manner can not only achieve efficient removal of antigenic components and mineral components in bone particles, but also make the decellularization and decalcification of all bone particles more uniform, without causing excessive or insufficient decalcification of some bone particles. In the present application, the stirring method is not limited, and the purpose of the present application can be achieved. For example, an overhead mixer can be used with a rotation speed of 200-300rpm.

[0044] In some embodiments of the present application, the mass volume ratio of the protein protective agent to the hydrochloric acid solution in the decalcification step is 5-15g:100mL. The inventors found in their research that the amount of the protein protective agent added is controlled within the scope of the present application, which can better protect BMP-2 and various cytokines from being destroyed during the decalcification process.

[0045] In some embodiments of the present application, the mass volume ratio of the bone particles to the decalcification solution is 1g:6-10mL.

[0046] In some embodiments of the present application, after the pretreatment, degreasing, disinfection, decellularization and decalcification steps are completed, each also includes cleaning with a cleaning agent; the cleaning agent is selected from at least one of purified water, 0.01-0.1 mol / L phosphate buffer (pH 7.4-7.8) and 0.02-0.1 mol / L MES buffer (pH 5.5-6.7).

[0047] In some embodiments of the present application, the cleaning step includes adding a cleaning agent according to a feed-liquid mass volume ratio of 1 g: 3-10 mL, stirring for 5-10 minutes, changing the liquid midway, and repeating the cleaning 5-15 times. In the present application, the stirring method is not limited, as long as it can achieve the purpose of the present application. For example, an overhead stirrer can be used with a rotation speed of 200-300 rpm.

[0048] In some embodiments of the present application, the cleaning step after decalcification is to use 0.01-0.1 mol / L phosphate (pH=7.4-7.8) buffer to clean the solution until it reaches neutrality, and then clean it with purified water.

[0049] In the present application, the oscillation is a conventional operation in the art, and the present application is not limited thereto. It is sufficient as long as the purpose of the present application can be achieved. For example, an air bath or a refrigerated shaker can be used for oscillation, and the oscillation frequency is 100-200 rpm.

[0050] The second aspect of the present application provides a decalcified bone matrix prepared by the preparation method described in the first aspect of the present application, which comprises BMP-2 and collagen, wherein the BMP-2 content based on the decalcified bone matrix is ​​17-35 ng / g.

[0051] The inventors found in their research that the decalcified bone matrix prepared by the preparation method of the present application retains the spatial structure of the bone intactly and has good biocompatibility, biodegradability and bone repair ability.

[0052] The technical solution of the present application is further explained below through specific embodiments.

[0053] The experimental materials and methods used in the following examples are conventional materials and methods unless otherwise specified.

[0054] Example 1

[0055] (1) Pretreatment: Take cortical bones of 20-30 month old cattle, remove the surface soft tissue and periosteum by physical scraping, cut into bone blocks with a thickness of 3-20 mm, and crush into bone particles with a particle size of 250-2000 μm. Mix the bone particles with purified water at a mass-to-volume ratio of 1 g:5 mL, and shake and wash 6 times, each time for 10 min;

[0056] (2) Disinfection: Mix the bone particles with 0.2 wt% peracetic acid solution at a material-liquid mass volume ratio of 1 g:5 mL, shake for 120 min, discard the peracetic acid solution, add purified water, and stir and wash 6 times, each time for 5 min;

[0057] (3) Degreasing: Mix the sterilized bone particles with isopropanol at a mass-to-volume ratio of 1 g:5 mL, shake for 5 h, discard the isopropanol, add 0.05 mol / L MES buffer (pH = 5.5), stir and wash 6 times, each time for 5 min;

[0058] (4) Decellularization: The defatted bone particles were mixed with 0.8 wt% sodium dodecyl ether sulfate solution at a feed-liquid mass volume ratio of 1 g:5 mL, placed in a low-temperature water tank (4°C), stirred for 24 h, and the sodium dodecyl ether sulfate solution was discarded. The bone particles were then mixed with 0.5 wt% Triton X-100 solution at a feed-liquid mass volume ratio of 1 g:5 mL, placed in a low-temperature water tank (4°C), stirred for 24 h, and the Triton X-100 solution was discarded. 0.05 mol / L MES buffer (pH = 5.5) was then added, and the mixture was stirred and washed for 6 times, each time for 10 min.

[0059] (5) Decalcification: Add 8 g of sucrose to 100 mL of hydrochloric acid solution (0.5 mol / L) to obtain a decalcified solution; mix the decellularized bone particles with the decalcified solution at a mass-to-volume ratio of 1 g:8 mL, place in a low-temperature water tank (4°C), stir for 4 h, and change the solution every hour; discard the decalcified solution, add 0.1 mol / L phosphate buffer (pH = 7.8), stir and wash for 3 times, each time for 10 min, wash until the solution reaches neutrality, and then stir and wash with purified water for 7 times, each time for 10 min;

[0060] (6) Freeze-drying: Spread the decalcified bone matrix flat in a steel plate mold, freeze-dry and package;

[0061] (7) Sterilization: The packaged decalcified bone matrix is ​​sterilized by irradiation at 20 kGy and then stored at room temperature.

[0062] Example 2

[0063] Except that 8 g of sucrose in step (5) was replaced by 8 g of mannose, the rest was the same as in Example 1.

[0064] Example 3

[0065] Except that 8 g of sucrose in step (5) was replaced by 8 g of polyethylene glycol, the rest was the same as in Example 1.

[0066] Comparative Example 1

[0067] Except that the decalcification solution in step (5) is replaced by 0.5 mol / L hydrochloric acid solution, the rest is the same as Example 1.

[0068] Effect measurement

[0069] 1. Biochemical composition and content analysis

[0070] BMP-2 content detection: BMP-2 was extracted according to the method in "BMP-2 detection in DBM to preliminarily determine the activity of bone bank bone donation", and then the BMP-2 content was detected using the bovine bone morphogenetic protein 2 (BMP-2) ELISA detection kit (Shanghai ELISA Biotechnology Co., Ltd.) according to the instructions.

[0071] Table 1

[0072] BMP-2 (ng / g) Example 1 31.05±2.55 Example 2 25.93±7.40 Example 3 26.30±1.72 Comparative Example 1 17.28±5.56

[0073] BMP-2 is one of the members of the transforming growth factor B superfamily. It has the ability to induce undifferentiated mesenchymal stem cells to differentiate and proliferate into chondrocytes and osteoblasts, promote osteoblast differentiation and maturation, participate in bone and cartilage growth and development and its reconstruction process, and then accelerate bone defect repair. As can be seen from Table 1, no protein protective agent is added in Comparative Example 1, and the BMP-2 content of decalcified bone matrix is ​​17.28 ± 5.56 ng / g; while Examples 1 to 3 add carbohydrates or polyol protein protective agents to traditional decalcification reagents, and the BMP-2 content of decalcified bone matrix is ​​31.05 ± 2.55 ng / g, 25.93 ± 7.40 ng / g, and 26.30 ± 1.72 ng / g, respectively. It is explained that the decalcification method of the present application is adopted, and carbohydrates and polyol protein protective agents are added to traditional decalcification reagents, which can reduce the damage to BMP-2 during the decalcification process, thereby improving bone repair ability.

[0074] 2. Analysis of immunogenic substances

[0075] Residual host cells: The sample to be tested was fixed with 5% neutral formalin, embedded in paraffin, cut into 0.4 μm slices, dewaxed with xylene, dehydrated with a series of alcohols, stained with HE, and observed under a microscope.

[0076] DNA content: Detected in accordance with YY / T 0606.25-2014 "Determination of DNA Residue in Animal-derived Biological Materials: Fluorescence Staining Method".

[0077] Fat content: tested by acid hydrolysis treatment as specified in the second method of GB / T 5009.6-2016.

[0078] Table 2

[0079] DNA content (ng / mg) Fat content (wt%) Example 1 2.95±0.14 0.16±0.10 Example 2 3.13±1.79 0.33±0.10 Example 3 2.87±0.12 0.30±0.14

[0080] The HE staining of the decalcified bone matrix particles of Example 1 is as follows Figure 1 As shown, from Figure 1 It can be seen that the decalcified bone matrix particles of Example 1 have no blue-dyed nuclear material, the basement membrane structure is clear, and no donor cells remain, indicating that the antigenic substances of the decalcified bone matrix particles of the present application are removed more thoroughly and have good biological safety.

[0081] As can be seen from Table 2, the DNA content in the decalcified bone matrix particles of Examples 1 to 3 is 2.95±0.14 ng / mg, 3.13±1.79 ng / mg, and 2.87±0.12 ng / mg, respectively; the fat content in the decalcified bone matrix particles of Examples 1 to 3 is 0.16±0.10 wt%, 0.33±0.10 wt%, and 0.30±0.14 wt%, respectively. The preparation method of the decalcified bone matrix provided by the present application is described, and the fat and DNA content in the decalcified bone matrix obtained by combining specific pretreatment, disinfection, defatting, decellularization, decalcification, freeze-drying and sterilization methods is lower, and the biosafety is better.

[0082] 3. Cell experiments

[0083] Cell adhesion experiment: (1) 10 mg of irradiated and sterilized decalcified bone matrix particles of Example 2 were added to each well of a 48-well plate, and MC3T3-E1 cells were cultured at 6×10 4 The cells were inoculated on the surface of irradiated decalcified bone matrix particles at a density of 100 cells / well, with 3 replicate wells in each group, and then placed in a cell culture incubator at 37°C and 5% CO2 (volume fraction, the rest is air); (2) After culturing for 1 day, the culture plates were removed from the incubator, washed with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde for 30 min. The cells were then stained with rhodamine-labeled phalloidin (Yisheng Biotechnology (Shanghai)) and 4',6-diamidino-2-phenylindole (DAPI, Solebol) according to the product instructions, and the cell morphology and cytoskeleton structure were observed under a fluorescence microscope. The results are shown in Table 2. Figure 2 .

[0084] from Figure 2 The results show that MC3T3-E1 cells spread and elongate effectively on the surface of decalcified bone matrix particles, and the MC3T3-E1 cells have good morphology, indicating that the cells can adhere and grow well on the surface of decalcified bone matrix particles.

[0085] 4. In vivo ectopic osteogenesis induction experiment

[0086] According to YY / T 1680-2020 "Evaluation of in vivo osteoinductive performance of allogeneic repair materials and demineralized bone materials", the decalcified bone matrix particles of Example 3 were implanted into the gluteus medius and gluteus maximus intermuscular space of healthy 6-8 week old male BALB / c nude mice. Goldner staining was performed 4 weeks after surgery. The results are shown in Figure 3 .

[0087] According to YY / T 1680-2020 "Evaluation of in vivo osteoinductive performance of allogeneic repair materials and demineralized bone materials", the decalcified bone matrix particles of comparative example 1 were implanted into the intermuscular space between the gluteus medius and gluteus maximus of healthy 6-8 week old male BALB / c nude mice. Goldner staining was performed 4 weeks after surgery. The results are shown in the figure. Figure 4 .

[0088] from Figure 3 It can be seen that the number of osteoblasts and chondrocytes growing at the interface between muscle and decalcified bone matrix particles and inside the decalcified bone matrix particles is large and the distribution is good. At the same time, the generation of osteoid can also be observed, which shows that the decalcified bone matrix particle material prepared by the present application has good ectopic bone induction ability. Figure 3 compared to, Figure 4 The number and growth distribution of osteoblasts and chondrocytes around and inside the decalcified bone matrix particles in the comparative example 1 are significantly poorer, indicating that the ectopic osteoinduction ability of the decalcified bone matrix particles in comparative example 1 is not as good as that of the decalcified bone matrix particles in example 3.

[0089] In summary, the preparation method of decalcified bone matrix provided by the present application adopts a combination of specific pre-treatment, disinfection, defatting, decellularization, decalcification, freeze-drying and sterilization methods, while effectively removing antigenic substances from animal tissues, it can also better retain the integrity of the bioactive components and structure in the material, especially by using the decalcification method of the present application, adding sugars or polyol protein protective agents to traditional decalcification reagents, while achieving efficient removal of mineral components, it can also reduce the damage to BMP-2 and various cytokines during the decalcification process. Further, the preparation conditions of the present application are simple, the quality is controllable, and the cost is low.

[0090] The decalcified bone matrix prepared by the present application retains the spatial structure of the bone intactly, has good biocompatibility, biodegradability and bone repair ability, and can be used for the repair and reconstruction of bone defects. Furthermore, the decalcified bone matrix prepared by the present application is low in price and has good market application prospects.

[0091] The above description is only a preferred embodiment of the present application and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application are included in the protection scope of the present application.

Claims

1. A method for preparing a decalcified bone matrix, comprising the following steps: Pretreatment: Take animal cortical bones, remove the surface soft tissue and periosteum, cut into bone blocks with a thickness of 3-20 mm, and crush into bone particles with a particle size of 100-3000 μm; Disinfection: Mix the bone particles with a disinfectant according to a material-liquid mass volume ratio of 1 g: 3-10 mL, and shake for 60-150 min; wherein the disinfectant is selected from at least one of a 0.1-1 wt% peracetic acid solution and a 0.5-3 wt% hydrogen peroxide solution; Degreasing: Mix the bone particles with a degreasing agent according to a material-liquid mass volume ratio of 1g:5-10mL, and shake for 3-6h; wherein the degreasing agent is selected from at least one of isopropanol, acetone and anhydrous ethanol; Decellularization: mixing the bone particles with a decellularization reagent according to a material-liquid mass volume ratio of 1 g: 5-10 mL, stirring at 0-10° C. for 16-48 hours; wherein the decellularization reagent is selected from at least one of a 0.2-1.0 wt % sodium dodecyl ether sulfate solution, a 0.1-1 wt % Triton X-100 solution, and a 0.5-2 wt % sodium deoxycholate solution; Decalcification: adding a protein protective agent to a 0.4-0.8 mol / L hydrochloric acid solution to obtain a decalcified solution; mixing the bone particles with the decalcified solution according to a material-liquid mass volume ratio of 1 g: 3-15 mL, stirring at 0-10° C. for 2-8 hours, and changing the solution every hour; wherein the mass volume ratio of the protein protective agent to the hydrochloric acid solution is 1-20 g: 100 mL; wherein the protein protective agent is selected from at least one of a carbohydrate and a polyol; wherein the carbohydrate is selected from at least one of sucrose, glucose, mannose and trehalose, and the polyol is selected from at least one of polyethylene glycol and glycerol; Freeze drying: freeze drying the bone particles; Sterilization: freezing the bone particles at low temperature and sterilizing them by irradiation to obtain decalcified bone matrix; wherein the irradiation sterilization is cobalt 60 irradiation sterilization with an irradiation dose of 18-25 kGy.

2. The preparation method according to claim 1, wherein The animal cortical bone is derived from cattle or pigs.

3. The preparation method according to claim 1, wherein The protein protective agent is selected from at least one of sucrose, glucose, trehalose and polyethylene glycol.

4. The preparation method according to claim 1, wherein The temperature in the decalcification step is 2-8°C and the time is 3-6h.

5. The preparation method according to claim 1, wherein In the decalcification step, the mass volume ratio of the protein protective agent to the hydrochloric acid solution is 5-15g:100mL; the mass volume ratio of the bone particles to the decalcification solution is 1g:6-10mL.

6. The preparation method according to claim 1, wherein After the pretreatment, degreasing, disinfection, decellularization and decalcification steps are completed, each step further comprises cleaning with a cleaning agent; the cleaning agent is selected from at least one of purified water, 0.01-0.1 mol / L phosphate buffer and 0.02-0.1 mol / L MES buffer.

7. The preparation method according to claim 6, wherein: The cleaning step includes adding a cleaning agent according to a material-liquid mass volume ratio of 1 g: 3-10 mL, stirring for 5-10 minutes, changing the liquid in the middle, and repeating the cleaning 5-15 times.

8. The preparation method according to claim 7, wherein: The cleaning step after decalcification is to use 0.01-0.1 mol / L phosphate buffer to wash until the solution reaches neutrality, and then wash with purified water.

9. The decalcified bone matrix prepared by the preparation method according to any one of claims 1 to 8, comprising bone morphogenetic protein 2 and collagen, wherein: The bone morphogenetic protein 2 content based on the decalcified bone matrix is ​​17-35 ng / g.

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