CN1456363A - Preparing method for heteroossein base materials - Google Patents

Abstract

The invention relates to the technical field of biomaterials such as bone surgery repair materials and bone tissue engineering extracellular matrix materials, in particular to a new method for preparing heterogeneous collagen matrix. This method selects heterogeneous bones such as cattle and pigs, and mainly undergoes heterogeneous bone source and processing—degreasing treatment—partial decalcification—decollagen removal—protease digestion, and finally sterilized and packaged to obtain a new type of heterogeneous bone collagen matrix, which retains the original bone The bone mineral component and most of the collagen, among which the collagen component is digested by protease, cut off the telopeptide that can cause immunogenicity, so the immunogenicity is greatly reduced. While having strong mechanical properties, the material has good porosity and pore connectivity, and is suitable for use as extracellular matrix materials for bone tissue engineering and bone defect transplantation materials, providing a cheap alternative for the development of bone transplantation surgery and bone tissue engineering. biomaterials.

Description

A New Method for Preparation of Heterogeneous Collagen Matrix

technical field

The invention relates to the technical field of biomaterials such as bone surgery repair materials and bone tissue engineering extracellular matrix materials. More precisely, the present invention relates to a method for processing heterogeneous cancellous bone and cortical bone by biological and chemical methods to obtain implantable heterogeneous bone repair materials and bone tissue engineering extracellular matrix materials.

Background technique

Due to trauma, infection, tumor and dysplasia, the bone loses some bone quality and forms a large gap, which is called bone defect. Due to the large gaps in most bone defects, it is difficult for osteoblasts to crawl through the gaps and normal healing cannot occur, eventually forming nonunions. In the United States, there are as many as 980,000 cases of bone grafting for various reasons each year, and in Germany, this figure is as high as 100,000 cases (Kessler S, Wohlfart U M, Ingnatius A, et al.Solventdehydrated bone transplants to bridge segmental bone defects: histomorphological and biomechanical investigations in an animal model. Arch Orthop TraumaSurg, 2001, 121: 472-475).

It has been clinically proven that autologous bone grafting has always been the best way to treat bone defects. There will be no immune reaction in the patient's body, and the cells and bioactive molecules in the transplanted bone can continue to survive at the recipient site and perform corresponding functions to promote the healing of the bone defect. Its size and shape are often limited. Bone allografts provide a wide variety of cortical or cancellous bone of various shapes and sizes. However, it is easy to cause immune reaction, the connection speed with the host bone is slow at the edge of the bone defect, and there is a risk of infectious viral diseases, and the cost of sample preparation, processing and storage is very high, and its application is greatly limited. Artificially synthesized degradable polymer materials, its composition, molecular weight, mechanical properties, degradation speed, etc. can be pre-designed and controlled, and it is also easy to shape and build a porous three-dimensional structure, but the degradation products of many such materials will make the body too acidic High, it is easy to induce an inflammatory response.

In order to overcome these shortcomings, people began to consider new bone substitute materials. The bone tissue engineering developed in the past ten years has opened up a new channel for the repair of large-scale bone defects. Among them, the development of matrix materials that can support cell adhesion, proliferation and differentiation, and release bioactive factors or seed cells is an important condition for the success of bone tissue engineering. Natural xenograft bone has the advantages of rich sources and low price, and is one of the potential sources of extracellular matrix materials for bone tissue engineering and bone defect graft materials. However, xenograft bone contains many organic components, and many acid-soluble proteins are immunogenic. After entering the human body, it can induce immune rejection and must be completely removed. Type I collagen in bone matrix has a milder immunogenicity due to its regular helical structure. 95% of the amino acids in the collagen molecule have the repeated sequence of Gly-X-Y and a triple helix structure, and about 5% of the amino acids at both ends do not have this sequence and cannot form a helical structure. These regions are related to the immunogenicity of collagen, except Removal of these regions can greatly reduce immunogenicity. Therefore, how to remove the antigenic substances of xenograft bone and retain its ability to induce osteogenesis is the primary problem to be solved in xenograft bone research.

There are many processing methods of heterogeneous bone at home and abroad. As early as 1937, Orell first applied deproteinized bone to clinical practice. This method is called Os Purum method. After removing soft tissue from heterogeneous bone, KOH is used to remove connective tissue, and then degreased with acetone. Deproteinize in saline solution. In the following decades, various deproteinized bones such as Kiel _bone , Oswestry bone _, and Anorganic bone appeared. other proteins. Its antigenicity can also be reduced or eliminated by methods such as decalcification, cryogenic freezing, boiling, and radiation irradiation. At present, most of the heterogeneous bones used in bone transplantation are calcined bone and inorganic bone treated by some physical and chemical methods. In order to avoid severe immune rejection, important organic components such as collagen are usually removed. The mechanical properties of bone are poor, thus limiting its use (Salama R. Xenogeneic bone grafting in humans. Clin Orthop, 1983, 174: 113-121). Luo Zhuojing et al. used a combination of centrifugal technology, ultrasonic technology and chemical treatment technology to process bovine cancellous bone, and obtained a large block-shaped xenograft bone graft material with no immunogenicity and certain mechanical support ability (Luo Zhuojing Jing and Hu Yunyu, Novel bulk defatted and antigen-removed xenograft material and its preparation method, Chinese Patent No. ZL97108538.2). Yang Zhiming and others used physical and chemical treatment methods to make bio-derived bone scaffolds from pig bones or heterogeneous bones, and introduced polymer materials or active ingredients to construct bio-derived tissue-engineered bones (Yang Zhiming and Qin Tingwu, Bio-derived tissue-engineered bone and its Preparation method, Chinese Patent No. ZL00132082.3). These processing methods can remove the antigenic substances of xenograft bone and retain its natural network structure, but the use of strong oxidizing reagents such as H ₂ O ₂ to remove the antigenic substances will greatly damage the organic components and will inevitably affect its mechanical strength , especially the cancellous bone material itself has very low mechanical strength. It has been reported in the literature that the mechanical strength of bovine cancellous bone is significantly reduced after being treated with H ₂ O ₂ , and its compressive ultimate strength is 5.3564MPa. , reduced by 92.8% in 96 hours, only 0.6896MPa (Luo Zhuojing, Hu Yunyu, Hou Demen, Zhao Ting. Correlation experiment between the mechanical strength of bovine cancellous bone and the time limit of antigen-removing treatment. Journal of Fourth Military Medical University, 1996; 17(6):434-436).

Contents of the invention

In order to overcome the shortcoming of existing method, the purpose of the present invention is to study a kind of preparation method of bone collagen matrix, while removing or reducing immunogenicity, keep the bone mineral structure and part organic collagen structure of natural bone as far as possible, to increase The overall mechanical strength of the grafted bone and retain proper osteogenic properties.

The invention mainly relates to a preparation method of a novel heterogeneous collagen matrix, and its main technical feature is that a series of biological and chemical methods are used to process the heterogeneous bones of cattle, pigs and the like. The technical route adopted is as follows:

(1) Source and processing of heterogeneous bone materials

Fresh bovine or pig leg bones were obtained from slaughterhouses. After fully removing soft tissue, cartilage and bone marrow, they were washed and placed in a -20°C refrigerator for 24 hours. Cutting with a low-speed saw to separate the metaphysis, the cancellous bone of the epiphysis is processed into a block, and its long axis is consistent with the alignment direction of the bone trabecula, which is used to prepare extracellular matrix materials for tissue engineering or bone defect filling materials; the middle part of the backbone Processed into cylinders or strips for the preparation of bone defect graft materials.

(2) Degreasing treatment

The processed bones were repeatedly washed with 40-60°C pressure water, then soaked in 0.01-5mol/L lye at room temperature for 24 hours, and then placed in a fat extractor containing a degreasing agent and degreased at 50-60°C for 48-48 hours. 72 hours, and every 24 hours, take out the bone block and centrifuge it at a speed of 4000 rpm for about 30 minutes in a centrifuge to remove cellular components, fat and other substances in the bone block. The degreasing agent can be 1:1-1:3 methanol/chloroform, Organic solvents such as ethyl formate, methyl acetate, isopropanol or acetone.

(3) Partial decalcification treatment

Bone fragments were partially decalcified in 0.1-5 mol/L acidic solution at room temperature for 5-10 minutes, and then dialyzed repeatedly with double distilled water until the pH value was 6 to remove residual acid.

(4) Decollagen

The bone pieces were put into the non-collagenous protein mixture containing pH 7.4, the mixture was composed of nitrogen-containing basic organic matter, Tris buffer and salt, and placed in a refrigerator at 4°C to remove the non-collagenous protein, and the time was 24h, urea or guanidine hydrochloride can be used as non-collagen removal agent.

(5) Protease digestion

Digest the bone block with 0.05-0.5% protease, add a small amount of thiolase inhibitor for preservation, use buffer solution to control the pH of the solution to 7.8-8.0, and the bone-to-liquid ratio is 1:1-1:6, and digest at 37°C for 48 hours . Then soak in 4mol/L NaCl for 12-24 hours to remove dissolved non-collagen and part of collagen.

After protease digestion, the non-collagen protein in the bone block can be further removed, and the remaining collagen in the bone block can be stripped of telopeptide and become antelopeptide collagen, eliminating the immunogenicity of collagen.

(6) Disinfection and packaging

The bone fragments were rinsed repeatedly with distilled water, dried in vacuum at 50°C for 24 hours, packaged with three layers of polyethylene film, sterilized with ethylene oxide for 2 hours, and stored in a refrigerator at 4°C for later use.

Compared with the prior art, the present invention has the following advantages:

1. The cancellous bone material prepared by biological and chemical methods retains the bone mineral components and most of the collagen of the original bone. After the collagen components are digested by protease, the telopeptides that can cause immunogenicity are excised, so the immune The originality is greatly reduced, and the existence of collagen greatly improves the mechanical strength of the material. At the same time, the material has good porosity and pore connectivity, which provides good conditions for the crawling replacement of osteoblasts and other seed cells, and is suitable for bone Extracellular matrix materials for tissue engineering.

2. The cortical bone material prepared by biological and chemical methods, in addition to low immunogenicity and certain porosity, also has excellent mechanical properties, such as the bovine cortical bone material prepared according to this method, its tensile Young's The modulus can reach about 6000Mpa, and the maximum stress can reach about 110Mpa, which is relatively close to the mechanical properties of human long bones. After implantation in the body, it eliminates the "stress" caused by the difference in mechanical properties between other bone graft materials such as metals and human bones. occlusion" effect. Suitable for bone defect grafting material.

3. The materials prepared by the present invention have various shapes, which can be cut into various shapes according to clinical needs, and can also be further treated with antibiotics or other drugs or introduced active peptides, Growth factors, degradable polymers, etc. are modified.

The significance of the invention lies in the combination of biological and chemical methods to develop a new type of heterogeneous collagen matrix, to provide cheap biological materials for the development of bone transplantation surgery and bone tissue engineering, and to meet the needs of the market.

Description of drawings

Fig. 1 is the schematic diagram of the processing process of the heterogeneous cortical bone collagen matrix material involved in the present invention, 1 is the fresh pig or beef leg bone obtained from the slaughterhouse, which is frozen after removing soft tissue and other components; 2 is the backbone part of the interception; 3 is Processed material.

Figure 2 is a photo of the appearance of cylindrical and strip materials prepared by the present invention.

Detailed ways

Embodiment 1 A kind of preparation method of bovine cortical bone collagen matrix material

The cortical bone material of the present invention should be taken from the middle of the diaphysis (see accompanying drawing 1). The processing process should be cut with a low-speed saw after freezing to avoid damage to collagen due to high temperature. The processed bone was repeatedly washed with 50°C pressure water, then soaked in 0.1mol/L NaOH solution at room temperature for 24 hours, and the bone was placed in a Soxhlet extractor containing ethyl formate at 60°C for 72 hours, and the bone was degreased every 24 hours. The block was taken out and centrifuged at a speed of 4000 rpm for about 30 min in a centrifuge to remove foreign matter in the bone block; the bone block was partially decalcified in 0.5mol/L HCl solution at room temperature for 10 min, and then dialyzed repeatedly with double distilled water until the pH value was 6. To remove the residual acid solution; put the bone into a solution containing 4mol/L HCl guanidine, 50mmol/L Tris-HCl and 0.5Mmol/L CaCl ₂ , with a pH of 7.4, and place it in a refrigerator at 4°C to remove non-toxic acid. Collagen, the time is 24h; the bone block is digested with 0.1% trypsin, a small amount of sodium azide solution is added for preservation, and the buffer solution of Na ₂ HPO ₄ and NaH ₂ PO ₄ is used to control the pH of the solution to 7.8, and the bone liquid ratio is 1:3, digested at 37°C for 48h. Soak in 4mol/L NaCl for 12-24 hours to remove the dissolved non-collagen and some collagen; the bone block was washed repeatedly with double-distilled water, dried in vacuum at 50°C for 24 hours, encapsulated with three layers of polyethylene film, ethylene oxide Sterilize for 2 hours and store in a 4°C refrigerator for later use.

Embodiment 2 A kind of preparation method of bovine cancellous bone collagen matrix material

The cancellous bone material of the present invention should be taken from the epiphysis, and its long axis should be consistent with the arrangement direction of bone trabeculae. The processing process should be cut into blocks with a low-speed saw after freezing to avoid high temperature damage to collagen. The processed bone was repeatedly washed with 50°C pressure water, then soaked in 0.05mol/L NaOH solution at room temperature for 24 hours, and placed in a Soxhlet extractor containing a 1:1 methanol/chloroform mixed solvent at 55-60°C. Degrease for 72 hours, take out the bone pieces every 24 hours, and centrifuge them at a speed of 4000 rpm for about 30 minutes in a centrifuge; partially decalcify the bone pieces in 0.1mol/L HCl solution at room temperature for 10 minutes, and then dialyze them repeatedly with double distilled water to reach the pH value 6, to remove the residual acid; put the bone into a solution containing 6mol/L urea, and place it in a refrigerator at 4°C to remove non-collagen for 24 hours; digest the bone with 0.1% trypsin, Add sodium azide solution for preservation, use a buffer solution of Na ₂ HPO ₄ and NaH ₂ PO ₄ to control the pH of the solution to 7.8, and the ratio of bone to fluid to 1:4, and digest at 37°C for 36 hours. Then soak in 4M NaCl solution for 12 hours; the bone fragments were rinsed repeatedly with double distilled water, dried in vacuum at 50°C for 24 hours, packaged with three layers of polyethylene film, sterilized with ethylene oxide for 2 hours, and stored in a refrigerator at 4°C for later use.

The appearance of the cylindrical and strip materials prepared by the present invention is shown in the photo shown in FIG. 2 .

Claims (4)

1. the new method of xenogenesis bone collagen matrix preparation, it is characterized in that: adopt a series of biological and chemical methods that xenogenesis bones such as cattle, pig are handled, process is as follows:

(1) source of xenogenesis bone material and processing

Choose the thigh bone of fresh cattle or pig, after fully removing soft tissue, cartilage and bone marrow, clean, freezing, the cutting and separating metaphysis is processed into bulk with epiphysis end spongy bone, its major axis and bone trabecular orientation are consistent, and cylinder or bar shaped are processed in key middle part;

(2) ungrease treatment

The bone piece that processes washes repeatedly with 40～60 ℃ of press waters, use 0.01～5mol/L alkali liquor soaking at room temperature 24h then, use degreasing agent in 50～60 ℃ of following defat 48～72h the bone piece, every 24h takes out the bone piece on the centrifuge with 4000 rev/mins speed centrifugal about 30 minutes, removes materials such as bone piece inner cell composition and fat;

(3) partly decalcifying is handled

The room temperature lower part decalcification 5～10 minutes in 0.1～5mol/L acid solution of bone piece, reuse distilled water dialyse repeatedly to pH value be 6, to remove residual acid solution;

(4) take off noncollagen protein

The bone piece puts into that to contain pH value be the mixed liquor that 7.4 noncollagen protein removes agent, this mixed liquor consist of nitrogenous base organic matter, Tris buffer and salt, place 4 ℃ refrigerator to remove noncollagen protein, the time is 24h;

(5) protease digestion

The bone piece is with 0.05～0.5% protease digestion, it is anticorrosion to add a small amount of sulfydryl enzyme inhibitor, pH with buffer solution control solution is 7.8～8.0, the bone liquid ratio is 1: 1～1: 6, under 37 ℃ of conditions, digest 48h, reuse 4mol/LNaCl soaks 12～24h, to remove dissolved noncollagen protein and part collagen protein;

(6) sterilization and packing

2, the new method of xenogenesis bone collagen matrix preparation according to claim 1, it is characterized in that: degreasing agent adopts 1: 1～1: 3 methanol/chloroform, Ethyl formate, methyl acetate, isopropyl alcohol or acetone.

3, the new method of xenogenesis bone collagen matrix preparation according to claim 1 is characterized in that: adopt carbamide or guanidine hydrochloride to remove agent as noncollagen protein.

4. according to the xenogenesis bone collagen matrix of claim 1, the preparation of 2 or 3 described methods, it is characterized in that: the immunogenicity that reduces or eliminate xenogenesis spongy bone such as cattle, pig and cortical bone, collagen protein becomes the ratio of branch to be about 1: 2 with the bone ore deposit, collagen protein has wherein excised the end peptide through Protease Treatment, the material of gained is block, the cylindric or strip of white, the mesh structural porous structure that has kept nature bone, adopt the young's modulus in tension of the collagen stroma material of cattle cortical bone preparation to reach about 6000Mpa, maximum stress reaches about 110Mpa.

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