CN118105545B - A natural biological bone material and preparation method thereof - Google Patents
A natural biological bone material and preparation method thereof Download PDFInfo
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- CN118105545B CN118105545B CN202410142221.6A CN202410142221A CN118105545B CN 118105545 B CN118105545 B CN 118105545B CN 202410142221 A CN202410142221 A CN 202410142221A CN 118105545 B CN118105545 B CN 118105545B
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- bone
- degreasing
- cancellous bone
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- 239000013527 degreasing agent Substances 0.000 claims abstract description 89
- 239000002904 solvent Substances 0.000 claims abstract description 59
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 44
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- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 claims abstract description 8
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Classifications
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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Abstract
本发明提出了一种天然生物骨材料及其制备方法，包括选取动物源的松质骨，依次加入脱脂剂和乙醇溶剂后进行抽真空，充入氮气至正压，维持正压，超声振荡，得脱脂松质骨；所述脱脂剂包括强碱试剂、渗透助剂、溶剂和表面活性剂1；渗透助剂包括六偏磷酸钠、焦磷酸钠、五水偏硅酸钠中的至少一种；所述溶剂包括三乙二醇丁醚和/或三乙二醇甲醚；所述表面活性剂1包括MF EH和/或CC9NS；在脱脂松质骨中加入脱杂蛋白液，浸泡，清洗后加入过氧化氢溶液继续浸泡，得预处理松质骨。本发明可以获得脂肪等组织含量低、免疫源性低、力学性能优异的大尺寸的松质骨材料，松质骨的压缩强度＞3MPa，孔隙率在60～80％之间，制备时间较短，适合工业化大规模生产。
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, and adding a hydrogen peroxide solution to continue soaking after washing to obtain 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
The invention relates to the technical field of medical materials, in particular to a natural biological bone material and a preparation method thereof.
Background
Repairing bone defects caused by wounds, infections and the like is an important subject in the orthopaedics field, and bone grafting is an extremely important treatment means except for reduction and fixation of equipment. Wherein, the autologous bone grafting is used as a gold standard of bone grafting, and has good healing effect clinically. However, autologous bone is mostly taken from the fibula or hip bone of the patient, the bone taking amount is limited, secondary wounds exist, and related complications may be caused, and these limitations have prompted the study of bone graft substitute materials.
In order to solve the problem of bone grafting, materials such as artificial bone, allogeneic bone, xenogeneic bone and the like are used for clinical treatment instead of autologous bone grafting at present. Most of the artificial bone materials are hydroxyapatite crystals calcined at a high temperature of more than 1000 ℃ or beta-tricalcium phosphate or calcium sulfate crystals synthesized by chemical reaction, compared with natural bone, the artificial bone cannot simulate the complex microstructure of the natural bone materials, and a small amount of carbonate existing in a weak crystal form is lacking, so that the degradability of the artificial bone materials has an important influence on the degradability of the bone, and therefore, the degradability of the artificial bone materials is always a bottleneck limiting the clinical application of the artificial bone materials. The allogenic bone is derived from human cadaver bone, and is prepared by removing fat and part of protein (mainly antigenic substances) during preparation, and sterilizing. Because the protein is not completely removed, the bone has strong compression resistance, good histocompatibility with human body, light immune rejection after implantation and relatively good bone induction performance. However, due to age differences of donors, the bone sources available for bone grafting are very limited and there is a certain risk of viral infection of the same bone.
The heterogeneous bones are mostly bovine bones or porcine bones, and have wide sources and good application prospect due to the similarity of the structure of the heterogeneous bones with human bones. The immune reaction-inducing substances in fresh bone are mainly non-collagen protein, glycoprotein on the surface of bone cell membrane and fat. How to effectively destroy cell surface antigens and remove antigenic substances, and obtaining heterogeneous bone materials with low immunogenicity is an important point in the heterogeneous bone preparation process. In the prior art, the antigen removing method mainly comprises deep low-temperature freezing, chemical reagents (such as methanol/chloroform mixed solution, 20% -30% hydrogen peroxide and ethylenediamine), surfactants (SDS, triton and the like), protease treatment, high-temperature calcination and the like. The calcination method is to heat the raw material of the heterogeneous bone at a temperature higher than 300 ℃ and thoroughly remove organic matters in the heterogeneous bone through carbonization and oxidization, the calcination method can effectively remove antigens, but the product has high brittleness, and the high temperature can raise the crystallinity of the heterogeneous bone to reduce the bioactivity and degradability of the heterogeneous bone, so that the calcined bone only has bone conductivity and loses inducibility. The most representative calcined bovine bone product is Bio-Oss bone powder, bulls bone ends are selected, defatted by toluene Soxhlet extraction, deproteinized by ethylenediamine solution, dried at 160 ℃, and calcined at 350 ℃ to obtain inorganic cancellous bone particles. Because of the use of the calcination process, the hydroxyapatite in the Bio-Oss bone powder is partially crystallized, so that the bone material has poor compression resistance, is only suitable for bone defects of oral cavity, maxillofacial bone and other parts, and has no ideal clinical use effect on repairing the bone defects which are large in defect parts and need a certain mechanical strength support. The method for treating cancellous bone by using chemical reagent or enzyme is effective and feasible at present, CN201310507012.9 is a deproteinized biological bone and its preparation method, and the method comprises the steps of treating bone particles by using irradiation, denaturing protein, treating the irradiated bone particles by using a mixed solution of a surfactant and hydrogen peroxide, treating by using a hypochlorite solution and a hydrogen peroxide solution, and sterilizing by using ethylene oxide to obtain the deproteinized biological bone. The prior art mainly uses small-sized bone particles, the prior art is adopted to treat large spongy bones, the prior treatment mode is to gradually treat the surface layer to the deep layer of the bone block, if the reaction time is too short, the deep layer part is difficult to effectively treat, the residue is relatively large, the treatment time is long, the internal treatment of the bone block is complete, the spongy bones on the surface layer can obviously reduce the mechanical property and even generate chalking phenomenon due to excessive treatment, and meanwhile, in the clinical application process, the trauma size of some patients is large or the shape is specific, the materials are required to be reduced, and the natural bones are cut into specific shapes, so that the spongy bones with larger sizes are required to be prepared. At present, the prior art mainly takes the preparation of small-sized bone particles as the main material, so that the development of a method for preparing a large-block bone cancellous material with low immunogenicity and excellent mechanical properties is particularly important.
Disclosure of Invention
In view of the above, the present invention aims to provide a natural biological bone material and a preparation method thereof, wherein the natural biological bone material can quickly penetrate into bone blocks by using a high-permeability degreasing fluid, so that lipid can be separated from and dissolved in the bone blocks.
The technical scheme of the invention is realized as follows:
a preparation method of a natural biological bone material comprises the following steps:
S1, degreasing, namely selecting cancellous bone of animal origin, sequentially adding a degreasing agent and an ethanol solvent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, and carrying out ultrasonic vibration 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, and the surfactant 1 comprises MF EH and/or CC9NS;
S2, removing non-collagen, namely adding impurity-removing protein liquid into the defatted cancellous bone, soaking, cleaning, and then adding hydrogen peroxide solution for continuous soaking to obtain pretreated cancellous bone;
the impurity-removed protein liquid comprises inorganic salt, chelating agent, surfactant 2 and buffer liquid;
The inorganic salt comprises at least one of sodium chloride, potassium chloride and disodium hydrogen phosphate, and the surfactant 2 comprises Triton-100 and/or sodium dodecyl sulfate;
And S3, drying and sterilizing the pretreated cancellous bone to obtain the target bone material.
The natural bone contains bone marrow tissue, bone matrix, adipose tissue, blood and other antigens, wherein the adipose tissue accounts for about 30% of the natural bone tissue, is filled in the pores of the three-dimensional structure of the inorganic bone, is a main antigen component, and is a key for preparing cancellous bone when the adipose tissue is deeply penetrated into the tissue in the process of preparing the large cancellous bone.
The degreasing agent is used for degreasing, the penetrating agent and the penetrating auxiliary agent are added into the degreasing agent, so that the degreasing agent can be quickly penetrated into tissues, adipose tissues are peeled off from the frameworks under the action of the alkali agent and are dissolved by high-efficiency surfactants MF EH, CC9NS and the like, and most of lipids in the bone tissues can be quickly removed by adding the degreasing agent.
In the spongy bone degreasing treatment stage, the degreasing agent with low concentration gradient and the ethanol solution are combined to remove residual lipid tissues, and the gradient concentration and the ethanol solution degreasing treatment are adopted through mutual cooperation of the components, so that the tissues such as bone marrow tissues, fat tissues and blood in the bone tissues can be effectively and deeply removed, excessive treatment is avoided, and the mechanical property of the spongy bone material is improved.
In addition, in order to improve the infiltration performance of the degreasing agent, the degreasing process is completed by matching vacuum glove box equipment, the internal gas of the bone tissue is pumped out, then the gas is introduced to enable the glove box to keep positive pressure, the degreasing agent can enter the bone tissue more easily under the condition of positive external pressure through the operation, after the degreasing agent is fully infiltrated, the degreasing agent is subjected to ultrasonic treatment by adopting an ultrasonic cleaning machine, molecular movement in the tissue is accelerated, so that the lipid tissue is more easily stripped and dissolved, an ice bag is added into the ultrasonic cleaning machine, the ultrasonic process temperature is prevented from being too high, collagen can be well protected from being damaged, after the treatment is completed, lipid substances are fully removed from bone tissue pores by adopting the high-permeability gradient degreasing agent to match with an ethanol solution for alternate degreasing, and the ultrasonic cleaning and centrifugation processes are used for matching with the processes of vacuum glove box for vacuumizing and pressurizing alternately, lipid and bone marrow in the bone tissue which are not combined with a bone scaffold can be effectively removed, and the collagen in the bone tissue can be effectively removed by using the dedoping solution and the hydrogen peroxide aqueous solution, and the collagen material with high-quality collagen content can be effectively removed through the operation performance of low-quality, and high-quality fat-quality materials can be obtained by the operation of low-quality and high-quality performance.
In step S1, the degreasing agent and the ethanol solvent are used alternately to degrease the cancellous bone, wherein the alternate use method comprises the steps of adding the degreasing agent to degrease the cancellous bone with a reagent, and then adding the ethanol solvent to degrease the cancellous bone with ethanol;
The reagent degreasing treatment comprises the steps of adding a degreasing agent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, carrying out ultrasonic vibration and centrifuging, wherein the ethanol degreasing treatment comprises the steps of adding an ethanol solvent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, carrying out ultrasonic vibration, and the ethanol solvent is absolute ethanol or ethanol solution with the volume concentration of 60-85%. When the follow-up stripped lipid gradually decreases, degreasing treatment can be performed by adopting ethanol solution with a certain volume concentration, so that the ethanol consumption can be reduced, the cost can be saved, the degreasing is milder, and the damage of bone fragments can be reduced.
Further, the degreasing agent comprises 0.5-4% of strong base reagent, 0.5-1.5% of penetration auxiliary agent, 1.5-5% of solvent, 1-3% of surfactant 1 and the balance of water according to weight percentage, wherein the strong base reagent comprises at least one of sodium hydroxide, potassium hydroxide and sodium carbonate;
The impurity-removed protein liquid comprises, by weight, 10-25% of inorganic salt, 0.1-0.3% of chelating agent, 1-3% of surfactant 2, 0.1-0.2 of buffer solution and the balance of water, wherein the chelating agent comprises EDTA-2Na and/or EDTA-4Na.
Still further, the degreasing agent further comprises 0.15-0.5% of penetrating agent, and the penetrating agent comprises oep-98 and/or oep-70.
Further, the vacuum degree is-0.10 to-0.05 MPa, the positive pressure is 0.02 to 0.05MPa when the nitrogen is filled, the positive pressure is maintained for 1 to 2 hours, the ultrasonic vibration power is 25 to 35kHz, the ultrasonic vibration time is 5 to 20 minutes, the centrifugal power is 2000 to 3000rpm, and the centrifugal time is 5 to 20 minutes.
Further, 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.
In the step S2, the weight ratio of the defatted cancellous bone to the impurity-removed protein liquid is 1:10-12, the soaking temperature of the impurity-removed protein liquid is 2-6 ℃, and the soaking time is 8-12 h.
Furthermore, the impurity-removed protein liquid is replaced every 2-4 hours.
In the 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 soaking time of adding the hydrogen peroxide solution is 15-25 h.
The natural biological bone material prepared by the preparation method provided by the invention has low fat content, low immunogenicity and excellent mechanical properties.
Compared with the prior art, the invention has the beneficial effects that:
The preparation method of the natural biological bone material can obtain large-size spongy bone material with low tissue content such as fat, low immunogenicity and excellent mechanical property, the compression strength of the spongy bone is more than 3MPa, the porosity is between 60 and 80 percent, and meanwhile, the method for removing the fat and the impurity protein of the spongy bone is mild and simple, does not excessively treat the spongy bone on the surface layer, has short preparation time and is suitable for industrialized mass production.
In addition, the invention adopts the high permeability gradient degreasing agent to match with ethanol solution for alternate degreasing, uses the vacuum glove box vacuumizing and pressurizing to alternately perform the processes, and matches with ultrasonic cleaning, centrifugation and other processes, so that the lipid, the bone marrow and other tissues which are not combined with the bone scaffold in bone tissues can be effectively and deeply removed, and the obtained spongy bone material has the fat content of only 0.032-0.062 percent.
Drawings
FIG. 1 is the results of the cancellous bone MTT test treated in example 1;
FIG. 2 is a photograph of untreated cancellous bone;
FIG. 3 is a photograph of cancellous bone treated in example 1;
FIG. 4 is a microscopic morphology of cancellous bone;
Fig. 5 is a comparative example 1 treatment of cancellous bone slice treatment.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
Oep-98 and oep-70 used in the invention are fatty alcohol polyoxyethylene ether and are purchased from Guangzhou Dongshi chemical engineering Co., ltd;
MF EH is a special non/cationic compound surfactant purchased from new chemical engineering, inc. In guangzhou, city;
CC9NS is a cationic surfactant purchased from Guangzhou City Tuo Xin chemical technology Co.
EXAMPLE 1 Natural bone Material
The degreasing agent used for preparing the natural bone material is as follows:
degreasing agent 1 is composed of the following components:
degreasing agent 2 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide | Alkali reagent | 1 |
| oep-70 | Alkali-resistant penetrant | 0.15 |
| Sodium hexametaphosphate: sodium pyrophosphate 1:1 | Penetration aid | 1 |
| Triethylene glycol butyl ether | Solvent(s) | 2 |
| MF EH:CC9NS1:1 | Surface active agent | 2 |
| Purified water | Solvent(s) | 93.85 |
Degreasing agent 3 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium carbonate | Alkali reagent | 3 |
| Sodium hexametaphosphate: sodium pyrophosphate 1:1 | Penetration aid | 1 |
| Triethylene glycol butyl ether | Solvent(s) | 1.5 |
| MF EH:CC9NS1:1 | Surface active agent | 1 |
| Purified water | Solvent(s) | 93.5 |
The impurity-removed protein solution consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium chloride potassium chloride 1:1 | Inorganic salt | 10 |
| EDTA-2Na | Chelating agent | 0.2 |
| Triton-100:SDS1:1 | Surface active agent | 2 |
| Tris | Buffering agents | 0.1 |
| Purified water | Solvent(s) | 87.7 |
A preparation method of a natural bone material comprises the following steps:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method comprises the following steps:
(1) Degreasing the bone block by using a degreasing agent 1, wherein the weight ratio of the bone block to the degreasing agent is 1:5, and the specific steps are as follows:
s1, putting the bone block added with the degreasing agent 1 into a vacuum glove box, vacuumizing to the vacuum degree of-0.07 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 10min;
S2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.02MPa, and keeping for 1h;
s3, placing the container with the bone pieces in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and keeping the ultrasonic operation at a lower temperature;
s4, centrifuging the bone block degreasing agent 1 mixture by using a centrifuge, wherein the centrifuging parameters are that the rotating speed is 3000rpm, and the centrifuging time is 7min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper.
(2) Degreasing by using absolute ethyl alcohol, wherein the volume ratio of the bone block to the absolute ethyl alcohol is 1:5, and the specific steps are as follows:
s1, putting the bone block added with the absolute ethyl alcohol into a vacuum glove box, vacuumizing to the vacuum degree of-0.05 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
s2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.03MPa, and keeping for 1h;
s3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 10min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically treating for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning for 5 times, and fully absorbing the water in the bone blocks by using dust-free paper.
(3) Degreasing the bone block by using a degreasing agent 2, wherein the weight ratio of the bone block to the degreasing agent is 1:5, and the specific steps are as follows:
S1, putting the bone block added with the degreasing agent 2 into a vacuum glove box, vacuumizing to the vacuum degree of-0.08 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
S2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.03MPa, and keeping for 1.5h;
S3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, centrifuging the bone block degreasing agent 2 mixture by using a centrifuge, wherein the centrifugation parameters are that the rotation speed is 2500rpm, and the centrifugation time is 10min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper.
(4) Degreasing the bone block by using an 80% ethanol solution with volume concentration, wherein the volume ratio of the bone block to the 80% ethanol solution with volume concentration is 1:5, and the specific steps are as follows:
S1, putting the bone block added with the absolute ethyl alcohol aqueous solution into a vacuum glove box, vacuumizing to the vacuum degree of-0.06 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
S2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.02MPa, and keeping for 1h;
S3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 5-15min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically treating for 10min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning for 4 times, and fully absorbing the water in the bone blocks by using dust-free paper.
(5) Degreasing the bone block by using a degreasing agent 3, wherein the weight ratio of the bone block to the degreasing agent 3 is 1:5, and the specific steps are as follows:
s1, putting the bone block added with the degreasing agent 3 into a vacuum glove box, vacuumizing to the vacuum degree of-0.09 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
s2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.04MPa, and keeping for 1h;
S3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, centrifuging the bone block degreasing agent 2 mixture by using a centrifuge, wherein the centrifuging parameters are that the rotating speed is 2000rpm, and the centrifuging time is 15min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper.
3. Bone block removal of non-collagen
The removing method comprises the following steps:
S1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:10, soaking for 12 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
S2, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the defatted bone in a 10% hydrogen peroxide solution for 24 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:5;
S4, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20.
4. Freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 2 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 24 hours;
5. Cobalt 60 irradiation sterilization
And (5) carrying out cobalt 60 radiation sterilization on the packaged bone blocks by using the radiation dose of 20 KGy.
EXAMPLE 2 Natural bone Material
The degreasing agent used for preparing the natural bone material is as follows:
Degreasing agent 4 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Potassium hydroxide | Alkali reagent | 1.5 |
| oep-98 | Alkali-resistant penetrant | 0.4 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 2:1 | Penetration aid | 1.2 |
| Triethylene glycol methyl ether | Solvent(s) | 4 |
| CC9NS | Surface active agent | 2 |
| Purified water | Solvent(s) | 90.9 |
Degreasing agent 5 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Potassium hydroxide | Alkali reagent | 0.75 |
| oep-98 | Alkali-resistant penetrant | 0.1 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 2:1 | Penetration aid | 0.8 |
| Triethylene glycol methyl ether | Solvent(s) | 2.5 |
| CC9NS | Surface active agent | 2.5 |
| Purified water | Solvent(s) | 93.35 |
The degreasing agent 6 consists of the following components:
| Component (A) | Action | Weight of (E) |
| Potassium carbonate | Alkali reagent | 4 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 2:1 | Penetration aid | 0.5 |
| Triethylene glycol methyl ether | Solvent(s) | 2 |
| CC9NS | Surface active agent | 1 |
| Purified water | Solvent(s) | 92.5 |
The impurity-removed protein solution consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium chloride potassium chloride 1:1 | Inorganic salt | 10 |
| EDTA-2Na | Chelating agent | 0.2 |
| Triton-100:SDS1:1 | Surface active agent | 2 |
| Tris | Buffering agents | 0.1 |
| Purified water | Solvent(s) | 87.7 |
The preparation method of the natural bone material of this embodiment has the same steps as those of embodiment 1, and the specific steps are as follows:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method is sequentially carried out:
| Sequence(s) | Solvent(s) | Bone block to solvent ratio | Vacuum degree | Pressure retention | Ultrasonic power, time | Centrifuging, and taking time |
| 1 | Degreasing agent 4 | Weight ratio of 1:5 | -0.07MPa | 0.02MPa,1h | 28kHz,15min | 3000rpm,7min |
| 2 | Absolute ethyl alcohol | Volume ratio 1:5 | -0.05MPa | 0.03MPa,1h | 28kHz,10min | - |
| 3 | Degreasing agent 5 | Weight ratio of 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 of 1:5 | -0.09MPa | 0.04MPa,1h | 28kHz,15min | 2000rpm,15min |
Note that 80% ethanol refers to an 80% ethanol solution by volume
Respectively placing the bone blocks with the solvent into a vacuum glove box, vacuumizing, slowly stirring to completely discharge bubbles on the surfaces of the bone blocks, standing for 10-15 min, opening an air charging valve, charging nitrogen to positive pressure, placing the container with the bone blocks into an ultrasonic cleaner, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic at a lower temperature, centrifuging the mixture by using a centrifuge (when absolute ethyl alcohol and 80% ethanol solution with volume concentration are used, no centrifuging step), finally ultrasonically cleaning by using purified water, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic temperature, repeatedly washing to neutral, and fully absorbing the moisture in the bone blocks by using dust-free paper.
3. Bone block removal of non-collagen
The removing method comprises the following steps:
S1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:10, soaking for 12 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
S2, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the defatted bone in a 10% hydrogen peroxide solution for 24 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:5;
s4, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
4. freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 2 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 24 hours;
5. Cobalt 60 irradiation sterilization
The packaged bone pieces were sterilized by cobalt 60 irradiation using 20 KGy.
EXAMPLE 3 Natural bone Material
The degreasing agent used for preparing the natural bone material is as follows:
The degreasing agent 7 consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide, potassium hydroxide 1:1 | Alkali reagent | 1 |
| oep-98:oep-70 1:1 | Alkali-resistant penetrant | 0.5 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 1:1 | Penetration aid | 1.5 |
| Triethylene glycol butyl ether-triethylene glycol methyl ether 2:1 | Solvent(s) | 5 |
| MF EH | Surface active agent | 1 |
| Purified water | Solvent(s) | 91 |
Degreasing agent 8 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide, potassium hydroxide 1:1 | Alkali reagent | 0.5 |
| oep-98:oep-70 1:1 | Alkali-resistant penetrant | 0.15 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 1:1 | Penetration aid | 1.5 |
| Triethylene glycol butyl ether-triethylene glycol methyl ether 2:1 | Solvent(s) | 2 |
| MF EH | Surface active agent | 3 |
| Purified water | Solvent(s) | 92.85 |
The degreasing agent 9 consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium carbonate and potassium carbonate 1:1 | Alkali reagent | 2 |
| Sodium pyrophosphate sodium metasilicate pentahydrate 1:1 | Penetration aid | 0.5 |
| Triethylene glycol butyl ether-triethylene glycol methyl ether 2:1 | Solvent(s) | 2.5 |
| MF EH | Surface active agent | 3 |
| Purified water | Solvent(s) | 92 |
The impurity-removed protein solution consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium chloride, potassium chloride and disodium hydrogen phosphate 2:1:0.5 | Inorganic salt | 25 |
| EDTA-2Na:EDTA-4Na 1:1 | Chelating agent | 0.2 |
| Triton-100:SDS1:1 | Surface active agent | 1 |
| Tris | Buffering agents | 0.1 |
| Purified water | Solvent(s) | 73.7 |
The preparation method of the natural bone material of the embodiment has the same bone block degreasing steps as those of the embodiment 1, and the parameters of bone block non-collagen removal are adjusted, specifically the steps are as follows:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method is sequentially carried out:
| Sequence(s) | Solvent(s) | Bone block to solvent ratio | Vacuum degree | Pressure retention | Ultrasonic power, time | Centrifuging, and taking time |
| 1 | Degreasing agent 7 | Weight ratio of 1:5 | -0.07MPa | 0.02MPa,1h | 28kHz,15min | 3000rpm,7min |
| 2 | Absolute ethyl alcohol | Volume ratio 1:5 | -0.05MPa | 0.03MPa,1h | 28kHz,10min | - |
| 3 | Degreasing agent 8 | Weight ratio of 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 of 1:5 | -0.09MPa | 0.04MPa,1h | 28kHz,15min | 2000rpm,15min |
Note that 80% ethanol refers to an 80% ethanol solution by volume
Respectively placing the bone blocks with the solvent into a vacuum glove box, vacuumizing, slowly stirring to completely discharge bubbles on the surfaces of the bone blocks, standing for 10-15 min, opening an air charging valve, charging nitrogen to positive pressure, placing the container with the bone blocks into an ultrasonic cleaner, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic at a lower temperature, centrifuging the mixture by using a centrifuge (when absolute ethyl alcohol and 80% ethanol solution with volume concentration are used, no centrifuging step), finally ultrasonically cleaning by using purified water, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic temperature, repeatedly washing to neutral, and fully absorbing the moisture in the bone blocks by using dust-free paper.
3. Bone block removal of non-collagen
The removing method comprises the following steps:
s1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:12, soaking for 8 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
s2, soaking and cleaning for 4 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the defatted bone in a hydrogen peroxide solution with the mass concentration of 12% for 18 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:8;
s4, soaking and cleaning for 4 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:15.
4. Freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 3 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 20 hours;
5. Cobalt 60 irradiation sterilization
And (5) carrying out cobalt 60 radiation sterilization on the packaged bone blocks by using the radiation dose of 22 KGy.
EXAMPLE 4 Natural bone Material
The preparation method of the natural bone material of this embodiment uses the same degreasing agent and deproteinized protein liquid as in embodiment 1, and the degreasing parameters of the bone block are different, and specifically comprises the following steps:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method is sequentially carried out:
| Sequence(s) | Solvent(s) | Bone block to solvent ratio | Vacuum degree | Pressure retention | Ultrasonic power, time | Centrifuging, and taking time |
| 1 | Degreasing agent 1 | Weight ratio of 1:7 | -0.08MPa | 0.03MPa,1h | 28kHz,15min | 2500rpm,10min |
| 2 | Absolute ethyl alcohol | Volume ratio 1:7 | -0.06MPa | 0.02MPa,1.5h | 28kHz,10min | - |
| 3 | Degreasing agent 2 | Weight ratio of 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 of 1:7 | -0.10MPa | 0.05MPa,2h | 32kHz,15min | 1500rpm,15min |
Note that 80% ethanol refers to an 80% ethanol solution by volume
The specific degreasing method comprises the following steps:
(1) Degreasing the bone block by using a degreasing agent 1, wherein the weight ratio of the bone block to the degreasing agent 1 is 1:7, and the specific steps are as follows:
s1, putting the bone block added with the degreasing agent 1 into a vacuum glove box, vacuumizing to the vacuum degree of-0.08 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 10min;
s2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.03MPa, and keeping for 1h;
s3, placing the container with the bone pieces in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and keeping the ultrasonic operation at a lower temperature;
s4, centrifuging the bone block degreasing agent 1 mixture by using a centrifuge, wherein the centrifuging parameters are that the rotating speed is 2500rpm, and the centrifuging time is 10min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper.
(2) Degreasing the femur by using absolute ethyl alcohol, wherein the volume ratio of the bone block to the absolute ethyl alcohol is 1:7, and the specific operation is as follows:
S1, putting the bone block added with the absolute ethyl alcohol into a vacuum glove box, vacuumizing to the vacuum degree of-0.06 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
S2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.02MPa, and keeping for 1.5h;
s3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 10min, wherein the power of the ultrasonic cleaner is 28kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically treating for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning for 5 times, and fully absorbing the water in the bone blocks by using dust-free paper.
(3) Degreasing the bone block by using a degreasing agent 2, wherein the weight ratio of the bone block to the degreasing agent 2 is 1:7, and the method comprises the following steps:
s1, putting the bone block added with the degreasing agent 2 into a vacuum glove box, vacuumizing to the vacuum degree of-0.09 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
s2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.04MPa, and keeping for 2 hours;
s3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 32kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, centrifuging the bone block degreasing agent 2 mixture by using a centrifuge, wherein the centrifuging parameters are that the rotating speed is 2000rpm, and the centrifuging time is 10min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper.
(4) Degreasing the bone block by using 80% ethanol solution with volume concentration, wherein the volume ratio of the bone block to the 80% ethanol solution with volume concentration is 1:7, and the specific operation is as follows:
s1, putting the bone block added with the absolute ethyl alcohol aqueous solution into a vacuum glove box, vacuumizing to the vacuum degree of-0.05 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
S2, opening an inflation valve, charging nitrogen to positive pressure, wherein the pressure is 0.03MPa, and keeping for 1.5h;
s3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 5-15min, wherein the power of the ultrasonic cleaner is 32kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically treating for 10min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning for 4 times, and fully absorbing the water in the bone blocks by using dust-free paper.
(5) Degreasing the bone block by using a degreasing agent 3, wherein the weight ratio of the bone block to the degreasing agent 3 is 1:7, and the method comprises the following steps:
S1, putting the bone block added with the degreasing agent 3 into a vacuum glove box, vacuumizing to the vacuum degree of-0.10 MPa, slowly stirring to completely discharge bubbles on the surface of the bone block, and standing for 15min;
s2, opening an inflation valve, charging nitrogen to positive pressure, keeping the pressure at 0.05MPa for 2 hours;
s3, placing the container with the bone blocks in an ultrasonic cleaner, performing ultrasonic oscillation for 15min, wherein the power of the ultrasonic cleaner is 32kHz, placing an ice bag in the ultrasonic cleaner, and maintaining the ultrasonic temperature;
S4, centrifuging the bone block degreasing agent 2 mixture by using a centrifuge, wherein the centrifuging parameters are that the rotating speed is 1500rpm, and the centrifuging time is 15min;
S5, ultrasonically cleaning with purified water, wherein the weight ratio of the bone blocks to the purified water is 1:10, ultrasonically cleaning for 15min, placing an ice bag in an ultrasonic cleaner, maintaining the ultrasonic temperature, repeatedly cleaning to be neutral, and fully absorbing the water in the bone blocks by using dust-free paper;
the degreasing agents 1,2 and 3 used in the above were the same as in example 1.
3. Bone block removal of non-collagen
The method comprises the following specific steps:
S1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:10, soaking for 12 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
S2, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the defatted bone in a 10% hydrogen peroxide solution for 24 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:5;
s4, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
the components of the above-used deproteinized protein solution were the same as those in example 1.
4. Freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 2 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 24 hours;
5. Cobalt 60 irradiation sterilization
And (5) carrying out cobalt 60 radiation sterilization on the packaged bone blocks by using the radiation dose of 20 KGy.
Performance testing
1. Compressive Strength
Reference standard GB/T1041-2008
The calculation formula is as follows:
σ=F/A
Wherein:
Sigma-stress parameter in megapascals (MPa);
f-force tested in newtons (N);
a—the original area of the sample in square millimeters (mm 2);
using the cancellous bone prepared in example 1, cancellous bone preparations were cut into cubes of 10X 10mm, 5 groups of samples, and test results are shown in Table 1;
TABLE 1 determination of cancellous bone compression Strength
| 1 | 2 | 3 | 4 | 5 | |
| Area (mm 2) | 107.231 | 109.381 | 113.935 | 108.779 | 112.536 |
| Maximum force (N) | 410 | 370 | 460 | 390 | 430 |
| Compressive Strength (MPa) | 3.82 | 3.38 | 4.04 | 3.59 | 3.82 |
As can be seen from Table 1, the spongy bone prepared by the method has the compressive strength of more than 3MPa, has higher compressive strength and meets the implantation requirement.
2. Porosity of the porous material
Reference standard YY/T1616-2018
Immersing cancellous bone in absolute ethyl alcohol to be fully soaked, receiving negative pressure, sucking residual air in a sample to be emptied, fully filling the absolute ethyl alcohol in a porous bracket, and finally obtaining the total volume V 2 of bone blocks and absolute ethyl alcohol until no air bubble escapes;
Calculation formula
E=(V1-V3)/(V2-V3)
In the middle of
E-porosity of
V 1 -initial absolute ethanol volume;
V 2 -absolute ethanol volume in the test tube after the bone pieces are fully filled with absolute ethanol;
v 3 -taking out the bone pieces filled with absolute ethyl alcohol, and leaving the volume of absolute ethyl alcohol;
Cancellous bone products were cut into cubes of 10x 10mm and the test results are shown in table 2.
TABLE 2 determination of cancellous bone porosities
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Porosity (%) | 68.2 | 70.1 | 72.5 | 66.9 |
As can be seen from the results of Table 2, the porosity of the prepared cancellous bone is 60-80%, and the pore diameter of the cancellous bone is 200-400 μm, which is determined from the scanning electron microscope of FIG. 4, and the cancellous bone is suitable for the growth and proliferation of cells in pores.
3. Cytotoxicity of cells
The potential cytotoxicity of the samples was determined by MTT method with reference to GB/T16886.5-2017 medical device biological evaluation in vitro cytotoxicity test, fifth section. It was confirmed whether or not a toxic reaction could be caused after the leaching solution of the material was contacted with the cultured cells.
Evaluation criteria
(1) 50% Of the sample extract is at least as active as 100% or more active than 100% cells, otherwise the experiment should be repeated;
(2) The lower the% cell viability, the greater the potential cytotoxicity;
(3) Cell viability < 70% of blank, indicating potential cytotoxicity of the samples;
(4) 100% of the cell viability of the test sample extract is the final result;
The result of MTT toxicity test on cancellous bone prepared in example 1 is shown in FIG. 1, and it can be confirmed that cancellous bone prepared in the present invention has very low cytotoxicity.
4. Fat tissue residue
The operation method comprises the steps of precisely weighing 1.0000g of a sample, adding 10mL of diethyl ether, standing at room temperature for 2h, shaking for 1 time every half hour, filtering diethyl ether into a constant-weight weighing bottle, adding 10mL of diethyl ether into residues, standing at room temperature for 2h, shaking for 1 time every half hour, and filtering diethyl ether into the constant-weight weighing bottle. Washing the residue with 5mL diethyl ether, filtering, and naturally volatilizing diethyl ether, drying the weighing bottle at 105deg.C for 2 hr, cooling to room temperature, and weighing with weight of m2;
The calculation method comprises the following steps:
Fat content (%) = (m 2-m 1)/sample weight×100%
TABLE 3 determination of Sony fructus Psoraleae fat residue
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Fat content (%) | 0.032 | 0.044 | 0.035 | 0.062 |
As can be seen from Table 3, the cancellous bone prepared by the method of the present invention has a very low fat content, substantially below 0.1%, and a remarkable degreasing effect.
5. DNA residual test
The operation method comprises grinding cancellous bone sample into powder, extracting DNA in the sample with DNA extraction kit, adding RNase to remove RNA in the solution, and detecting double-stranded DNA concentration with Nanodrop Lite ultramicro spectrophotometer
And (3) calculating results:
the evaluation criterion is that the DNA residual quantity is lower than 50 mug/g;
TABLE 4 determination of cancellous bone DNA residue
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Sampling volume (mg) | 0.033 | 0.038 | 0.042 | 0.045 |
| DNA concentration (ng/. Mu.L) | 4.8 | 4.9 | 4.6 | 5.1 |
| DNA content (μg/g) | 14.45 | 12.89 | 10.95 | 8.824 |
As can be seen from the table, the residual concentration of cancellous bone DNA prepared by the present invention is very low, indicating that cells in cancellous bone can be effectively destroyed and DNA in cells can be cleared by the method of the present invention.
Comparative example 1-
The comparative example differs from example 1 in the composition of the degreasing agent and the deproteinized liquid, specifically as follows:
the degreasing agent 10 consists of the following components:
degreasing agent 11 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide, potassium hydroxide 1:1 | Alkali reagent | 0.5 |
| Triethylene glycol butyl ether-triethylene glycol methyl ether 2:1 | Solvent(s) | 2 |
| MF EH | Surface active agent | 3 |
| Purified water | Solvent(s) | 94.5 |
Degreasing agent 12 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium carbonate and potassium carbonate 1:1 | Alkali reagent | 2 |
| Triethylene glycol butyl ether-triethylene glycol methyl ether 2:1 | Solvent(s) | 2.5 |
| MF EH | Surface active agent | 3 |
| Purified water | Solvent(s) | 92.5 |
The impurity-removed protein solution consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium chloride, potassium chloride and disodium hydrogen phosphate 2:1:0.5 | Inorganic salt | 25 |
| EDTA-2Na:EDTA-4Na 1:1 | Chelating agent | 0.2 |
| Triton-100:SDS1:1 | Surface active agent | 1 |
| Tris | Buffering agents | 0.1 |
| Purified water | Solvent(s) | 73.7 |
The preparation method of the natural bone material of the comparative example comprises the following steps, wherein the step of degreasing the bone block is the same as that of the example 1:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method is sequentially carried out:
note that 80% ethanol refers to an 80% ethanol solution by volume
Respectively placing the bone blocks with the solvent into a vacuum glove box, vacuumizing, slowly stirring to completely discharge bubbles on the surfaces of the bone blocks, standing for 10-15 min, opening an air charging valve, charging nitrogen to positive pressure, placing the container with the bone blocks into an ultrasonic cleaner, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic at a lower temperature, centrifuging the mixture by using a centrifuge (when absolute ethyl alcohol and 80% ethanol solution with volume concentration are used, no centrifuging step), finally ultrasonically cleaning by using purified water, placing an ice bag into the ultrasonic cleaner, keeping the ultrasonic temperature, repeatedly washing to neutral, and fully absorbing the moisture in the bone blocks by using dust-free paper.
3. Bone block removal of non-collagen
The removing method comprises the following steps:
s1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:12, soaking for 8 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
s2, soaking and cleaning for 4 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the defatted bone in a hydrogen peroxide solution with the mass concentration of 12% for 18 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:8;
s4, soaking and cleaning for 4 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:15.
4. Freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 3 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 20 hours;
5. Cobalt 60 irradiation sterilization
And (5) carrying out cobalt 60 radiation sterilization on the packaged bone blocks by using the radiation dose of 22 KGy.
As a result of examination, the fat content of comparative example 1 was 1.532%, and although most of the lipid could be removed without adding any penetrant or penetrant aid in comparative example 1, the lipid was hardly removed in the middle portion of the cancellous bone, and as shown in FIG. 5, which is a case in the center portion of the cancellous bone treated in comparative example 1 after slicing, the lipid remained in the interior of the cancellous bone, and the treatment effect on the center portion was poor, as can be seen from FIG. 5.
Comparative example 2-
The difference between this comparative example and example 1 is the step of adjusting the bone block degreasing, specifically as follows:
The degreasing agent used for preparing the natural bone material is as follows:
degreasing agent 1 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide | Alkali reagent | 2 |
| oep-70 | Alkali-resistant penetrant | 0.3 |
| Sodium hexametaphosphate: sodium pyrophosphate 1:1 | Penetration aid | 1 |
| Triethylene glycol butyl ether | Solvent(s) | 3 |
| MF EH:CC9NS1:1 | Surface active agent | 1 |
| Purified water | Solvent(s) | 92.7 |
Degreasing agent 2 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium hydroxide | Alkali reagent | 1 |
| oep-70 | Alkali-resistant penetrant | 0.15 |
| Sodium hexametaphosphate: sodium pyrophosphate 1:1 | Penetration aid | 1 |
| Triethylene glycol butyl ether | Solvent(s) | 2 |
| MF EH:CC9NS1:1 | Surface active agent | 2 |
| Purified water | Solvent(s) | 93.85 |
Degreasing agent 3 is composed of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium carbonate | Alkali reagent | 3 |
| Sodium hexametaphosphate: sodium pyrophosphate 1:1 | Penetration aid | 1 |
| Triethylene glycol butyl ether | Solvent(s) | 1.5 |
| MF EH:CC9NS1:1 | Surface active agent | 1 |
| Purified water | Solvent(s) | 93.5 |
The impurity-removed protein solution consists of the following components:
| Component (A) | Action | Weight of (E) |
| Sodium chloride potassium chloride 1:1 | Inorganic salt | 10 |
| EDTA-2Na | Chelating agent | 0.2 |
| Triton-100:SDS1:1 | Surface active agent | 2 |
| Tris | Buffering agents | 0.1 |
| Purified water | Solvent(s) | 87.7 |
The preparation method of the natural bone material of the comparative example, degreasing agents 1,2 and 3 and the deproteinized protein solution are the same as those of example 1, and the specific steps are as follows:
1. cutting distal cancellous bone of tibial epiphysis by using a bone cutting machine, then cutting off outer cortical bone, and flushing out blood stains and cutting residues on the surface by purified water;
2. Bone block degreasing
The degreasing method comprises the following steps:
(1) Degreasing the bone blocks by using a degreasing agent 1, wherein the weight ratio of the bone blocks to the degreasing agent 1 is 1:5, S1 is to put a container with the bone blocks into an ultrasonic cleaner, ultrasonically shake for 30min, the power of the ultrasonic cleaner is 28kHz, and an ice bag is put into the ultrasonic cleaner to keep ultrasonic at a lower temperature;
(2) Degreasing a femur by using absolute ethyl alcohol, wherein the volume ratio of a bone block to the absolute ethyl alcohol is 1:5, and the specific operation comprises the following steps of S1, putting a container with the bone block into an ultrasonic cleaner, carrying out ultrasonic vibration for 20min, setting the power of the ultrasonic cleaner to be 28kHz, putting an ice bag into the ultrasonic cleaner, and keeping the ultrasonic temperature;
(3) Degreasing the bone block by using a degreasing agent 2, wherein the weight ratio of the bone block to the degreasing agent 2 is 1:5, and the method specifically comprises the following steps of S1, placing a container with the bone block in an ultrasonic cleaner, carrying out ultrasonic vibration for 30min, setting the power of the ultrasonic cleaner at 28kHz, placing an ice bag in the ultrasonic cleaner, and keeping the ultrasonic temperature;
(4) Degreasing the bone block by using an absolute ethyl alcohol aqueous solution with the volume concentration of 80 percent, wherein the volume ratio of the bone block to the absolute ethyl alcohol aqueous solution is 1:5, and the specific operation comprises the following steps of S1, putting a container with the bone block into an ultrasonic cleaner, carrying out ultrasonic vibration for 15min, wherein the power of the ultrasonic cleaner is 28kHz, putting an ice bag into the ultrasonic cleaner, and keeping the ultrasonic temperature;
(5) Degreasing the bone blocks by using a degreasing agent 3, wherein the weight ratio of the bone blocks to the degreasing agent 3 is 1:5, and the method specifically comprises the following steps of S1, placing a container with the bone blocks in an ultrasonic cleaner, carrying out ultrasonic vibration for 30min, setting the power of the ultrasonic cleaner at 28kHz, placing an ice bag in the ultrasonic cleaner, and keeping the ultrasonic temperature;
3. Bone block removal of non-collagen
The removing method comprises the following steps:
S1, adding impurity-removing protein liquid into defatted bone, wherein the weight ratio of the defatted bone to the impurity-removing protein liquid is 1:10, soaking for 12 hours at the temperature of 4 ℃, and replacing the impurity-removing protein liquid every 2 hours;
S2, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20;
S3, soaking the bone with 10% hydrogen peroxide solution for 24 hours, wherein the weight ratio of the defatted bone to the hydrogen peroxide solution is 1:5;
S4, soaking and cleaning for 3 times by using purified water, wherein the weight ratio of the defatted bone to the purified water is 1:20.
4. Freeze-drying of bone pieces
Freezing the immersed bone blocks in a refrigerator at-20 ℃ for 2 hours, then transferring the immersed bone blocks into a refrigerator at-80 ℃ for deep cooling treatment for 1 hour, and finally freeze-drying the bone blocks in a freeze dryer for 24 hours;
5. Cobalt 60 irradiation sterilization
And (5) carrying out cobalt 60 radiation sterilization on the packaged bone blocks by using the radiation dose of 20 KGy.
According to detection, the fat content of the comparative example 2 is 0.315%, and the processes of vacuum pressurization, centrifugation and the like are not adopted in the comparative example 2, so that the fat removal efficiency of the degreasing agent is reduced.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (7)
1. The preparation method of the natural biological bone material is characterized by comprising the following steps:
S1, degreasing, namely selecting cancellous bone of animal origin, sequentially adding a degreasing agent and an ethanol solvent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, and carrying out ultrasonic vibration to obtain defatted cancellous bone;
The degreasing agent comprises 0.5-4% of strong alkali reagent, 0.5-1.5% of penetration auxiliary agent, 1.5-5% of solvent, 1-3% of surfactant 1 and the balance of water according to weight percentage, wherein the strong alkali reagent comprises at least one of sodium hydroxide, potassium hydroxide and sodium carbonate, and the solvent comprises triethylene glycol butyl ether and/or triethylene glycol methyl ether;
the penetration aid comprises at least one of sodium hexametaphosphate, sodium pyrophosphate and sodium metasilicate pentahydrate, and the surfactant 1 comprises MF EH and/or CC9NS;
In the step S1, the degreasing agent and the ethanol solvent are used for degreasing the cancellous bone in an alternative mode, wherein the alternative mode comprises the steps of adding the degreasing agent to perform reagent degreasing treatment on the cancellous bone, and then adding the ethanol solvent to perform ethanol degreasing treatment on the cancellous bone;
the degreasing treatment of the reagent comprises the steps of adding a degreasing agent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, carrying out ultrasonic oscillation and centrifuging;
the ethanol degreasing treatment comprises the steps of adding an ethanol solvent, vacuumizing, charging nitrogen to positive pressure, maintaining the positive pressure, and carrying out ultrasonic oscillation, wherein the ethanol solvent is absolute ethanol or ethanol solution with the volume concentration of 60-85%;
The vacuum degree is-0.10 to-0.05 MPa, the positive pressure is 0.02 to 0.05MPa when nitrogen is filled, the positive pressure is maintained for 1 to 2 hours, the ultrasonic vibration power is 25 to 35kHz, the ultrasonic vibration time is 5 to 20 minutes, the centrifugal power is 2000 to 3000rpm, and the centrifugal time is 5 to 20 minutes;
S2, removing non-collagen, namely adding impurity-removing protein liquid into the defatted cancellous bone, soaking, cleaning, and then adding hydrogen peroxide solution for continuous soaking to obtain pretreated cancellous bone;
The impurity-removed protein liquid comprises 10-25% of inorganic salt, 0.1-0.3% of chelating agent, 1-3% of surfactant 2, 0.1-0.2 of buffer solution and the balance of water according to weight percentage, wherein the chelating agent comprises EDTA-2Na and/or EDTA-4Na;
The inorganic salt comprises at least one of sodium chloride, potassium chloride and disodium hydrogen phosphate, and the surfactant 2 comprises Triton-100 and/or sodium dodecyl sulfate;
And S3, drying and sterilizing the pretreated cancellous bone to obtain the target bone material.
2. The method for preparing a natural biological bone material according to claim 1, wherein the degreasing agent further comprises 0.15-0.5% penetrating agent, and the penetrating agent comprises oep-98 and/or oep-70.
3. The method for preparing a natural biological bone material according to claim 1, wherein 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 a natural biological bone material according to claim 1, wherein in the step S2, the weight ratio of the defatted cancellous bone to the impurity-removed protein liquid is 1:10-12, the soaking temperature of the impurity-removed protein liquid is 2-6 ℃, and the soaking time is 8-12 h.
5. The method for preparing a natural biological bone material according to claim 4, wherein the deproteinized liquid is replaced every 2 to 4 hours.
6. The method for preparing a natural biological bone material according to claim 1, wherein in the 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 soaking time of adding the hydrogen peroxide solution is 15-25 h.
7. The natural biological bone material according to any one of claims 1 to 6.
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