CN116212114A

CN116212114A - Preparation method of decalcified bone matrix

Info

Publication number: CN116212114A
Application number: CN202310448181.3A
Authority: CN
Inventors: 吴静; 欧阳澳利; 张静逸; 郑梁杰; 冉永峰; 江涛; 李志宏
Original assignee: Hangzhou Huamai Medical Technology Co ltd
Current assignee: Hangzhou Huamai Medical Technology Co ltd
Priority date: 2023-04-24
Filing date: 2023-04-24
Publication date: 2023-06-06

Abstract

The application provides a preparation method of a decalcified bone matrix, which combines specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods, can better retain the integrity of bioactive components and structures in materials, and particularly adopts the decalcification method, and the saccharide and polyalcohol protein protectant is added into the traditional decalcified reagent, so that the high-efficiency removal of mineral components can be realized, and the damage to BMP-2 and various cytokines in the decalcification process can be reduced. The decalcified bone matrix prepared by the method well reserves the space structure of bones, has good biocompatibility, biodegradability and bone repair capability, can be used for repairing and reconstructing bone defects, and has wide application prospect.

Description

Preparation method of decalcified bone matrix

Technical Field

The application relates to the technical field of biomedical materials, in particular to a preparation method of a decalcified bone matrix.

Background

Bone tissue defect caused by severe fracture trauma, bone surgery and the like causes serious loss of the function of a patient's movement system, and the quality of life is drastically reduced or even threatens life. With the continuous improvement of living standard and the further aggravation of the aging trend, the current demand for bone tissue repair is increasing, and the demands for bone tissue repair are great in clinical application and bone tissue reconstruction repair research fields.

Natural bone graft materials, represented by decalcified bone matrix (Demineralized bone matrix, DBM), have been a major focus in the research field of current bone graft materials due to their excellent osteoinductive, osteoconductive and bone-promoting capabilities. Decalcification bone matrix is a matrix material which is composed of collagen, non-collagen, growth factors with low concentration and the like after removing part or all of mineral matters in bone tissues, and the activity of promoting bone formation mainly depends on the abundant bone morphogenetic proteins (Bone Morphogenetic Proteins, BMPs) and various osteogenic factors. BMPs and various osteogenic factors in the decalcified bone matrix are tightly combined with collagen in the bone matrix, so that mesenchymal cells can be induced to differentiate into cartilage tissues and osteoblasts, and finally the cartilage tissues and the bone tissues are formed. However, in normal intact bones, BMPs are isolated by dense mineral component Bao Rao, and it is difficult to effectively exert the function of inducing osteogenesis. After decalcification, bao Rao on BMPs is removed, so that the BMPs can be smoothly released to exert the osteogenesis inducing effect, and on the other hand, the special pore structure of the decalcified bone ensures that the BMPs can be permanently and stably released slowly, so that the BMPs can play a role for a long time, so that decalcification is an important step for determining the effectiveness of the decalcified bone matrix. The difficulty with the decalcification process is that it minimizes damage to BMPs and various cytokines while removing minerals. Therefore, how to remove minerals and improve the retention of BMPs and various cytokines and the osteogenesis induction capability of the decalcified bone matrix is the focus of the current research for preparing the decalcified bone matrix.

Disclosure of Invention

The aim of the application is to provide a preparation method of a decalcified bone matrix, which is used for realizing efficient removal of mineral components in the decalcified bone matrix and reducing damage to BMP-2 and various cytokines.

The first aspect of the present application provides a method for preparing a decalcified bone matrix comprising the steps of:

pretreatment: taking animal cortical bone, removing surface soft tissue and periosteum, cutting into bone pieces with thickness of 3-20mm, and pulverizing into bone particles with particle size of 100-3000 μm;

and (3) disinfection: according to the mass volume ratio of 1g: mixing the bone particles with disinfectant in 3-10mL, and oscillating for 60-150min; wherein the disinfectant is selected from at least one of 0.1-1wt% peracetic acid solution and 0.5-3wt% hydrogen peroxide solution;

degreasing: mixing the bone particles with degreasing agent according to the mass-volume ratio of 1g to 5-10mL, and oscillating for 3-6h; wherein the degreasing agent is at least one selected from isopropanol, acetone and absolute ethyl alcohol;

decellularization: according to the mass volume ratio of 1g: mixing 5-10mL of bone particles with a decellularization reagent, and stirring for 16-48h at 0-10 ℃; wherein the decellularization reagent is at least one selected from 0.2-1.0wt% of sodium dodecyl ether sulfate solution, 0.1-1wt% of triton X-100 solution and 0.5-2wt% of sodium deoxycholate solution;

decalcification: adding a protein protecting agent into a hydrochloric acid solution with the concentration of 0.4-0.8mol/L to obtain decalcification solution; according to the mass volume ratio of 1g:3-15mL of the bone particles are mixed with decalcification liquid, the temperature is 0-10 ℃, and the mixture is stirred for 2-8h, and the liquid is changed every hour; wherein the mass volume ratio of the protein protectant to the hydrochloric acid solution is 1-20 g/100 mL; wherein the protein protectant is selected from at least one of saccharides and polyols; wherein the saccharide 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;

and (3) freeze-drying: freeze-drying the bone particles;

and (3) sterilization: freezing the bone particles at low temperature, and performing irradiation sterilization treatment to obtain decalcified bone matrix; wherein the irradiation sterilization is cobalt 60 irradiation sterilization, and the irradiation dose is 18-25kGy.

In a second aspect, the present application provides a decalcified bone matrix prepared by the method of the first aspect of the present application, comprising bone morphogenic protein 2 and collagen, wherein the content of bone morphogenic protein 2 based on said decalcified bone matrix is 17-35ng/g.

The preparation method of the decalcified bone matrix provided by the application combines specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods, can effectively remove animal tissue antigenic substances, and simultaneously can better retain the integrity of bioactive components and structures in the materials, and particularly by adopting the decalcified method, the saccharide and polyalcohol protein protectant are added into the traditional decalcified reagent, so that the high-efficiency removal of mineral components can be realized, and simultaneously, the damage to BMP-2 and various cytokines in the decalcified process can be reduced. Furthermore, the preparation condition of the preparation method is simple, the quality is controllable, and the cost is low.

The decalcified bone matrix prepared by the method well maintains the space structure of bones, has good biocompatibility, biodegradability and bone repair capability, and can be used for repairing and reconstructing bone defects. Furthermore, the decalcified bone matrix prepared by the method is low in price and has good market application prospect.

Of course, not all of the above-described advantages need be achieved simultaneously in practicing any one of the products or methods of the present application.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other embodiments may also be obtained according to these drawings to those skilled in the art.

FIG. 1 is a hematoxylin-eosin staining (HE staining) diagram of decalcified bone matrix particles of example 1;

FIG. 2 is a fluorescence plot of the adherent growth of cells on decalcified bone matrix particles of example 2;

FIG. 3 is a Goldner staining pattern of decalcified bone matrix particles of example 3 in vivo ectopic osteoinduction;

fig. 4 is a Goldner staining pattern of decalcified bone matrix particles of comparative example 1 in vivo ectopic osteoinduction.

Detailed Description

The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present application. Based on the embodiments herein, a person of ordinary skill in the art would be able to obtain all other embodiments based on the disclosure herein, which are within the scope of the disclosure herein.

and (3) freeze-drying: freeze-drying the bone particles;

The inventors found in the study that the bone fragments were crushed to particles of 100-3000 μm size, and the finally prepared decalcified bone matrix induced bone formation activity was higher.

The inventors have found in the study that the decalcified bone matrix prepared by the degreasing method and degreasing agent of the present application has a low fat content of 0.05-0.5wt%. The purity of isopropyl alcohol and acetone in the degreasing agent of the present application is 99% or more.

The inventor finds that the sterilization mode of the application is adopted, and the peroxyacetic acid and the hydrogen peroxide are selected as the sterilization agents, so that the sterilization purpose is realized, and the osteogenesis induction capability of the decalcified bone matrix can be reserved.

The inventor finds in the study that the sterilization mode of the application is adopted to freeze the bone particles at low temperature, and the irradiation sterilization treatment with the irradiation dose of 18-25kGy is adopted, so that the bone particles can be stably and effectively sterilized, and the induced osteogenesis capability of the decalcified bone matrix is not damaged. The method of low-temperature freezing is not limited in the present application, and the object of the present application may be achieved, for example, dry ice may be filled around the sample to keep the temperature low.

The inventor finds in the research that the prepared decalcified bone matrix basically maintains the complete structure by adopting the decellularization method and the decellularization reagent, and matrix components are not affected basically, and further, the damage to BMP-2 and various cytokines in the decalcification process is reduced by adopting the decellularization method.

The inventor finds that by adopting the decalcification method, the saccharide and the polyalcohol protein protective agent are added into the traditional decalcification reagent, and the decalcification temperature and decalcification time are controlled within the scope of the application, so that the damage to BMP-2 and various cytokines in the decalcification process is reduced.

Lyophilization is a routine procedure in the art, and is not limited herein, and the bone particles may be lyophilized at-50 to-30℃for 48 to 72 hours, for example.

In the application, the specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods are combined, so that the structural integrity of the material and the bioactive components, especially BMP-2 and various osteogenic factors for promoting bone formation can be well reserved, and the osteogenesis induction capability of the decalcified bone matrix is improved.

In some embodiments of the present application, the animal cortical bone is derived from bovine or porcine; preferably, the animal cortical bone is derived from a cow of 20-30 months of age or a pig of 4-6 months of age.

In some embodiments of the present application, the protein protectant is selected from at least one of sucrose, glucose, trehalose, and polyethylene glycol. The inventor finds that the dissolution or denaturation of BMP-2 and various cytokines can be reduced by adding sucrose, glucose, trehalose or polyethylene glycol as a protein protective agent in the decalcification process, and the stable protective effect can be achieved on the BMP-2 and various cytokines in a solution or in a dry state.

In some embodiments of the present application, the temperature in the decalcification step is 2-8deg.C for 3-6 hours.

In some embodiments of the present application, the temperature in the decellularization and decalcification step is 2-8 ℃. The inventors have found in the study that controlling the decellularization and decalcification temperatures within the scope of the present application reduces the damage to BMP-2 and various cytokines by the decellularization and decalcification process.

The decellularization and decalcification steps in the present application are both performed by stirring. The inventor finds that the method of stirring is adopted to remove cells and calcium, so that not only can the antigen components and mineral components in bone grains be efficiently removed, but also the degree of the cell removal and the calcium removal of all the bone grains can be more uniform, and partial bone grains can not be excessively or insufficiently removed. In the present application, the stirring mode is not limited, and the object of the present application can be achieved, for example, a top-mounted stirrer with a rotation speed of 200 to 300rpm may be used.

In some embodiments of the present application, the mass to volume ratio of the protein protectant to the hydrochloric acid solution in the decalcification step is 5-15g:100ml. The inventor discovers in the research that the addition amount of the protein protectant is controlled within the scope of the application, so that BMP-2 and various cytokines can be better protected from being destroyed in the decalcification process.

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

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

In some embodiments of the application, the cleaning step comprises adding cleaning agent according to the mass-volume ratio of 1g to 3-10mL, stirring for 5-10min, changing the liquid halfway, and repeating the cleaning for 5-15 times. In the present application, the stirring mode is not limited, and the object of the present application can be achieved, for example, a top-mounted stirrer with a rotation speed of 200 to 300rpm may be used.

In some embodiments of the present application, the post decalcification washing step is a washing with 0.01-0.1mol/L phosphate buffer (ph=7.4-7.8) until the solution is neutral, followed by a washing with purified water.

In the present application, the shaking is a conventional operation in the art, and the present application is not limited thereto, and the purpose of the present application can be achieved, for example, an air bath or a freezing shaking table can be used for shaking, and the shaking frequency is 100-200rpm.

A second aspect of the present application provides a decalcified bone matrix prepared by the method of preparation of the first aspect of the present application comprising BMP-2 and collagen, wherein the BMP-2 content based on the decalcified bone matrix is 17-35ng/g.

The inventor finds in the research that the decalcified bone matrix prepared by the preparation method of the application well maintains the space structure of bones and has good biocompatibility, biodegradability and bone repair capability.

The technical scheme of the application is further explained by specific examples.

The experimental materials and methods used in the examples below are conventional materials and methods unless otherwise specified.

Example 1

(1) Pretreatment: taking cortical bone of cattle of 20-30 months old, removing surface soft tissues and periosteum by a physical scraping mode, cutting into bone blocks with the thickness of 3-20mm, crushing into bone particles with the particle size of 250-2000 mu m, mixing the bone particles with purified water according to the mass-volume ratio of feed liquid of 1g to 5mL, oscillating and cleaning for 6 times, and 10min each time;

(2) And (3) disinfection: mixing bone particles with 0.2wt% of peracetic acid solution according to the mass-volume ratio of 1g to 5mL of the feed liquid, oscillating for 120min, discarding the peracetic acid solution, adding purified water, stirring and cleaning for 6 times, and 5min each time;

(3) Degreasing: mixing the sterilized bone particles with isopropanol according to the mass-volume ratio of 1g to 5ml of the feed liquid, oscillating for 5 hours, discarding the isopropanol, adding 0.05mol/L MES buffer solution (pH=5.5), stirring and cleaning for 6 times, and 5 minutes each time;

(4) Decellularization: mixing defatted bone particles with 0.8wt% of sodium dodecyl ether sulfate solution according to the mass volume ratio of 1g to 5ml of feed liquid, placing into a low-temperature water tank (4 ℃), stirring for 24 hours, discarding the sodium dodecyl ether sulfate solution, mixing with 0.5wt% of triton X-100 solution according to the mass volume ratio of 1g to 5ml of feed liquid, placing into a low-temperature water tank (4 ℃) for 24 hours, stirring, discarding the triton X-100 solution, adding 0.05mol/L MES buffer solution (pH=5.5), stirring and cleaning for 6 times, and 10 minutes each time;

(5) Decalcification: 8g of sucrose is added into 100mL of hydrochloric acid solution (0.5 mol/L) to obtain decalcification solution; according to the mass volume ratio of 1g:8mL of the bone particles after cell removal are mixed with decalcification liquid, and the mixture is placed into a low-temperature water tank (4 ℃), stirred for 4 hours, and the liquid is changed every hour; discarding decalcification solution, adding 0.1mol/L phosphate buffer solution (pH=7.8), stirring and cleaning for 3 times each for 10min, cleaning until the solution is neutral, stirring and cleaning for 7 times each for 10min with purified water;

(6) And (3) freeze-drying: spreading the decalcified bone matrix in a steel plate mold, freeze-drying and packaging;

(7) And (3) sterilization: the decalcified bone matrix after packaging is preserved at normal temperature after irradiation sterilization of 20 kGy.

Example 2

Example 1 was repeated except that 8g of sucrose in step (5) was replaced with 8g of mannose.

Example 3

Example 1 was repeated except that 8g of sucrose in step (5) was replaced with 8g of polyethylene glycol.

Comparative example 1

Example 1 was repeated except that the decalcification solution in step (5) was replaced with a 0.5mol/L hydrochloric acid solution.

Effect measurement

1. Biochemical composition and content analysis

BMP-2 content detection, namely extracting BMP-2 according to a method in a method for preliminary judging bone supplying activity of a bone stock in DBM (bone marrow-derived management System), and then detecting the BMP-2 content by using a bovine bone morphogenetic protein 2 (BMP-2) ELISA detection kit (Shanghai enzyme-linked biotechnology Co., ltd.) instruction manual.

TABLE 1

	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

BMP-2 is one of members of transforming growth factor B superfamily, has the capability of inducing the directional differentiation and proliferation of undifferentiated mesenchymal stem cells to chondroblasts and osteoblasts, promotes the differentiation and maturation of osteoblasts, participates in the growth and development of bones and cartilage and the reconstruction process thereof, and further accelerates the repair of bone defects. As can be seen from Table 1, comparative example 1, in which no protein protectant was added, had a BMP-2 content of 17.28.+ -. 5.56ng/g for the decalcified bone matrix; whereas examples 1 to 3 add saccharide or polyalcohol protein protectants to conventional decalcification agents, the decalcified bone matrix had BMP-2 contents of 31.05.+ -. 2.55ng/g, 25.93.+ -. 7.40ng/g, and 26.30.+ -. 1.72ng/g, respectively. The method for decalcification of the bone repair agent is adopted, and saccharides and polyalcohol protein protective agents are added into the traditional decalcification agent, so that damage to BMP-2 in the decalcification process can be reduced, and the bone repair capability is improved.

2. Analysis of immunogenic substances

Residual host cell amount: the sample to be tested was fixed with 5% neutral formalin, paraffin-embedded, cut into 0.4 μm slices, dewaxed with xylene, dehydrated with a series of alcohols, HE stained and observed under a microscope.

DNA content: according to YY/T0606.25-2014 method for measuring animal derived biological Material DNA residual quantity: fluorescence staining method.

Fat content: the acid hydrolysis treatment specified in the second method of GB/T5009.6-2016 was carried out.

TABLE 2

	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

The HE staining chart of the decalcified bone matrix particles of example 1 is shown in fig. 1, and it can be seen from fig. 1 that the decalcified bone matrix particles of example 1 have no blue-stained nuclear material, clear basement membrane structure, no donor cell residue, and the application shows that the decalcified bone matrix particles have thorough removal of antigen material and good biological safety.

As can be seen from Table 2, the DNA content in the decalcified bone matrix particles of examples 1 to 3 was 2.95.+ -. 0.14ng/mg, 3.13.+ -. 1.79ng/mg, 2.87.+ -. 0.12ng/mg, respectively; the fat content in the decalcified bone matrix particles of examples 1 to 3 was 0.16.+ -. 0.10wt%, 0.33.+ -. 0.10wt% and 0.30.+ -. 0.14wt%, respectively. The preparation method of the decalcified bone matrix provided by the application is illustrated, and the specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods are combined, so that the obtained decalcified bone matrix has lower fat and DNA content and better biological safety.

3. Cell experiment

Cell adhesion experiments: (1) 10mg of radiation sterilized decalcified bone matrix particles of example 2 were added to each well of a 48-well plate, and MC3T3-E1 cells were plated at 6X 10 ⁴ Density of individual holes/holes is inoculated on the surface of the irradiated decalcified bone matrix particles, 3 compound holes are arranged in each group, and then the mixture is put into 37 ℃ and 5% CO ₂ Culturing in a cell incubator (volume fraction, balance air); (2) After 1 day of incubation, the plates were removed from the incubator, washed with Phosphate Buffered Saline (PBS), fixed with 4% paraformaldehyde for 30min, and then labeled with rhodamine with phalloidin (next holy biotechnology (shanghai)) and 4', 6-diamidino-2-phenylindole (DAPI, soribao) and stained according to the product instructions, and the cell morphology and cytoskeletal structure were observed under a fluorescence microscope, as shown in fig. 2.

From the results of FIG. 2, MC3T3-E1 cells can be effectively spread and elongated on the surface of the decalcified bone matrix particles, and the MC3T3-E1 cells have good morphology, which indicates that the cells can be well adhered and grown on the surface of the decalcified bone matrix particles.

4. In vivo ectopic osteogenesis induction experiment

The decalcified bone matrix particles of example 3 were implanted into the gluteus medius and gluteus maximus spaces of healthy 6-8 week old male BALB/c nude mice according to the experiment of YY/T1680-2020 on evaluation of in vivo osteogenic Induction Performance of allogeneic prosthetic Material demineralized bone Material. The material was Goldner stained 4 weeks after surgery and the results are shown in FIG. 3.

The decalcified bone matrix particles of comparative example 1 were implanted into the gluteus medius and gluteus maximus spaces of healthy 6-8 week old male BALB/c nude mice according to the experiment of YY/T1680-2020, evaluation of in vivo osteogenic Induction Performance of allogeneic repair Material demineralized bone Material. The material was Goldner stained 4 weeks after surgery and the results are shown in FIG. 4.

From fig. 3, it can be seen that osteoblasts and chondrocytes grow in a large number and are well distributed at the interface between muscle and decalcified bone matrix particles and in the inside of decalcified bone matrix particles, and meanwhile, the osteoid can be generated, which indicates that the decalcified bone matrix particle material prepared by the method has good ectopic osteoinduction capability. The significantly poorer numbers and growth distribution of osteoblasts and chondrocytes around and within the decalcified bone matrix particles in fig. 4 as compared with fig. 3, shows that the decalcified bone matrix particles of comparative example 1 have inferior ectopic osteoinductive capacity as those of the decalcified bone matrix particles of example 3.

In summary, the preparation method of the decalcified bone matrix provided by the application combines specific pretreatment, disinfection, degreasing, decellularization, decalcification, freeze-drying and sterilization methods, so that bioactive components and structural integrity in the material can be well reserved while animal tissue antigenic substances are effectively removed, and especially by adopting the decalcified method, saccharide or polyalcohol protein protectant is added into the traditional decalcified reagent, so that the efficient removal of mineral components can be realized, and simultaneously, the damage to BMP-2 and various cytokines in the decalcified process can be reduced. Furthermore, the preparation condition of the preparation method is simple, the quality is controllable, and the cost is low.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. that are within the spirit and principles of the present application are intended to be included within the scope of the present application.

Claims

1. A method of preparing a decalcified bone matrix comprising the steps of:

decalcification: adding a protein protecting agent into a hydrochloric acid solution with the concentration of 0.4-0.8mol/L to obtain decalcification solution; according to the mass volume ratio of 1g:3-15mL of the bone particles are mixed with decalcification liquid, the temperature is 0-10 ℃, and the mixture is stirred for 2-8h, and the liquid is changed every hour; wherein the mass volume ratio of the protein protectant to the hydrochloric acid solution is 1-20g:100mL; wherein the protein protectant is selected from at least one of saccharides and polyols; wherein the saccharide 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;

and (3) freeze-drying: freeze-drying the bone particles;

2. The method of claim 1, wherein the animal cortical bone is derived from bovine or porcine; preferably, the animal cortical bone is derived from a cow of 20-30 months of age or a pig of 4-6 months of age.

3. The preparation method of claim 1, wherein the protein protectant is at least one selected from sucrose, glucose, trehalose and polyethylene glycol.

4. The preparation method according to claim 1, wherein the decalcification step is carried out at a temperature of 2-8deg.C for a period of 3-6 hours.

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

6. The method of claim 1, wherein the pretreatment, degreasing, sterilization, decellularization and decalcification steps are performed, each further comprising washing with a detergent; the cleaning agent is at least one selected from purified water, 0.01-0.1mol/L phosphate buffer and 0.02-0.1mol/LMES buffer.

7. The preparation method of claim 6, wherein the cleaning step comprises the steps of adding a cleaning agent according to the mass-volume ratio of the feed liquid of 1g to 3-10mL, stirring for 5-10min, changing the feed liquid halfway, and repeatedly cleaning for 5-15 times.

8. The process according to claim 7, wherein the decalcification is carried out by washing with 0.01-0.1mol/L phosphate buffer until the solution becomes neutral, and washing with purified water.

9. A decalcified bone matrix prepared according to any one of claims 1 to 8 comprising bone morphogenic protein 2 and collagen, wherein the content of bone morphogenic protein 2 based on said decalcified bone matrix is 17-35ng/g.