CN108578773B - Method for preparing porous block-shaped heterogeneous bone repair material in short process and bone repair material prepared by method - Google Patents
Method for preparing porous block-shaped heterogeneous bone repair material in short process and bone repair material prepared by method Download PDFInfo
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- 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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- A61L27/3641—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 site of application in the body
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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
- A61L27/3683—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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- A61L27/00—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
- A61L27/3683—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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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention provides a preparation method of a porous blocky heterogeneous bone repair material, which comprises the following steps: 1) cutting mammal cancellous bone into blocks, washing with water, drying, soaking in hydrogen peroxide solution and sodium hydroxide solution respectively to primarily remove organic matters, washing with water, and drying; 2) placing the spongy bone with the organic matters removed preliminarily and an alkaline solution into a pressure-bearing closed container, heating and preserving heat, washing with water, and drying; 3) placing the cancellous bone and distilled water in a pressure-bearing closed container, heating and preserving heat, washing with water, and drying; 4) calcining and sterilizing. The invention also provides the porous blocky allogenic bone repair material prepared by the preparation method. The preparation method of the invention can completely remove fat and protein which cause immune antigen reaction at lower temperature, has no pollution of toxic and harmful organic solvent in the treatment process, and the treated heterogeneous bone material has weak crystal structure and contains a large amount of carbonate, thus better promoting the regeneration of bone tissue.
Description
Technical Field
The invention belongs to the field of biomedical engineering materials, and particularly relates to a method for preparing a porous block-shaped allogenic bone repair material for repairing bone tissues in a surgical operation and a bone repair material prepared by the method.
Background
Bone tissue defect caused by trauma, tumor, infection and other factors is a common clinical disease in orthopedics department. Bone grafting is a commonly used method of repair, and is mainly classified into autologous bone grafting, allogeneic bone grafting, and xenogeneic bone grafting. The main limitations of the former two methods are supply shortage, easily occurring supply area complications and existence of disease infection potential, and the heterogeneous bone transplantation provides a new idea and method for bone defect repair.
In bone grafting operation, the requirement on heterogeneous bone repair materials is high, and the heterogeneous bone repair materials have the following characteristics: (1) free of fats and proteins that elicit immune responses; (2) can be prepared into a certain size, and is convenient to fill; (3) has osteoinductive and osteoconductive properties; (4) good biocompatibility and moderate biodegradability, the degradation speed needs to be matched with the regeneration speed of bone tissues, and degradation products in a human body are harmless to the human body; (5) has surface chemistry and microstructure that supports bone cell growth and functional differentiation; (6) easy to process, sterilize and store.
The key to the preparation of the allogenic bone repair material is to remove organic matters which are easy to cause antigenicity, such as fat, protein and the like. At present, the methods of chemical soaking treatment and high-temperature calcination are mainly adopted for degreasing and deproteinizing the heterogeneous bone repair material. The chemical soaking treatment usually uses toxic solvents such as ethylenediamine, acetone, chloroform, methanol, and diethyl ether. The use of toxic solvents in large amounts causes a great environmental stress. In addition, since the heterogeneous bone repairing material has a porous structure, toxic solvents are difficult to clean by cleaning, toxic chemical reagents are left, and the biological activity of the heterogeneous bone is damaged. The combined treatment method of sodium hydroxide and hydrogen peroxide in the chemical soaking treatment method is also commonly used, but the method can only partially eliminate the antigenicity of the heterogeneous bone, and the strength and the chemical composition of the method can be greatly changed along with the prolonging of the soaking time. The high-temperature calcination refers to the calcination of the heterogeneous bone under the high-temperature condition (more than 600 ℃) to remove organic matters, the high-temperature calcination can destroy the weak crystal structure of the heterogeneous bone repair material, and the carbonate in the bone can also be decomposed, so that the heterogeneous bone repair material loses bioactivity. Therefore, the existing toxic solvent soaking method and high-temperature calcining method can not meet the preparation requirement of the heterogeneous bone.
Murugan R et al (Murugan R, Ramakrishna S, Rao K P. nanoporous hydroxy-carbonate scaffold of mineral bone [ J. Materials Letters, 2006, 60 (23): 2844-. The method uses two toxic solvents of acetone and diethyl ether in the preparation process, and can cause the residue of the toxic solvent. In addition, the NaOH treatment and the higher temperature calcination temperature significantly destroy the weak crystalline structure, bioactivity and unique microstructure of the allogenic bone.
Barakat N A M et al (Barakat N A M, Khil M S, Omran A M, et al. extraction of pure natural hydroxyl from the bone bones bio-way by way of a steam differential method [ J ]. Journal of materials processing technology, 2009, 209 (7): 3408-3415.) cattle bones were used as raw materials, fat was removed using an acetone solution, and prepared into powders of 450 μ M or less. Then, the nano hydroxyapatite powder is prepared by using 25 percent NaOH solution for alkali heating for 5 hours at the temperature of 250 ℃. The obtained heterogeneous bone material is highly crystallized nano-grade hydroxyapatite powder, and completely loses the multidimensional hole structure and the weakly crystallized crystal structure characteristics of bovine bone (especially cancellous bone). In addition, the acetone solution used in the preparation process can cause toxic dissolution residues in the prepared heterogeneous bone repair material.
Manufactured by Geistlich corporation
(Geistlich, Switzerland) is a heterogeneous bone graft material widely used in bone graft surgery. U.S. patent No. 5,167,961 (lucsi, Heinz; Geistlich, peter. process for preparing high purity bone mineral. U.S. patent No. 5,167,961, 1992) of this company prepares the xenogenic bone material by first removing organic matter with a toxic solvent, ethylenediamine, then repeatedly washing with its specially prepared solution and deionized water, and finally calcining at 250-600 ℃. Toxic solvent residues may occur in the process, and the process is run for over 15 days, producing a streamThe process is too long. Zimmermann et al (Zimmermann C E, Gierloff M, Hedderich J, et al, biocompatibility of bone graft subsistents: effects on survivals and promotion of bone multilineage cells in vitro [ J]A Folia morphohoga, 2011, 70 (3): 154-160.) found that
The leachate was incubated with cells and only 1/3 adherent cells survived, as illustrated in the side section
And part of the toxic chemical solvent remains.
Therefore, the problems to be solved in the preparation of heterogeneous bone are: (1) toxic solvents are not used in the preparation process, so that the biological activity of the heterogeneous bone is prevented from being damaged; (2) the heterogeneous bone block with a certain size and a heterogeneous bone original micro-nano hole structure can be prepared, and bone defect repair under specific conditions is facilitated; (3) the temperature in the preparation process is lower, and the influence on the poor crystallinity and the carbonate content of the heterogeneous bone is reduced.
Disclosure of Invention
The invention aims to provide a novel method for preparing a heterogeneous bone, and the heterogeneous bone repair material which has a rich porous structure and is in a weak crystallization state can be prepared by the method.
The invention also aims to provide the porous blocky heterogeneous bone repair material prepared by the preparation method.
The technical scheme is as follows: the invention provides a preparation method of a porous blocky heterogeneous bone repair material, which comprises the following steps:
1) soaking the blocky cancellous bone subjected to preliminary organic matter removal in an alkaline solution, placing the blocky cancellous bone in a pressure-bearing closed container, heating to 100-240 ℃, then preserving heat at 100-240 ℃ for 1-12 hours, cooling, taking out the cancellous bone, washing with water, and drying;
2) soaking the cancellous bone treated in the step 1) in distilled water or deionized water, placing the cancellous bone in a pressure-bearing closed container, heating to 100-240 ℃, then preserving heat at 100-240 ℃ for 1-12 hours, cooling, taking out the cancellous bone, washing with water, and drying;
3) calcining the cancellous bone treated in the step 2) at the temperature of 300-480 ℃ for 4-24 hours, and then sterilizing to obtain the porous block-shaped dissimilar bone repairing material.
The method for primarily removing the organic matters comprises the following steps:
a) washing the blocky mammal cancellous bone with water until no bone marrow tissue is visible, drying, soaking in hydrogen peroxide solution, washing, and drying;
b) soaking the spongy bone treated in the step a) in a sodium hydroxide solution, then washing with water and drying.
The method for primarily removing the organic matters can primarily remove fat and protein in the cancellous bone through water washing, hydrogen peroxide solution soaking and sodium hydroxide solution soaking.
Step 1), fat and protein in cancellous bone can be basically removed through alkali heat treatment; step 2) through hydrothermal treatment, alkali adhered to the spongy bone after treatment in the step 1) can be removed; and 3) through low-temperature calcination and sterilization treatment, the pathogenic bacteria carried in the cancellous bone of the mammal can be eliminated.
The block-shaped cancellous bone of the present invention may be obtained from common mammals, such as pigs, sheep, horses, and the like.
In the above method for primarily removing organic matter, in the step a), the cancellous bone of the mammal is cut into 0.1cm3～50cm3Washing the bone blocks with water, preferably washing the bone blocks with a high-pressure water gun until the bone tissues are white and yellow and no bone marrow tissues are visible, and drying the bone blocks in a drying oven for 12 hours; the mass fraction of the hydrogen peroxide in the hydrogen peroxide solution is preferably 10-70%, and the hydrogen peroxide solution is soaked for 12-48 hours at the temperature of 5-70 ℃; the cancellous bone after soaking in the hydrogen peroxide solution was repeatedly washed with distilled water and then dried in a drying oven for 12 hours.
In the step b), soaking the dried cancellous bone in a sodium hydroxide solution at the temperature of 5-70 ℃ for 12-48 hours, wherein the concentration is preferably 0.5-4M; repeatedly cleaning the spongy bone soaked in the sodium hydroxide solution by using distilled water, and drying the spongy bone in a drying oven for 12 hours;
in the step 1), the pH value of the alkaline solution is preferably 8-13, and the alkaline solution is a solution of one compound or a mixed solution of a plurality of compounds of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium silicate and sodium silicate; the volume ratio of the cancellous bone to the alkaline solution is 1: 20-1: 80; the pressure-bearing closed container can be a reaction kettle, the reaction kettle is kept warm in a heating box for 1-12 hours at the temperature of 100-240 ℃, and after the reaction kettle is cooled to the room temperature, the spongy bone is taken out and repeatedly cleaned and dried by using distilled water for 1-4 times.
In the step 2), the dried cancellous bone and distilled water are placed in a closed container together, the volume ratio of the cancellous bone to the distilled water is 1: 20-1: 80, the reaction kettle is insulated in a heating box for 1-12 hours at the temperature of 100-240 ℃, the reaction kettle is cooled to the room temperature, the cancellous bone is taken out, washed and dried repeatedly by the distilled water, and the operation is repeated for 1-4 times.
In the step 1) and the step 2), the heating and heat preservation temperature in the pressure-bearing closed container is preferably 180-240 ℃.
Preferably, the calcination temperature is 300-400 ℃, and the irradiation sterilization is cobalt 60 irradiation sterilization or ethylene oxide sterilization. The invention also provides a porous block-shaped heterogeneous bone repair material which is prepared by the preparation method.
Has the advantages that: the porous block-shaped allogenic bone repair material prepared by the method has chemical components, material hole structures and weak crystal structures which are consistent with those of human bones, maximally simulates the structures and the components of human bone tissues, maintains the biological activity of the allogenic bone repair material, and can promote the growth and repair of the bone tissues. The preparation method disclosed by the invention is simple, the preparation time is greatly shortened, the preparation method is a short-flow preparation method, the raw material source is wide, the preparation method is suitable for large-scale industrial production, the production cost can be obviously reduced, and the bone repair material with low price and good curative effect is provided for patients.
Drawings
FIG. 1 is a macroscopic photograph of a bulk allogenic bone repair material prepared by the method of the present invention;
FIG. 2 is a scanning electron microscope (magnified 100 times) of a porous blocky heterogeneous bone repair material prepared by the method of the present invention;
FIG. 3 is an X-ray diffraction image of a porous block-shaped allogenic bone repair material prepared by different preparation methods;
FIG. 4 is a Fourier transform infrared spectroscopy picture of a porous bulk xenogenic bone material prepared by the method of the present invention.
Detailed Description
Example 1
The preparation method of the porous blocky heterogeneous bone material comprises the following steps:
(1) extracting cancellous bone:
(a) collecting fresh bovine vertebra, avoiding exposure to pollutants, and freezing for storage.
(b) Thawing frozen bovine vertebra, cleaning, cutting bovine cancellous bone by bone sawing machine, and cutting cancellous bone into 2cm3Washing the blocks with high pressure water gun repeatedly until the bone tissue is white and yellow and no bone marrow tissue is visible, and drying in a drying oven for 12 hr.
(2) Pretreatment of cancellous bone:
(a) treatment of aqueous hydrogen peroxide solution
Soaking the spongy bone treated and dried in the step (1) in hydrogen peroxide with the mass fraction of 10% at 25 ℃ for 12 hours, repeatedly washing the soaked spongy bone with distilled water, and drying the spongy bone in a drying oven for 12 hours.
(b) Sodium hydroxide solution treatment
Soaking the dried cancellous bone in the step (a) for 12 hours in a sodium hydroxide solution with the concentration of 1M at the temperature of 25 ℃, repeatedly washing the soaked cancellous bone with distilled water, and drying the cancellous bone in a drying oven for 12 hours.
(3) Alkali heat treatment of cancellous bone:
mixing the cancellous bone treated in the step (2) with a sodium hydroxide solution, putting the mixture into a closed reaction kettle, keeping the pH value of the sodium hydroxide solution at 13 and the volume ratio of the cancellous bone to the sodium hydroxide solution at 1: 20, keeping the reaction kettle in a heating box at 180 ℃ for 2 hours, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly washing and drying the cancellous bone with distilled water, and repeating the steps for 3 times.
(4) Hydrothermal treatment of cancellous bone:
mixing the cancellous bone treated in the step (3) with distilled water, putting the mixture into a closed reaction kettle, keeping the volume ratio of the cancellous bone to the distilled water at 1: 20, keeping the reaction kettle in a heating box for 2 hours at the temperature of 180 ℃, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly cleaning and drying the cancellous bone by distillation, and repeating the operation for 2 times.
(5) Calcining cancellous bone:
and (4) placing the spongy bone treated in the step (4) into a high-temperature furnace for calcining, heating at the rate of 10 ℃/min to 400 ℃, preserving the heat for 12 hours, and naturally cooling and taking out the spongy bone for later use.
(6) And (3) sterilizing the cancellous bone:
the cancellous bone is irradiated and sterilized by cobalt 60 (the irradiation dose is 30KGy), and the heterogeneous bone repair material is obtained.
The heterogeneous bone repair material is characterized by structure and components, and the characterization results are shown in table 1 and fig. 1-4.
Table 1 shows the porous block-shaped heterogeneous bone material prepared by the method of the present invention and the commercially available same type bone repair material Geistlich
It can be seen that the prepared xenogeneic bone does not contain organic matter and contains more CO3 2-Zakaaria F A et al (Zakaaria F A, Marsad N H, ManafA Y A, et a1.Characterisation of carbonated application for positional application in biological assays [ J]Materials Research Innovations, 2009, 13 (3): 309-312.) found that carbonate contributes to the improvement of the bioactivity, so the heterogeneous bone repair material prepared by the method is more beneficial to the repair of bone tissues.
TABLE 1
FIG. 1 is a macroscopic photograph of the heterogeneous bone repairing material prepared by the method of the present invention, and it can be seen from FIG. 1 that the obtained heterogeneous bone material is milky white peculiar to hydroxyapatite, which indicates that there is no residual organic matter on the surface.
FIG. 2 is a scanning electron microscope (magnified 100 times) picture of the porous block-shaped bone repairing material prepared by the method of the present invention, and it can be seen from FIG. 2 that the prepared bone repairing material has a three-dimensional pore structure, the size of the macropores is larger than 100um, and a plurality of small pore structures are distributed in the three-dimensional pore structure, such a pore structure is beneficial to the bone tissue repair.
Fig. 3 is an X-ray diffraction image of the porous block-shaped heterogeneous bone repair material prepared by the method and the calcination method of the present invention, and it can be seen from fig. 3 that the heterogeneous bone repair material prepared by the method and the calcination method of the present invention is a hydroxyapatite component, but the bone repair material prepared by the method of the present invention has a wide peak shape and a weak crystallization state, and the degree of crystallization is calculated to be 42%, which is far smaller than that of the heterogeneous bone repair material prepared by the calcination method. Wherein, the calcination method specifically comprises the following steps:
(1) extraction of cancellous bone
Collecting fresh bovine vertebra, avoiding exposure to pollutants, and freezing for storage. The frozen bovine vertebrae were thawed and the muscle and connective tissue attached thereto were removed by oral scraping.
Cutting bovine cancellous bone by bone sawing machine to 2cm3Repeatedly washing the blocks with a high-pressure water gun until the bone tissue is white and yellow and no bone marrow tissue is visible, and drying in a drying oven for 12 hours.
(2) Pretreatment of
Putting dried bovine cancellous bone into 30% H at room temperature2O2Soaking in the solution for 12 hr, repeatedly cleaning the soaked Os bovis Seu Bubali with distilled water, and drying in a drying oven for 12 hr.
And (3) soaking the dried bovine cancellous bone in a NaOH solution with the concentration of 1mol/L for 12 hours at room temperature, repeatedly washing the soaked bovine cancellous bone with distilled water, and drying in a drying oven for 12 hours.
(3) Calcination treatment
And (3) placing the dried bovine cancellous bone into a crucible, placing the crucible into a tubular furnace, calcining at 900 ℃, wherein the calcining atmosphere is air, the heating rate is 5 ℃/min, the heat preservation time is 3 hours, and then cooling along with the furnace and taking out the sample.
FIG. 4 is a Fourier transform infrared spectrum of a porous block bone material prepared by the method of the present invention, which can be found at 1043cm in FIG. 4-1、606cm-1And 569cm-1A typical PO appears4 3-The peak of (a) indicates that the prepared heterogeneous bone material has an apatite structure at 1418cm-1Two splitting peaks exist, which indicates that CO exists in the crystal structure of the prepared heterogeneous bone repair material3 2-. Therefore, it was confirmed that the prepared xenogenic bone material was typical hydroxyapatite carbonate.
Example 2
The preparation method of the porous blocky heterogeneous bone material comprises the following steps:
(1) extracting cancellous bone:
cutting the cancellous bone of the pig into 8cm3Washing the blocks with high pressure water gun repeatedly until the bone tissue is white and yellow and no bone marrow tissue is visible, and drying in a drying oven for 12 hr.
(2) Pretreatment of cancellous bone:
(a) treatment of aqueous hydrogen peroxide solution
Soaking the spongy bone treated and dried in the step (1) in hydrogen peroxide with the mass fraction of 70% at the temperature of 5 ℃ for 48 hours, repeatedly washing the soaked spongy bone with deionized water, and drying the spongy bone in a drying oven for 12 hours.
(b) Sodium hydroxide solution treatment
Soaking the dried cancellous bone in the step (a) for 12 hours in a 70 ℃ environment by using potassium hydroxide with the concentration of 0.5M, repeatedly washing the soaked cancellous bone by using distilled water, and drying the cancellous bone in a drying oven for 12 hours.
(3) Alkali heat treatment of cancellous bone:
mixing the cancellous bone treated in the step (2) with a potassium bicarbonate solution, putting the mixture into a closed reaction kettle, keeping the pH value of a sodium hydroxide solution at 8 and the volume ratio of the cancellous bone to the potassium bicarbonate solution at 1: 80, keeping the temperature of the reaction kettle in a heating box for 12 hours, keeping the temperature at 240 ℃, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly washing and drying the cancellous bone with distilled water, and repeating the washing and drying processes for 3 times.
(4) Hydrothermal treatment of cancellous bone:
mixing the cancellous bone treated in the step (3) with distilled water, putting the mixture into a closed reaction kettle, keeping the volume ratio of the cancellous bone to the distilled water at 1: 80, keeping the reaction kettle in a heating box for 12 hours at the temperature of 240 ℃, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly cleaning and drying the cancellous bone by distillation, and repeating the operation for 4 times.
(5) Calcining cancellous bone:
and (4) placing the cancellous bone treated in the step (4) into a high-temperature furnace for calcining, wherein the heating rate is 1 ℃/min, heating to 300 ℃, preserving heat for 24 hours, and naturally cooling to take out the cancellous bone for later use.
(6) And (3) sterilizing the cancellous bone:
sterilizing the cancellous bone by using ethylene oxide to obtain the dissimilar bone repair material.
Example 3
The preparation method of the porous blocky heterogeneous bone material comprises the following steps:
(1) extracting cancellous bone:
cutting cancellous bone of sheep to 0.1cm3Washing the blocks with high pressure water gun repeatedly until the bone tissue is white and yellow and no bone marrow tissue is visible, and drying in a drying oven for 12 hr.
(2) Pretreatment of cancellous bone:
(a) treatment of aqueous hydrogen peroxide solution
Soaking the spongy bone treated and dried in the step (1) in 30% hydrogen peroxide by mass fraction at 70 ℃ for 12 hours, repeatedly washing the soaked spongy bone with deionized water, and drying the spongy bone in a drying oven for 12 hours.
(b) Sodium hydroxide solution treatment
Soaking the dried cancellous bone in the step (a) in sodium hydroxide with the concentration of 4M at the temperature of 5 ℃ for 48 hours, repeatedly washing the soaked cancellous bone with distilled water, and drying the cancellous bone in a drying oven for 12 hours.
(3) Alkali heat treatment of cancellous bone:
mixing the cancellous bone treated in the step (2) with a sodium carbonate solution, putting the mixture into a closed reaction kettle, keeping the pH value of the sodium hydroxide solution at 10 and the volume ratio of the cancellous bone to the sodium hydroxide solution at 1: 50, keeping the reaction kettle in a heating box at the temperature of 240 ℃ for 1 hour, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly washing and drying the cancellous bone by using distilled water, and repeating the washing and drying for 1 time.
(4) Hydrothermal treatment of cancellous bone:
mixing the cancellous bone treated in the step (3) with deionized water, placing the mixture in a closed reaction kettle, wherein the volume ratio of the cancellous bone to the distilled water is 1: 60, keeping the reaction kettle in a heating box for 1 hour at the temperature of 240 ℃, cooling the reaction kettle to room temperature, taking out the cancellous bone, repeatedly cleaning and drying the cancellous bone by distillation, and repeating the step for 1 time.
(5) Calcining cancellous bone:
and (3) placing the spongy bone treated in the step (4) into a high-temperature furnace for calcining, raising the temperature to 400 ℃ at the rate of 0.1 ℃/min, preserving the heat for 4 hours, and naturally cooling and taking out the spongy bone for later use.
(6) And (3) sterilizing the cancellous bone:
the cancellous bone is irradiated and sterilized by cobalt 60 (the irradiation dose is 30KGy), and the heterogeneous bone repair material is obtained.
The method solves the problem of thoroughly removing fat and protein from porous block-shaped heterogeneous bone material at lower temperature. Firstly, the block-shaped heterogeneous bone material is pretreated by using an improved chemical method, then fat and protein are further removed by an alkali thermal method, and finally, the weakly-crystallized porous block-shaped heterogeneous bone repairing material is obtained by calcining and irradiating or ethylene oxide sterilization. The porous blocky allogenic bone repair material prepared by the invention has the same porosity, pore traffic, pore size and the like as human cancellous bone, has a weak crystalline structure, is beneficial to the adhesion growth of cells and the growth of blood vessels and nerves, and promotes the repair of bone tissues. The invention has wide and cheap raw material sources, simple preparation method and no toxic chemical reagent used in the process, is suitable for large-scale industrialized production and reduces the economic burden of patients.
Claims (7)
1. A preparation method of a porous blocky heterogeneous bone repair material is characterized by comprising the following steps:
1) soaking the blocky cancellous bone subjected to preliminary organic matter removal in an alkaline solution, placing the blocky cancellous bone in a pressure-bearing closed container, heating to 100-240 ℃, then preserving heat at 100-240 ℃ for 1-12 hours, cooling, taking out the cancellous bone, washing with water, and drying; the pressure-bearing closed container is a reaction kettle, the pH value of the alkaline solution is 8-13, and the volume ratio of the cancellous bone to the alkaline solution is 1: 20-1: 80; washing with water, wherein the repetition times of drying is 1-4 times;
2) soaking the cancellous bone treated in the step 1) in distilled water or deionized water, placing the cancellous bone in a pressure-bearing closed container, heating to 100-240 ℃, then preserving heat at 100-240 ℃ for 1-12 hours, cooling, taking out the cancellous bone, washing with water, and drying;
3) calcining the cancellous bone treated in the step 2) at the temperature of 300-480 ℃ for 4-24 hours, and then sterilizing to obtain the porous block-shaped dissimilar bone repairing material.
2. The method for producing a porous block-shaped allogenic bone repair material according to claim 1, wherein in the step 1), the volume of the block-shaped cancellous bone is 0.1cm3～50cm3(ii) a The method for primarily removing the organic matters comprises the following steps: washing the blocky cancellous bone with water until the bone tissue is white and yellow and no bone marrow tissue is visible, then sequentially soaking the blocky cancellous bone with a hydrogen peroxide solution and a strong alkali solution, cleaning and drying.
3. The method for preparing the porous blocky heterogeneous bone repair material according to claim 1, wherein in the step 1), the alkaline solution is a solution of one compound or a mixed solution of a plurality of compounds of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, potassium silicate and sodium silicate.
4. The method for preparing a porous block-shaped allogenic bone repair material according to claim 1, wherein in the step 2), the volume ratio of the cancellous bone to the distilled or deionized water is 1: 20-1: 80; and washing with water, wherein the repetition frequency of drying is 1-4 times.
5. The method for preparing the porous blocky heterogeneous bone repair material according to claim 1, wherein in the step 1) and the step 2), the temperature for the heating and the heat preservation in the pressure-bearing closed container is 180-240 ℃.
6. The method for preparing the porous blocky heterogeneous bone repair material according to claim 1, wherein in the step 3), the calcination temperature is 300-400 ℃, and the sterilization treatment method is radiation sterilization or ethylene oxide sterilization.
7. A porous block-shaped heterogeneous bone repair material, which is prepared by the preparation method according to any one of claims 1 to 6.
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