A natural biological bone material and preparation method thereof

Abstract

The present invention proposes a natural biological bone material and a preparation method thereof, comprising selecting cancellous bone of animal origin, adding a degreasing agent and an ethanol solvent in sequence, vacuumizing, filling with nitrogen to positive pressure, maintaining positive pressure, and ultrasonically oscillating to obtain defatted cancellous bone; the degreasing agent comprises a strong alkali reagent, a penetration aid, a solvent and a surfactant 1; the penetration aid comprises at least one of sodium hexametaphosphate, sodium pyrophosphate, and sodium metasilicate pentahydrate; the solvent comprises triethylene glycol butyl ether and / or triethylene glycol methyl ether; the surfactant 1 comprises MF EH and / or CC9NS; adding a deproteinizing solution to the defatted cancellous bone, soaking, adding a hydrogen peroxide solution after washing and continuing to soak, and obtaining pretreated cancellous bone. The present invention can obtain a large-sized cancellous bone material with low content of tissues such as fat, low immunogenicity, and excellent mechanical properties, the compressive strength of the cancellous bone is greater than 3MPa, the porosity is between 60% and 80%, the preparation time is short, and it is suitable for industrial large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a natural biological bone material and a preparation method thereof. Background Art

[0002] Repairing bone defects caused by trauma, infection and other reasons is an important topic in the field of orthopedics. In addition to reduction and external fixation with equipment, bone transplantation is an extremely important treatment method. Among them, autologous bone transplantation, as the gold standard of bone transplantation, has a good healing effect in clinical practice. However, autologous bone is mostly taken from the patient's fibula or hip bone, the amount of bone taken is limited, there is secondary trauma, and it may cause related complications. These limitations have promoted the research of bone transplant substitute materials.

[0003] In order to solve the problem of bone transplantation, artificial bone, allogeneic bone, xenogeneic bone and other materials are currently used to replace autologous bone transplantation for clinical treatment. Artificial bone materials are mostly hydroxyapatite crystals calcined at a high temperature of more than 1000°C or β-tricalcium phosphate or calcium sulfate crystals synthesized by chemical reaction. Compared with natural bone, artificial bone cannot simulate the complex microstructure of natural bone materials, and lacks a small amount of carbonate in the form of weak crystals, which has an important impact on the degradability of bone. Therefore, the degradability of artificial bone materials has always been a bottleneck restricting its clinical application. Allogeneic bone comes from human cadaver bones. During the preparation process, only fat and part of the protein (mainly antigenic substances) need to be removed, and then disinfected and sterilized. Since the protein is not completely removed, the compressive resistance of the allogeneic bone is strong, the material has good compatibility with the human body, the immune rejection is mild after implantation, and the bone induction performance is relatively good. However, due to the age difference of the donors, the bone source that can be used for bone transplantation is very limited, and the allogeneic bone has a certain risk of viral infection.

[0004] Heterogeneous bones are mostly cattle bones or pig bones. Because they are similar to human bone structures and have a wide range of sources, they have good application prospects. The substances that cause immune reactions in fresh bones are mainly non-collagen proteins and glycoproteins and fats on the surface of bone cell membranes. How to effectively destroy cell surface antigens, remove antigenic substances, and obtain low immunogenicity heterogeneous bone materials is the focus of heterogeneous bone preparation technology. In the prior art, the methods for removing antigens mainly include deep low temperature freezing, chemical reagents (such as methanol / chloroform mixed solution, 20% to 30% hydrogen peroxide, ethylenediamine), surfactants (SDS, Triton, etc.), protease treatment, and high temperature calcination. The calcination method refers to heating the heterogeneous bone raw material at a temperature above 300°C to completely remove the organic matter in the heterogeneous bone through carbonization and oxidation. The calcination method can effectively remove antigens, but its product is relatively brittle, and the high temperature will increase the crystallinity of the heterogeneous bone, reduce its biological activity and degradability, so the calcined bone only has osteoconductivity and loses its inductivity. The most representative calcined bovine bone product is Bio-Oss bone powder. The end of the bovine femur is selected, degreased by toluene Soxhlet extraction, deproteinized by ethylenediamine solution, dried at 160°C, and then calcined at 350°C to obtain inorganic cancellous bone particles. Due to the use of the calcination process, the hydroxyapatite in the Bio-Oss bone powder is partially crystallized, resulting in poor compression resistance of the bone material, which is only suitable for bone defects in the oral cavity, maxillofacial bones, etc., and has no ideal clinical use effect for repairing bone defects with large defect sites that require a certain mechanical strength support. Using chemical reagents or enzyme treatment is currently a relatively effective and feasible method for treating cancellous bone. CN201310507012.9 A deantigenized biological bone and its preparation method discloses using irradiation to treat bone particles, denaturing the protein, and then using a mixed solution of a surfactant and hydrogen peroxide to treat the irradiated bone particles, and then using hypochlorite solution and hydrogen peroxide solution to treat, and sterilizing with ethylene oxide to obtain deproteinized biological bone. Existing products are mainly small-sized bone particles. When using existing technologies to process large pieces of cancellous bone, the following problems will occur: the current processing method is to gradually process from the surface to the deep layer of the bone block. If the reaction time is too short, the deep layer is difficult to be effectively processed and the residue will be relatively large; the processing time is long, and when the inside of the bone block is completely processed, the surface cancellous bone will be over-processed, resulting in a significant reduction in mechanical properties, and even powdering; at the same time, in the process of clinical application, some patients have large traumas or special shapes, which require subtractive manufacturing to cut natural bones into specific shapes, so it is necessary to prepare larger cancellous bones. At present, the existing technology is mainly based on the preparation of small-sized bone particles, so it is particularly important to develop methods for preparing large pieces of cancellous bone materials with low immunogenicity and excellent mechanical properties. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a natural biological bone material and a preparation method thereof, which uses a highly permeable defatting liquid that can quickly penetrate into the interior of the bone block and dissolve lipids from the bone block. The entire process of this method is short in time, and large-sized cancellous bone materials can be prepared. The material has excellent mechanical properties and is easy to process later. It also has the characteristics of low fat content and low immunogenicity.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A method for preparing a natural biological bone material comprises the following steps:

[0008] S1 defatting: select cancellous bone from animal sources, add degreasing agent and ethanol solvent in sequence, evacuate, fill with nitrogen to positive pressure, maintain positive pressure, and perform ultrasonic oscillation to obtain defatted cancellous bone;

[0009] The degreasing agent comprises a strong alkaline agent, a penetration aid, a solvent and a surfactant 1;

[0010] The penetration aid includes at least one of sodium hexametaphosphate, sodium pyrophosphate, and sodium metasilicate pentahydrate; the surfactant 1 includes MF EH and / or CC9NS;

[0011] S2: removing non-collagen proteins: adding deproteinizing solution to the defatted cancellous bone, soaking, and adding hydrogen peroxide solution to continue soaking after washing to obtain pretreated cancellous bone;

[0012] The deproteinized solution comprises an inorganic salt, a chelating agent, a surfactant 2 and a buffer;

[0013] The inorganic salt includes at least one of sodium chloride, potassium chloride and disodium hydrogen phosphate; the surfactant 2 includes Triton-100 and / or sodium dodecyl sulfate;

[0014] S3 finished product: The pretreated cancellous bone is dried and sterilized to obtain the target bone material.

[0015] Natural bone contains antigens such as bone marrow tissue, bone matrix, adipose tissue, and blood. Adipose tissue accounts for about 30% of natural bone tissue and fills the pores in the three-dimensional structure of inorganic bone. It is the main antigen component. In the process of preparing large pieces of cancellous bone, how to penetrate deep into the tissue and quickly remove the adipose tissue is the key to preparing cancellous bone.

[0016] The present invention adopts a novel degreasing agent for degreasing, and a penetrant and a penetration aid are added to the degreasing agent, so that the degreasing agent can quickly penetrate into the tissue. Under the action of the alkaline reagent, the fat tissue is peeled off from the skeleton and dissolved by the high-efficiency surfactant MF EH, CC9NS, etc. By adding the degreasing agent first, most of the lipids in the bone tissue can be quickly removed.

[0017] During the cancellous bone degreasing treatment stage, the present invention also combines a low-concentration gradient degreasing agent and an ethanol solution to remove remaining lipid tissue. Through the mutual synergy between the components and the degreasing treatment using a gradient concentration and an ethanol solution, bone marrow tissue, adipose tissue, blood and other tissues in the bone tissue can be effectively and deeply removed, over-treatment can be avoided, and the mechanical properties of the cancellous bone material can be improved.

[0018] In addition, in order to improve the penetration performance of the degreasing agent, the present invention uses a vacuum glove box device to complete the degreasing process. First, vacuum is drawn to remove the gas inside the bone tissue, and then gas is introduced to keep the glove box at a positive pressure. Through the above operation, the degreasing agent can more easily enter the interior of the bone tissue under the condition of external positive pressure; after the degreasing agent is fully infiltrated, the present invention uses an ultrasonic cleaning machine to perform ultrasonic treatment to accelerate the molecular movement in the tissue, making it easier to peel and dissolve the lipid tissue. Adding an ice bag to the ultrasonic cleaning machine can prevent the temperature of the ultrasonic process from being too high, and can better protect the collagen from being destroyed; after the treatment is completed, Then centrifuge the mixture to fully remove lipids from the pores of the bone tissue. The present invention uses a high-permeability gradient degreasing agent in combination with an ethanol solution for alternate degreasing, and uses a vacuum glove box for alternate vacuuming and pressurization, combined with ultrasonic cleaning, centrifugation and other processes, to effectively remove lipids, bone marrow and other tissues not combined with the bone scaffold in the bone tissue. The present invention can effectively remove antigenic substances in collagen through a deproteinization process using a deproteinized solution and a hydrogen peroxide aqueous solution. Through the above operations, a large-sized cancellous bone material with low content of tissues such as fat, low immunogenicity and excellent mechanical properties can be obtained.

[0019] Furthermore, in step S1, the degreasing agent and the ethanol solvent are used alternately to degrease the cancellous bone, and the alternating use includes: first adding the degreasing agent to perform a reagent degreasing treatment on the cancellous bone, and then adding the ethanol solvent to perform an ethanol degreasing treatment on the cancellous bone;

[0020] The reagent degreasing treatment includes adding a degreasing agent to evacuate, filling nitrogen to positive pressure, maintaining positive pressure, ultrasonic oscillation, and centrifugation; the ethanol degreasing treatment includes adding an ethanol solvent to evacuate, filling nitrogen to positive pressure, maintaining positive pressure, and ultrasonic oscillation; the ethanol solvent is anhydrous ethanol or an ethanol solution with a volume concentration of 60-85%. When the lipids to be peeled off gradually decrease in the subsequent process, the degreasing treatment can also be performed using an ethanol solution with a certain volume concentration, which can reduce the amount of ethanol used and save costs on the one hand, and on the other hand, the degreasing is more gentle and reduces the damage to the bone.

[0021] Further, the degreasing agent comprises, by weight percentage, 0.5-4% of a strong alkali agent, 0.5-1.5% of a penetration aid, 1.5-5% of a solvent, 1-3% of a surfactant 1 and the balance of water; the strong alkali agent comprises at least one of sodium hydroxide, potassium hydroxide and sodium carbonate; the solvent comprises triethylene glycol butyl ether and / or triethylene glycol methyl ether;

[0022] In terms of weight percentage, the deproteinized solution comprises 10-25% inorganic salt, 0.1-0.3% chelating agent, 1-3% surfactant 2, 0.1-0.2 buffer solution and the balance water; the chelating agent comprises EDTA-2Na and / or EDTA-4Na.

[0023] Furthermore, the degreasing agent also includes 0.15-0.5% of a penetrant, and the penetrant includes oep-98 and / or oep-70.

[0024] Furthermore, the vacuum is evacuated to a vacuum degree of -0.10 to -0.05 MPa; the nitrogen is filled to a positive pressure of 0.02 to 0.05 MPa, and the positive pressure is maintained for 1 to 2 hours; the power of the ultrasonic oscillation is 25 to 35 kHz, and the time of the ultrasonic oscillation is 5 to 20 minutes; the power of the centrifugation is 2000 to 3000 rpm, and the time of the centrifugation is 5 to 20 minutes.

[0025] Furthermore, the weight ratio of the cancellous bone to the degreasing agent is 1:5-7, and the volume ratio of the cancellous bone to the ethanol solvent is 1:5-7.

[0026] Furthermore, in step S2, the weight ratio of the defatted cancellous bone to the deproteinized solution is 1:10-12; the soaking temperature of the deproteinized solution is 2-6°C, and the soaking time is 8-12h.

[0027] Furthermore, the deproteinized solution is replaced every 2 to 4 hours.

[0028] Furthermore, in step S2, the weight ratio of the defatted cancellous bone to the hydrogen peroxide solution is 1:5-8, the mass concentration of the hydrogen peroxide solution is 10-12%; and the immersion time of the hydrogen peroxide solution is 15-25 hours.

[0029] The present invention provides a natural biological bone material prepared by the above-mentioned preparation method. The natural biological bone material has low fat content, low immunogenicity and excellent mechanical properties.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The invention uses a strong alkaline agent, a penetration aid, a solvent and a surfactant 1 as a degreasing agent and an ethanol solvent for degreasing treatment. Through vacuuming and pressurizing processes, combined with ultrasonic cleaning, centrifugation and other processes, bone marrow tissue, fat tissue, blood and other tissues in the bone tissue can be effectively removed. The deproteinization process of combining a deproteinizing liquid with a hydrogen peroxide aqueous solution can effectively remove antigenic substances in collagen. The preparation method of the natural biological bone material provided by the invention can obtain a large-sized cancellous bone material with low content of fat and other tissues, low immunogenicity and excellent mechanical properties. The compression strength of the cancellous bone is greater than 3MPa, and the porosity is between 60% and 80%. At the same time, the method for removing fat and foreign proteins from the cancellous bone of the invention is mild and simple, without excessive treatment of the surface cancellous bone, and the preparation time is short, which is suitable for industrial large-scale production.

[0032] In addition, the present invention adopts a high-permeability gradient degreasing agent and an ethanol solution for alternate degreasing, and uses a vacuum glove box to alternately evacuate and pressurize, in combination with ultrasonic cleaning, centrifugation and other processes, which can effectively and deeply remove lipids, bone marrow and other tissues not combined with the bone scaffold in the bone tissue, and the obtained cancellous bone material has a fat content of only 0.032-0.062%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The MTT test results of cancellous bone treated in Example 1;

[0034] Figure 2 This is a picture of untreated cancellous bone;

[0035] Figure 3 This is a picture of cancellous bone treated in Example 1;

[0036] Figure 4 It is the microscopic morphology of cancellous bone;

[0037] Figure 5 The cancellous bone slices were processed as in Comparative Example 1. DETAILED DESCRIPTION

[0038] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0039] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0040] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0041] The oep-98 and oep-70 used in the present invention are fatty alcohol polyoxyethylene ethers purchased from Guangzhou Dongshi Chemical Technology Co., Ltd.

[0042] MF EH is a special non- / cationic compound surfactant purchased from Guangzhou Tuoxin Chemical Technology Co., Ltd.;

[0043] CC9NS is a cationic surfactant purchased from Guangzhou Tuoxin Chemical Technology Co., Ltd.

[0044] Example 1 - Natural bone material

[0045] The degreasing agents used in the preparation of natural bone materials are as follows:

[0046] Degreasing agent 1 is composed of the following components:

[0047]

[0048]

[0049] Degreasing agent 2 consists of the following components:

[0050] Components effect weight Sodium hydroxide Alkaline reagent 1 oep-70 Alkali resistant penetrant 0.15 Sodium hexametaphosphate: sodium pyrophosphate 1:1 Penetration aids 1 Triethylene glycol butyl ether Solvents 2 MF EH:CC9NS1:1 Surfactants 2 Purified water Solvents 93.85

[0051] Degreasing agent 3 consists of the following components:

[0052] Components effect weight Sodium carbonate Alkaline reagent 3 Sodium hexametaphosphate: sodium pyrophosphate 1:1 Penetration aids 1 Triethylene glycol butyl ether Solvents 1.5 MF EH:CC9NS1:1 Surfactants 1 Purified water Solvents 93.5

[0053] The deproteinized solution is composed of the following components:

[0054] Components effect weight Sodium chloride:potassium chloride 1:1 Inorganic salt 10 EDTA-2Na Chelating agents 0.2 Triton-100: SDS1:1 Surfactants 2 Tris Buffer 0.1 Purified water Solvents 87.7

[0055] A method for preparing a natural bone material, comprising the following steps:

[0056] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0057] 2.Degreasing bone pieces

[0058] The degreasing method is:

[0059] (1) Degreasing agent 1 is used to degrease the bone block, and the weight ratio of the bone block to the degreasing agent is 1:5. The specific steps are as follows:

[0060] S1: Put the bone block with degreasing agent 1 into a vacuum glove box, evacuate the block to a vacuum degree of -0.07 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 10 minutes;

[0061] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.02MPa, which is maintained for 1h;

[0062] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 15 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to keep the ultrasound at a relatively low temperature.

[0063] S4 uses a centrifuge to centrifuge the bone block degreasing agent 1 mixture, and the centrifugation parameters are: speed 3000rpm, centrifugation time 7min;

[0064] S5 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 min, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The washing was repeated until neutral, and dust-free paper was used to fully absorb the moisture in the bone.

[0065] (2) Degreasing with anhydrous ethanol, the volume ratio of bone block to anhydrous ethanol is 1:5, and the specific steps are as follows:

[0066] S1: Put the bone block with anhydrous ethanol into a vacuum glove box, evacuate the box to a vacuum degree of -0.05 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0067] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.03 MPa, which is maintained for 1 hour;

[0068] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 10 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0069] S4 was ultrasonically cleaned with purified water, with a weight ratio of bone block to purified water of 1:10, for 15 min, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The cleaning was repeated 5 times, and dust-free paper was used to fully absorb the moisture in the bone block.

[0070] (3) Degreasing agent 2 is used to degrease the bone block, and the weight ratio of the bone block to the degreasing agent is 1:5. The specific steps are:

[0071] S1 put the bone block with degreasing agent 2 into a vacuum glove box, evacuate the block to a vacuum degree of -0.08 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0072] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.03 MPa, which is maintained for 1.5 h;

[0073] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 15 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0074] S4 uses a centrifuge to centrifuge the bone block degreasing agent 2 mixture, and the centrifugation parameters are: speed 2500rpm, centrifugation time 10min;

[0075] S5 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 min, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The washing was repeated until neutral, and dust-free paper was used to fully absorb the moisture in the bone.

[0076] (4) Degreasing the bone block using an 80% ethanol solution, wherein the volume ratio of the bone block to the 80% ethanol solution is 1:5. The specific steps are as follows:

[0077] S1: Put the bone block with anhydrous ethanol aqueous solution into a vacuum glove box, evacuate the solution to a vacuum degree of -0.06MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0078] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.02MPa, which is maintained for 1h;

[0079] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 5-15 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature;

[0080] S4 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 10 minutes, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The cleaning was repeated 4 times, and dust-free paper was used to fully absorb the moisture in the bone.

[0081] (5) Degreasing the bone block using degreasing agent 3, the weight ratio of the bone block to the degreasing agent 3 being 1:5, the specific steps are:

[0082] S1 put the bone block with degreasing agent 3 into a vacuum glove box, evacuate the block to a vacuum degree of -0.09 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0083] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.04 MPa, which is maintained for 1 hour;

[0084] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 15 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0085] S4 uses a centrifuge to centrifuge the bone block degreasing agent 2 mixture, and the centrifugation parameters are: speed 2000rpm, centrifugation time 15min;

[0086] S5 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 min, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The washing was repeated until neutral, and dust-free paper was used to fully absorb the moisture in the bone.

[0087] 3. Removal of non-collagen from bone

[0088] The removal method is:

[0089] S1: adding deproteinized solution to defatted bones, with a weight ratio of defatted bones to deproteinized solution of 1:10, soaking for 12 hours at 4°C, and changing the deproteinized solution every 2 hours;

[0090] S2 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0091] S3 was soaked in 10% hydrogen peroxide solution for 24 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:5;

[0092] S4 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20.

[0093] 4. Freeze-drying of bone blocks

[0094] The washed bone blocks were placed in a -20℃ refrigerator for 2 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 24 hours;

[0095] 5. Cobalt 60 irradiation sterilization

[0096] The packaged bone blocks were sterilized by cobalt 60 irradiation at a dose of 20 KGy.

[0097] Example 2 - Natural bone material

[0098] The degreasing agents used in the preparation of natural bone materials are as follows:

[0099] Degreasing agent 4 is composed of the following components:

[0100] Components effect weight Potassium hydroxide Alkaline reagent 1.5 oep-98 Alkali resistant penetrant 0.4 Sodium pyrophosphate: sodium metasilicate pentahydrate 2:1 Penetration aids 1.2 Triethylene glycol methyl ether Solvents 4 CC9NS Surfactants 2 Purified water Solvents 90.9

[0101] Degreasing agent 5 is composed of the following components:

[0102] Components effect weight Potassium hydroxide Alkaline reagent 0.75 oep-98 Alkali-resistant penetrant 0.1 Sodium pyrophosphate: sodium metasilicate pentahydrate 2:1 Penetration aids 0.8 Triethylene glycol methyl ether Solvents 2.5 CC9NS Surfactants 2.5 Purified water Solvents 93.35

[0103] Degreasing agent 6 consists of the following components:

[0104] Components effect weight Potassium carbonate Alkaline reagent 4 Sodium pyrophosphate: sodium metasilicate pentahydrate 2:1 Penetration aids 0.5 Triethylene glycol methyl ether Solvents 2 CC9NS Surfactants 1 Purified water Solvents 92.5

[0105] The deproteinized solution is composed of the following components:

[0106] Components effect weight Sodium chloride:potassium chloride 1:1 Inorganic salt 10 EDTA-2Na Chelating agents 0.2 Triton-100: SDS1:1 Surfactants 2 Tris Buffer 0.1 Purified water Solvents 87.7

[0107] The preparation method of the natural bone material in this embodiment, the bone block defatting step is the same as that in Example 1, and the specific steps are as follows:

[0108] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0109] 2.Degreasing bone pieces

[0110] The degreasing method is carried out in the following order:

[0111] sequence Solvents Bone block:solvent ratio Vacuum degree Pressure maintenance Ultrasonic power, time Centrifugal, time 1 Degreasing agent 4 Weight ratio 1:5 -0.07MPa 0.02MPa,1h 28kHz,15min 3000rpm,7min 2 Anhydrous ethanol Volume ratio 1:5 -0.05MPa 0.03MPa,1h 28kHz,10min - 3 Degreasing agent 5 Weight ratio 1:5 -0.08MPa 0.03MPa,1.5h 28kHz,15min 2500rpm,10min 4 80% ethanol Volume ratio 1:5 -0.06MPa 0.02MPa,1h 28kHz,15min - 5 Degreasing agent 6 Weight ratio 1:5 -0.09MPa 0.04MPa,1h 28kHz,15min 2000rpm,15min

[0112] Note: 80% ethanol refers to 80% ethanol solution by volume

[0113] The bone blocks with the above solvents are placed in a vacuum glove box respectively, vacuumed, slowly stirred to completely expel the bubbles on the surface of the bone blocks, and left to stand for 10 to 15 minutes; then the inflation valve is opened and nitrogen is filled to positive pressure; the container with the bone blocks is placed in an ultrasonic cleaning machine, and an ice bag is placed in the ultrasonic cleaning machine to keep the ultrasound at a low temperature; the mixture is centrifuged using a centrifuge (when anhydrous ethanol and an ethanol solution with a volume concentration of 80% are used, this centrifugation step is omitted); finally, purified water is used for ultrasonic cleaning, and an ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature, and the washing is repeated until neutral, and the moisture in the bone blocks is fully absorbed using dust-free paper.

[0114] 3. Removal of non-collagen from bone

[0115] The removal method is:

[0116] S1: adding deproteinized solution to defatted bones, with a weight ratio of defatted bones to deproteinized solution of 1:10, soaking for 12 hours at 4°C, and changing the deproteinized solution every 2 hours;

[0117] S2 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0118] S3 was soaked in 10% hydrogen peroxide solution for 24 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:5;

[0119] S4 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0120] 4. Freeze-drying of bone blocks

[0121] The washed bone blocks were placed in a -20℃ refrigerator for 2 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 24 hours;

[0122] 5. Cobalt 60 irradiation sterilization

[0123] The packaged bone blocks were sterilized by cobalt 60 irradiation at 20 KGy.

[0124] Example 3 - Natural bone material

[0125] The degreasing agents used in the preparation of natural bone materials are as follows:

[0126] Degreasing agent 7 is composed of the following components:

[0127] Components effect weight Sodium hydroxide:potassium hydroxide 1:1 Alkaline reagent 1 oep-98:oep-70 1:1 Alkali-resistant penetrant 0.5 Sodium pyrophosphate: sodium metasilicate pentahydrate 1:1 Penetration aids 1.5 Triethylene glycol butyl ether: triethylene glycol methyl ether 2:1 Solvents 5 MF E Surfactants 1 Purified water Solvents 91

[0128] Degreasing agent 8 is composed of the following components:

[0129] Components effect weight Sodium hydroxide:potassium hydroxide 1:1 Alkaline reagent 0.5 oep-98:oep-70 1:1 Alkali-resistant penetrant 0.15 Sodium pyrophosphate: sodium metasilicate pentahydrate 1:1 Penetration aids 1.5 Triethylene glycol butyl ether: triethylene glycol methyl ether 2:1 Solvents 2 MF E Surfactants 3 Purified water Solvents 92.85

[0130] Degreasing agent 9 is composed of the following components:

[0131] Components effect weight Sodium carbonate:potassium carbonate 1:1 Alkaline reagent 2 Sodium pyrophosphate: sodium metasilicate pentahydrate 1:1 Penetration aids 0.5 Triethylene glycol butyl ether: triethylene glycol methyl ether 2:1 Solvents 2.5 MF E Surfactants 3 Purified water Solvents 92

[0132] The deproteinized solution is composed of the following components:

[0133] Components effect weight Sodium chloride:potassium chloride:disodium hydrogen phosphate 2:1:0.5 Inorganic salt 25 EDTA-2Na:EDTA-4Na 1:1 Chelating agents 0.2 Triton-100: SDS1:1 Surfactants 1 Tris Buffer 0.1 Purified water Solvents 73.7

[0134] The method for preparing the natural bone material in this embodiment, the step of bone block defatting is the same as that in Example 1, and the parameters for removing non-collagen from the bone block are adjusted. The specific steps are as follows:

[0135] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0136] 2.Degreasing bone pieces

[0137] The degreasing method is carried out in the following order:

[0138] sequence Solvents Bone block:solvent ratio Vacuum degree Pressure maintenance Ultrasonic power, time Centrifugal, time 1 Degreasing agent 7 Weight ratio 1:5 -0.07MPa 0.02MPa,1h 28kHz,15min 3000rpm,7min 2 Anhydrous ethanol Volume ratio 1:5 -0.05MPa 0.03MPa,1h 28kHz,10min - 3 Degreasing agent 8 Weight ratio 1:5 -0.08MPa 0.03MPa,1.5h 28kHz,15min 2500rpm,10min 4 80% ethanol Volume ratio 1:5 -0.06MPa 0.02MPa,1h 28kHz,15min - 5 Degreasing agent 9 Weight ratio 1:5 -0.09MPa 0.04MPa,1h 28kHz,15min 2000rpm,15min

[0139] Note: 80% ethanol refers to 80% ethanol solution by volume

[0140] The bone blocks with the above solvents are placed in a vacuum glove box respectively, vacuumed, slowly stirred to completely expel the bubbles on the surface of the bone blocks, and left to stand for 10 to 15 minutes; then the inflation valve is opened and nitrogen is filled to positive pressure; the container with the bone blocks is placed in an ultrasonic cleaning machine, and an ice bag is placed in the ultrasonic cleaning machine to keep the ultrasound at a low temperature; the mixture is centrifuged using a centrifuge (when anhydrous ethanol and an ethanol solution with a volume concentration of 80% are used, this centrifugation step is omitted); finally, purified water is used for ultrasonic cleaning, and an ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature, and the washing is repeated until neutral, and the moisture in the bone blocks is fully absorbed using dust-free paper.

[0141] 3. Removal of non-collagen from bone

[0142] The removal method is:

[0143] S1: adding deproteinized liquid to defatted bones, with a weight ratio of defatted bones to deproteinized liquid of 1:12, soaking at 4°C for 8 hours, and changing the deproteinized liquid every 2 hours;

[0144] S2 was soaked and washed 4 times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0145] S3 was soaked in 12% hydrogen peroxide solution for 18 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:8;

[0146] S4 was soaked and washed with purified water 4 times, and the weight ratio of defatted bone to purified water was 1:15.

[0147] 4. Freeze-drying of bone blocks

[0148] The washed bone blocks were placed in a -20℃ refrigerator for 3 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 20 hours;

[0149] 5. Cobalt 60 irradiation sterilization

[0150] The packaged bone blocks were sterilized by cobalt 60 irradiation at a dose of 22 KGy.

[0151] Example 4 - Natural bone material

[0152] The method for preparing the natural bone material of this embodiment uses the same degreasing agent and deproteinizing solution as in Example 1, but the parameters for degreasing the bone blocks are different. The specific steps are as follows:

[0153] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0154] 2.Degreasing bone pieces

[0155] The degreasing method is carried out in the following order:

[0156] sequence Solvents Bone block:solvent ratio Vacuum degree Pressure maintenance Ultrasonic power, time Centrifugal, time 1 Degreasing agent 1 Weight ratio 1:7 -0.08MPa 0.03MPa,1h 28kHz,15min 2500rpm,10min 2 Anhydrous ethanol Volume ratio 1:7 -0.06MPa 0.02MPa,1.5h 28kHz,10min - 3 Degreaser 2 Weight ratio 1:7 -0.09MPa 0.04MPa,2h 32kHz,15min 2000rpm,10min 4 80% ethanol Volume ratio 1:5 -0.05MPa 0.03MPa,1.5h 32kHz,15min - 5 Degreasing agent 3 Weight ratio 1:7 -0.10MPa 0.05MPa,2h 32kHz,15min 1500rpm,15min

[0157] Note: 80% ethanol refers to 80% ethanol solution by volume

[0158] The specific degreasing method is as follows:

[0159] (1) Degreasing agent 1 is used to degrease the bone block, and the weight ratio of the bone block to the degreasing agent 1 is 1:7. The specific steps are as follows:

[0160] S1: Put the bone block with degreasing agent 1 into a vacuum glove box, evacuate the block to a vacuum degree of -0.08 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 10 minutes;

[0161] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.03 MPa, which is maintained for 1 hour;

[0162] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 15 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to keep the ultrasound at a relatively low temperature.

[0163] S4 uses a centrifuge to centrifuge the bone block degreasing agent 1 mixture, and the centrifugation parameters are: speed 2500rpm, centrifugation time 10min;

[0164] S5 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 minutes, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The washing was repeated until neutral, and dust-free paper was used to fully absorb the moisture in the bone.

[0165] (2) Degrease the femur using anhydrous ethanol. The volume ratio of bone block to anhydrous ethanol is 1:7. The specific operation is as follows:

[0166] S1: Put the bone block with anhydrous ethanol into a vacuum glove box, evacuate the box to a vacuum degree of -0.06MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0167] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.02MPa, which is maintained for 1.5h;

[0168] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 10 minutes, and the power of the ultrasonic cleaning machine is 28kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0169] S4 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 minutes, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The cleaning was repeated 5 times, and dust-free paper was used to fully absorb the moisture in the bone.

[0170] (3) Degreasing the bone block using degreasing agent 2, the weight ratio of the bone block to the degreasing agent 2 being 1:7, specifically in the following steps:

[0171] S1 put the bone block with degreasing agent 2 into a vacuum glove box, evacuate the block to a vacuum degree of -0.09 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0172] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.04 MPa, which is maintained for 2 h;

[0173] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic vibration for 15 minutes, and the power of the ultrasonic cleaning machine is 32kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0174] S4 uses a centrifuge to centrifuge the bone block degreasing agent 2 mixture, and the centrifugation parameters are: speed 2000rpm, centrifugation time 10min;

[0175] S5 was ultrasonically cleaned with purified water, with a weight ratio of bone to purified water of 1:10, for 15 minutes, and ice packs were placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The washing was repeated until neutral, and dust-free paper was used to fully absorb the moisture in the bone.

[0176] (4) Degreasing the bone block using an 80% ethanol solution by volume, wherein the volume ratio of the bone block to the 80% ethanol solution by volume is 1:7. The specific operation is as follows:

[0177] S1: Put the bone block with anhydrous ethanol aqueous solution into a vacuum glove box, evacuate the solution to a vacuum degree of -0.05 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0178] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.03 MPa, which is maintained for 1.5 h;

[0179] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic oscillation for 5-15 minutes, and the power of the ultrasonic cleaning machine is 32kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0180] S4 uses purified water for ultrasonic cleaning. The weight ratio of bone block to purified water is 1:10. Ultrasonic cleaning is performed for 10 minutes. Ice bags are placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature. The cleaning is repeated 4 times. Dust-free paper is used to fully absorb the moisture in the bone block.

[0181] (5) Degreasing the bone block using the degreasing agent 3, the weight ratio of the bone block to the degreasing agent 3 being 1:7, and the specific steps are as follows:

[0182] S1: Put the bone block with degreasing agent 3 into a vacuum glove box, evacuate the box to a vacuum degree of -0.10 MPa, stir slowly to completely expel the bubbles on the surface of the bone block, and let it stand for 15 minutes;

[0183] S2 opens the inflation valve and fills in nitrogen to a positive pressure of 0.05 MPa, which is maintained for 2 h;

[0184] S3 puts the container containing the bone block into an ultrasonic cleaning machine, performs ultrasonic vibration for 15 minutes, and the power of the ultrasonic cleaning machine is 32kHz. An ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature.

[0185] S4 uses a centrifuge to centrifuge the bone block degreasing agent 2 mixture, and the centrifugation parameters are: speed 1500rpm, centrifugation time 15min;

[0186] S5 uses purified water for ultrasonic cleaning, the weight ratio of bone block to purified water is 1:10, ultrasonic cleaning for 15 minutes, and puts ice bags in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeatedly washes until neutral, and uses dust-free paper to fully absorb the moisture in the bone block;

[0187] The components of the degreasing agent 1, degreasing agent 2 and degreasing agent 3 used above are the same as those in Example 1.

[0188] 3. Removal of non-collagen from bone

[0189] The specific steps are:

[0190] S1: adding deproteinized solution to defatted bones, with a weight ratio of defatted bones to deproteinized solution of 1:10, soaking for 12 hours at 4°C, and changing the deproteinized solution every 2 hours;

[0191] S2 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0192] S3 was soaked in 10% hydrogen peroxide solution for 24 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:5;

[0193] S4 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0194] The components of the deproteinized solution used above are the same as those in Example 1.

[0195] 4. Freeze-drying of bone blocks

[0196] The washed bone blocks were placed in a -20℃ refrigerator for 2 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 24 hours;

[0197] 5. Cobalt 60 irradiation sterilization

[0198] The packaged bone blocks were sterilized by cobalt 60 irradiation at a dose of 20 KGy.

[0199] Performance Testing

[0200] 1. Compression strength

[0201] Reference standard GB / T1041-2008

[0202] The calculation formula is:

[0203] σ=F / A

[0204] Where:

[0205] σ—stress parameter, in megapascals (MPa);

[0206] F—test force, in Newton (N);

[0207] A—Original area of ​​the sample, in square millimeters (mm 2 );

[0208] The cancellous bone prepared in Example 1 was used, and the cancellous bone product was cut into 10×10×10 mm cubes. Five groups of samples were tested, and the performance test results are shown in Table 1;

[0209] Table 1 Cancellous bone compression strength determination

[0210] 1 2 3 4 5 <![CDATA[Area (mm 2 )]]> 107.231 109.381 113.935 108.779 112.536 Maximum force (N) 410 370 460 390 430 Compression strength(MPa) 3.82 3.38 4.04 3.59 3.82

[0211] It can be seen from Table 1 that the compressive strength of the cancellous bones prepared by the present invention is greater than 3 MPa, which has a relatively high compressive strength and meets the implantation requirements.

[0212] 2. Porosity

[0213] Reference standard YY / T 1616-2018

[0214] Select a test tube with a scale and fill a certain amount of anhydrous ethanol in the test tube to measure the volume of anhydrous ethanol V1; immerse the cancellous bone in anhydrous ethanol and fully immerse it, connect negative pressure to suck out the remaining air in the sample, so that the anhydrous ethanol is fully filled in the porous support until no bubbles escape, and obtain the total volume of the bone block and anhydrous ethanol V2; take out the bone block soaked with anhydrous ethanol and read the value to obtain the remaining anhydrous ethanol volume V3; test each sample 5 times;

[0215] Calculation formula

[0216] E=(V1-V3) / (V2-V3)

[0217] In the formula

[0218] E—Porosity

[0219] V1—initial volume of anhydrous ethanol;

[0220] V2—the volume of anhydrous ethanol in the test tube after the bone block is fully filled with anhydrous ethanol;

[0221] V3—the volume of anhydrous ethanol remaining after the bone block filled with anhydrous ethanol is taken out;

[0222] The cancellous bone products were cut into cubes of 10×10×10 mm, and the performance test results are shown in Table 2.

[0223] Table 2 Determination of cancellous bone porosity

[0224] Example 1 Example 2 Example 3 Example 4 Porosity (%) 68.2 70.1 72.5 66.9

[0225] From the results in Table 2, we can see that the porosity of the prepared cancellous bone is between 60% and 80%. Figure 4 It can be determined from the scanning electron microscope image that the pore size of cancellous bone is between 200 and 400 μm, which is suitable for cell growth and proliferation in the pores.

[0226] 3. Cytotoxicity

[0227] The potential cytotoxicity of the samples was determined by MTT method in accordance with GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5 In Vitro Cytotoxicity Test". It was determined whether the material extract could induce toxic reactions after contact with cultured cells.

[0228] Evaluation Criteria

[0229] (1) The viability of 50% of the sample extract is at least equal to or higher than that of 100% of the cells; otherwise, the experiment should be repeated;

[0230] (2) The lower the cell viability %, the greater the potential cytotoxicity;

[0231] (3) Cell viability < 70% of the blank group, indicating that the sample has potential cytotoxicity;

[0232] (4) The cell viability % of the 100% test sample extract is the final result;

[0233] The cancellous bone prepared in Example 1 was subjected to an MTT toxicity test, and the results were as follows: Figure 1 As shown, it can be confirmed that the cancellous bone cells prepared by the present invention have very low toxicity.

[0234] 4. Residual fat tissue

[0235] Operation method: accurately weigh 1.0000g of sample, add 10mL of ether, place at room temperature for 2h, shake once every half hour, filter the ether into a constant-weight weighing bottle, the initial mass of the weighing bottle is m1, add 10mL of ether to the residue, place at room temperature for 2h, shake once every half hour, filter the ether into a constant-weight weighing bottle. Wash the residue with 5mL of ether, filter, and evaporate naturally; dry the weighing bottle at 105℃ for 2 hours, and weigh the mass m2 after cooling to room temperature;

[0236] Calculation method:

[0237] Fat content (%) = (m2-m1) / sample weight × 100%

[0238] Table 3 Determination of cancellous bone fat residue

[0239] Example 1 Example 2 Example 3 Example 4 Fat content (%) 0.032 0.044 0.035 0.062

[0240] It can be seen from Table 3 that the cancellous bone prepared by the method of the present invention has a very low fat content, basically below 0.1%, and has a significant fat removal effect.

[0241] 5. DNA residue test

[0242] Operation method: Grind the cancellous bone sample into powder, use the DNA extraction kit to extract the DNA in the sample, then add RNase to remove the RNA in the solution, and finally use the Nanodrop Lite ultra-micro spectrophotometer to detect the double-stranded DNA concentration.

[0243] Calculation results:

[0244]

[0245] Evaluation criteria: DNA residue less than 50 μg / g;

[0246] Table 4 Determination of residual DNA in cancellous bone

[0247] Example 1 Example 2 Example 3 Example 4 Sampling volume (mg) 0.033 0.038 0.042 0.045 DNA concentration (ng / μL) 4.8 4.9 4.6 5.1 DNA content (μg / g) 14.45 12.89 10.95 8.824

[0248] It can be seen from the table that the residual DNA concentration of the cancellous bone prepared by the present invention is very low, indicating that the method of the present invention can effectively destroy the cells in the cancellous bone and remove the DNA in the cells.

[0249] Comparative Example 1-

[0250] The difference between this comparative example and Example 1 is that the compositions of the degreasing agent and the deproteinizing liquid are different, as follows:

[0251] Degreasing agent 10 is composed of the following components:

[0252]

[0253]

[0254] Degreasing agent 11 is composed of the following components:

[0255] Components effect weight Sodium hydroxide:potassium hydroxide 1:1 Alkaline reagent 0.5 Triethylene glycol butyl ether: triethylene glycol methyl ether 2:1 Solvents 2 MF E Surfactants 3 Purified water Solvents 94.5

[0256] Degreasing agent 12 is composed of the following components:

[0257] Components effect weight Sodium carbonate:potassium carbonate 1:1 Alkaline reagent 2 Triethylene glycol butyl ether: triethylene glycol methyl ether 2:1 Solvents 2.5 MF E Surfactants 3 Purified water Solvents 92.5

[0258] The deproteinized solution is composed of the following components:

[0259] Components effect weight Sodium chloride:potassium chloride:disodium hydrogen phosphate 2:1:0.5 Inorganic salt 25 EDTA-2Na:EDTA-4Na 1:1 Chelating agents 0.2 Triton-100: SDS1:1 Surfactants 1 Tris Buffer 0.1 Purified water Solvents 73.7

[0260] The preparation method of the natural bone material in this comparative example, the bone block defatting step is the same as that in Example 1, and the specific steps are as follows:

[0261] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0262] 2.Degreasing bone pieces

[0263] The degreasing method is carried out in the following order:

[0264]

[0265]

[0266] Note: 80% ethanol refers to 80% ethanol solution by volume

[0267] The bone blocks with the above solvents are placed in a vacuum glove box respectively, vacuumed, slowly stirred to completely expel the bubbles on the surface of the bone blocks, and left to stand for 10 to 15 minutes; then the inflation valve is opened and nitrogen is filled to positive pressure; the container with the bone blocks is placed in an ultrasonic cleaning machine, and an ice bag is placed in the ultrasonic cleaning machine to keep the ultrasound at a low temperature; the mixture is centrifuged using a centrifuge (when anhydrous ethanol and an ethanol solution with a volume concentration of 80% are used, this centrifugation step is omitted); finally, purified water is used for ultrasonic cleaning, and an ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic temperature, and the washing is repeated until neutral, and the moisture in the bone blocks is fully absorbed using dust-free paper.

[0268] 3. Removal of non-collagen from bone

[0269] The removal method is:

[0270] S1: adding deproteinized liquid to defatted bones, with a weight ratio of defatted bones to deproteinized liquid of 1:12, soaking at 4°C for 8 hours, and changing the deproteinized liquid every 2 hours;

[0271] S2 was soaked and washed 4 times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0272] S3 was soaked in 12% hydrogen peroxide solution for 18 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:8;

[0273] S4 was soaked and washed with purified water 4 times, and the weight ratio of defatted bone to purified water was 1:15.

[0274] 4. Freeze-drying of bone blocks

[0275] The washed bone blocks were placed in a -20℃ refrigerator for 3 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 20 hours;

[0276] 5. Cobalt 60 irradiation sterilization

[0277] The packaged bone blocks were sterilized by cobalt 60 irradiation at a dose of 22 KGy.

[0278] After testing, the fat content of Comparative Example 1 was 1.532%. In Comparative Example 1, no penetrant or penetrant aid was added. Although most of the lipids could be removed, the lipids in the middle part of the cancellous bone were difficult to remove. Figure 5 The central part of the cancellous bone treated in Comparative Example 1 is sliced. Figure 5 It can be seen that some lipids are still remaining inside the cancellous bone, and the treatment effect on the central part is poor.

[0279] Comparative Example 2

[0280] The difference between this comparative example and Example 1 is that the steps of adjusting the bone block defatting are as follows:

[0281] The degreasing agents used in the preparation of natural bone materials are as follows:

[0282] Degreasing agent 1 is composed of the following components:

[0283] Components effect weight Sodium hydroxide Alkaline reagent 2 oep-70 Alkali-resistant penetrant 0.3 Sodium hexametaphosphate: sodium pyrophosphate 1:1 Penetration aids 1 Triethylene glycol butyl ether Solvents 3 MF EH:CC9NS1:1 Surfactants 1 Purified water Solvents 92.7

[0284] Degreasing agent 2 consists of the following components:

[0285] Components effect weight Sodium hydroxide Alkaline reagent 1 oep-70 Alkali-resistant penetrant 0.15 Sodium hexametaphosphate: sodium pyrophosphate 1:1 Penetration aids 1 Triethylene glycol butyl ether Solvents 2 MF EH:CC9NS1:1 Surfactants 2 Purified water Solvents 93.85

[0286] Degreasing agent 3 consists of the following components:

[0287] Components effect weight Sodium carbonate Alkaline reagent 3 Sodium hexametaphosphate: sodium pyrophosphate 1:1 Penetration aids 1 Triethylene glycol butyl ether Solvents 1.5 MF EH:CC9NS1:1 Surfactants 1 Purified water Solvents 93.5

[0288] The deproteinized solution is composed of the following components:

[0289] Components effect weight Sodium chloride:potassium chloride 1:1 Inorganic salt 10 EDTA-2Na Chelating agents 0.2 Triton-100: SDS1:1 Surfactants 2 Tris Buffer 0.1 Purified water Solvents 87.7

[0290] The preparation method of the natural bone material in this comparative example, the degreasing agents 1, 2, 3 and the deproteinized solution are the same as those in Example 1, and the specific steps are as follows:

[0291] 1. Select bovine tibia as the raw material for preparing cancellous bone; use a bone cutter to cut the cancellous bone at the distal end of the tibial epiphysis, then remove the outer cortical bone, and rinse the surface blood stains and cutting residues with purified water;

[0292] 2.Degreasing bone pieces

[0293] Degreasing method:

[0294] (1) Degreasing the bone block using degreasing agent 1, the weight ratio of the bone block to degreasing agent 1 is 1:5, S1 puts the container containing the bone block into an ultrasonic cleaning machine, ultrasonically vibrates for 30 minutes, the power of the ultrasonic cleaning machine is 28kHz, and an ice bag is placed in the ultrasonic cleaning machine to maintain the ultrasonic at a low temperature; S2 uses purified water for ultrasonic cleaning, the weight ratio of the bone block to purified water is 1:10, ultrasonically cleans for 15 minutes, and puts an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeats the washing until neutral, and uses dust-free paper to fully absorb the moisture in the bone block;

[0295] (2) Degreasing the femur using anhydrous ethanol, with the volume ratio of the bone block to anhydrous ethanol being 1:5. The specific operation includes the following steps: S1 placing the container containing the bone block into an ultrasonic cleaning machine, ultrasonically oscillating for 20 minutes, with the power of the ultrasonic cleaning machine at 28kHz, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature; S2 using purified water for ultrasonic cleaning, with the weight ratio of the bone block to purified water being 1:10, ultrasonically cleaning for 15 minutes, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeating the cleaning 5 times, and using dust-free paper to fully absorb the moisture in the bone block;

[0296] (3) Degreasing the bone block using degreasing agent 2, with the weight ratio of the bone block to the degreasing agent 2 being 1:5, specifically comprising the following steps: S1 placing the container containing the bone block into an ultrasonic cleaning machine, ultrasonically oscillating for 30 minutes, with the power of the ultrasonic cleaning machine being 28kHz, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature; S2 using purified water for ultrasonic cleaning, with the weight ratio of the bone block to the purified water being 1:10, ultrasonically cleaning for 15 minutes, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeatedly washing until neutral, and using dust-free paper to fully absorb the moisture in the bone block;

[0297] (4) Degreasing the bone blocks using an 80% volume concentration anhydrous ethanol aqueous solution, with the volume ratio of the bone blocks to the anhydrous ethanol aqueous solution being 1:5. The specific operation includes the following steps: S1 placing the container containing the bone blocks in an ultrasonic cleaning machine, ultrasonically oscillating for 15 minutes, the power of the ultrasonic cleaning machine being 28kHz, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature; S4 using purified water for ultrasonic cleaning, with the weight ratio of the bone blocks to purified water being 1:10, ultrasonically cleaning for 10 minutes, placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeating the cleaning 4 times, and using dust-free paper to fully absorb the moisture in the bone blocks;

[0298] (5) Degreasing the bone block using degreasing agent 3, with the weight ratio of the bone block to the degreasing agent 3 being 1:5, specifically comprising the following steps: S1 placing the container containing the bone block into an ultrasonic cleaning machine, ultrasonically oscillating for 30 minutes, with the power of the ultrasonic cleaning machine being 28kHz, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature; S2 using purified water for ultrasonic cleaning, with the weight ratio of the bone block to the purified water being 1:10, ultrasonically oscillating for 15 minutes, and placing an ice bag in the ultrasonic cleaning machine to maintain the ultrasonic temperature, repeatedly washing until neutral, and using dust-free paper to fully absorb the moisture in the bone block;

[0299] 3. Removal of non-collagen from bone

[0300] The removal method is:

[0301] S1: adding deproteinized solution to defatted bones, with a weight ratio of defatted bones to deproteinized solution of 1:10, soaking for 12 hours at 4°C, and changing the deproteinized solution every 2 hours;

[0302] S2 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20;

[0303] S3 was soaked in 10% hydrogen peroxide solution for 24 h, with the weight ratio of defatted bone to hydrogen peroxide solution being 1:5;

[0304] S4 was soaked and washed three times with purified water, and the weight ratio of defatted bone to purified water was 1:20.

[0305] 4. Freeze-drying of bone blocks

[0306] The washed bone blocks were placed in a -20℃ refrigerator for 2 hours, then transferred to a -80℃ refrigerator for deep freezing for 1 hour, and finally placed in a freeze dryer for 24 hours;

[0307] 5. Cobalt 60 irradiation sterilization

[0308] The packaged bone blocks were sterilized by cobalt 60 irradiation at a dose of 20 KGy.

[0309] After testing, the fat content of Comparative Example 2 is 0.315%. Comparative Example 2 does not use vacuum pressurization and centrifugal processes, which reduces the efficiency of fat removal by the degreasing agent.

[0310] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a natural biological bone material, characterized in that: The steps include: S1 defatting: select cancellous bone from animal sources, add degreasing agent and ethanol solvent in sequence, evacuate, fill with nitrogen to positive pressure, maintain positive pressure, and perform ultrasonic oscillation to obtain defatted cancellous bone; In terms of weight percentage, the degreasing agent comprises 0.5-4% of a strong alkali agent, 0.5-1.5% of a penetration aid, 1.5-5% of a solvent, 1-3% of a surfactant 1 and the balance of water; the strong alkali agent comprises at least one of sodium hydroxide, potassium hydroxide and sodium carbonate; the solvent comprises triethylene glycol butyl ether and / or triethylene glycol methyl ether; The penetration aid includes at least one of sodium hexametaphosphate, sodium pyrophosphate, and sodium metasilicate pentahydrate; the surfactant 1 includes MF EH and / or CC9NS; In step S1, the degreasing agent and the ethanol solvent are used alternately to degrease the cancellous bone, and the alternating use method includes: first adding the degreasing agent to perform a reagent degreasing treatment on the cancellous bone, and then adding the ethanol solvent to perform an ethanol degreasing treatment on the cancellous bone; The reagent degreasing treatment comprises adding a degreasing agent to evacuate, filling with nitrogen to positive pressure, maintaining positive pressure, ultrasonic oscillation, and centrifugation; The ethanol degreasing treatment comprises adding an ethanol solvent to evacuate, filling with nitrogen to positive pressure, maintaining positive pressure, and ultrasonic oscillation; the ethanol solvent is anhydrous ethanol or an ethanol solution with a volume concentration of 60-85%; The vacuum is evacuated to a vacuum degree of -0.10 to -0.05 MPa; the nitrogen is filled to a positive pressure of 0.02 to 0.05 MPa, and the positive pressure is maintained for 1 to 2 hours; the power of the ultrasonic oscillation is 25 to 35 kHz, and the time of the ultrasonic oscillation is 5 to 20 minutes; the power of the centrifugation is 2000 to 3000 rpm, and the time of the centrifugation is 5 to 20 minutes; S2: removing non-collagen proteins: adding deproteinizing solution to the defatted cancellous bone, soaking, and adding hydrogen peroxide solution to continue soaking after washing to obtain pretreated cancellous bone; In terms of weight percentage, the deproteinized solution comprises 10-25% inorganic salt, 0.1-0.3% chelating agent, 1-3% surfactant 2, 0.1-0.2 buffer and the balance water; the chelating agent comprises EDTA-2Na and / or EDTA-4Na; The inorganic salt includes at least one of sodium chloride, potassium chloride and disodium hydrogen phosphate; the surfactant 2 includes Triton-100 and / or sodium dodecyl sulfate; S3 finished product: The pretreated cancellous bone is dried and sterilized to obtain the target bone material.

2. The method for preparing the natural biological bone material according to claim 1, characterized in that: The degreasing agent also includes 0.15-0.5% penetrant, and the penetrant includes oep-98 and / or oep-70.

3. The method for preparing the natural biological bone material according to claim 1, characterized in that: The weight ratio of the cancellous bone to the degreasing agent is 1:5-7, and the volume ratio of the cancellous bone to the ethanol solvent is 1:5-7.

4. The method for preparing the natural biological bone material according to claim 1, characterized in that: In step S2, the weight ratio of the defatted cancellous bone to the deproteinized solution is 1:10-12; the soaking temperature of the deproteinized solution is 2-6°C, and the soaking time is 8-12h.

5. The method for preparing the natural biological bone material according to claim 4, characterized in that: Replace the deproteinized solution every 2 to 4 hours.

6. The method for preparing the natural biological bone material according to claim 1, characterized in that: In step S2, the weight ratio of the defatted cancellous bone to the hydrogen peroxide solution is 1:5-8, the mass concentration of the hydrogen peroxide solution is 10-12%; and the immersion time of the hydrogen peroxide solution is 15-25 hours.

7. The natural biological bone material prepared according to the method for preparing the natural biological bone material according to any one of claims 1 to 6.

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