Preparation method of acellular matrix for removing alpha-galactosyl antigen
阅读说明：本技术 一种去α-半乳糖基抗原脱细胞基质的制备方法 (Preparation method of acellular matrix for removing alpha-galactosyl antigen ) 是由 张小宁 肖成 夏莹 于 2020-06-01 设计创作，主要内容包括：本发明所涉及的是一种去α-半乳糖基抗原的脱细胞基质制备方法,属于再生医学生物材料制备和应用领域。在脱去引起排斥反应的抗原细胞成分的同时去除α-半乳糖基抗原(Strattice),提高了脱细胞基质的生物相容性,降低其抗原性,提高其促进组织再生能力,本项发明的核心是去除引起免疫排斥反应的物质而保留细胞外结构基质组成和天然结构,应用该方法制备的脱细胞基质生物材料,作为组织修复和再生的全新材料,是开发再生医学产品的理想而优越的生物医学材料。(The invention relates to a preparation method of an acellular matrix for removing alpha-galactosyl antigen, belonging to the field of preparation and application of regenerative medicine biomaterials. The core of the invention is to remove the substance causing immune rejection reaction and keep the composition and natural structure of extracellular structural matrix, and the acellular matrix biomaterial prepared by applying the method is used as a brand new material for tissue repair and regeneration, and is an ideal and superior biomedical material for developing regenerative medical products.)
1. The invention relates to a method for preparing an acellular matrix for removing alpha-galactosyl antigen.
2. The acellular matrix of claim 1 wherein the source of the starting material is allogeneic or xenogeneic.
3. The raw material of claim 2, wherein said raw material is selected from the group consisting of allogeneic or xenogeneic dermis, small intestinal submucosa, urinary bladder submucosa, amniotic membrane, placenta, blood vessels, heart valves, omentum majus, cartilage, cornea, sclera, iris.
4. The use of the acellular matrix of claim 1 in regenerative medicine biomaterials.
5. The process of claim 1, wherein: treating said starting material of claim 3 with a concentration of sodium hydroxide, and passing the treated starting material through a low concentration of an iodine-containing salt, NaH, in a phosphate buffer2PO4And sequentially treating the boron-containing salt solution to obtain the acellular matrix material.
6. The iodine-containing salts as claimed in claim 5, wherein the iodine-containing salts include potassium and sodium salts of hypoiodic acid, iodic acid, n-periodic acid, and meta-periodic acid.
7. The boron-containing salt of claim 5, wherein the boron-containing salt is selected from the group consisting of borate, borohydride, potassium borohydride, and sodium borohydride.
8. The phosphate buffer of claim 5, wherein the pH of the phosphate buffer is 5.5 to 9.5.
The concentration range of Tris is 0.01-1M, and the concentration range of Titon-100 is 0.1% -5%.
1. Field of the invention
The invention relates to a preparation method of an acellular matrix for removing alpha-galactosyl antigen, belonging to the field of preparation and application of regenerative medicine biomaterials.
The invention has the advantages that: the special technology of removing alpha-galactosyl antigen is used to effectively and completely remove tissue rejection reaction factors, so that the matrix has good mechanical strength and biocompatibility and excellent tissue regeneration capability.
The proper tissue of the pig with high tissue structure and immunological similarity to human is selected as the material source for preparing the acellular matrix, the antigen cell component causing rejection reaction is removed, and the alpha-galactosyl antigen (Strattece) is removed at the same time, so that the biocompatibility of the acellular matrix is improved, the antigenicity is reduced, the tissue regeneration promoting capability is improved, the allogeneic acellular matrix biomaterial is replaced, and the acellular matrix biomaterial with multiple purposes and dosage forms can be prepared. The acellular matrix with excellent performance provides a new good way for solving the problem of clinical tissue repair.
The core of the invention is to remove the material causing immunological rejection reaction and keep the extracellular structure matrix composition and natural structure, and the acellular matrix biomaterial prepared by the method is used as a brand new material for tissue repair and regeneration, and is an ideal biomedical material for developing regenerative medical products. The excellent performance of the acellular matrix biomaterial prepared by the method enables the acellular matrix biomaterial to be in an advanced level in the field of tissue repair and regeneration medical materials.
2. Background of the invention
The research and application of medical biomaterials are divided into natural materials, synthetic high molecular materials, inorganic materials and composite materials, wherein the natural materials are most widely applied, and comprise cotton fibers, chitin/chitosan, alginate, gelatin, collagen, animal (xenogenic) tissues, allogeneic/xenogenic tissues and the like, and are used for tissue transplantation, tissue replacement and tissue repair of human diseases and injuries.
The current international research on the use of human autologous/allogeneic and animal (xenogenic) tissues for decellularized regenerative medical biomaterials involves:
acellular matrix product: mainly uses cells and tissue materials with different sources and different cell removal process technical methods in the manufacturing process.
Utilization of various animal and human derived products: animal derived products (transplants) are obtained from different donor animals (e.g. pigs, sheep, horses or cattle), and from different age stages by harvesting fresh living tissue (e.g. dermis, small intestine submucosa, pericardium, etc.).
Animal-derived collagen matrix-derived product: such asA composite product made of Collagen, polysaccharide and synthetic materials of Biomatrix Collagen wound dressing (Synovis): such as
Human products, donated human cadaver skin (transplantation), use various processes to remove cells and pathogens: such asLifecell；
Foreign countries have earlier application of Acellular tissue Matrix biomaterials (ADM) in clinic, and the Acellular tissue Matrix biomaterials are researched from 1992 to burn patients, used in periodontal surgery and plastic repair surgery in 1994, and are widely applied to the fields of burns, oral cavity, ophthalmology, otolaryngology, abdominal surgery and the like. Imported products of the same kind are introduced from 2000 years in China.
Through the technical progress in recent years, the heterogenous acellular biomaterial becomes a mainstream product for tissue regeneration and repair, antigen cell components causing rejection reaction are removed, an extracellular matrix and a three-dimensional space frame structure are reserved, the antigenicity is low, the biocompatibility is good, no rejection reaction exists, the xenogenic acellular biomaterial is sterile, non-toxic and non-irritant, the mechanical strength of the reconstructed tissue is high, the elasticity is good, the tissue is not deformed, the material can be made into any clinically required dosage form and specification without limitation. The research direction of the regenerative biomedical materials is to improve the defects of the existing materials, enhance various performances of the existing materials, develop a proper three-dimensional porous structure, provide nutrition and metabolic environment for the growth and the propagation of cells, regulate the growth and the arrangement of the cells, load various medicines and growth factors on the materials, effectively control the release of the medicines, simultaneously achieve the healing and the treatment effects, and finally degrade the tissue engineering biomaterial to achieve the purpose of permanent replacement of the tissue by a modification or composite method.
In China, a biological type dura mater (spinal) membrane patch (meningeal patch for short), a thoracic general surgery repairing membrane and a sterile biological wound protecting membrane are prepared by using a decellularized bovine pericardium, are used for repairing meningeal defects and cardiac capsule defects and the like, and are approved by the national food and drug administration for clinical use. The heterogenic acellular dermal matrix dressing prepared by using the piglet dermis is specially used for over 70 percent of key burn (wound) departments in China and is approved by the national food and drug administration for clinical application. The fourth military medical university has made a great progress in preparing artificial cornea substitutes by taking porcine corneas as raw materials and utilizing a complex comprehensive technology of cell removal and antigen removal, has completed clinical research and enters the clinical application stage.
Zhongshan ophthalmological center of Zhongshan university, a method for preparing acellular matrix (Chinese patent invention, publication No. CN101274106A), discloses a method for preparing acellular matrix by using phospholipase, which has the advantage of reducing the damage to cell structure, but the product has enzyme residue.
American Isikang company 'a method for preparing acellular matrix' (Chinese patent application No. CN102170920A) discloses a method for preparing acellular matrix, which utilizes incision enzyme and physical conditions to remove cell components and has the defect of damaging the natural structure of cells.
In China tissue engineering research and clinical rehabilitation, volume 14, stage 38, China's tissue engineering research and clinical rehabilitation, Shenmanru, Yuanming, Zhang Yongzhen, and other "safety performance of extracellular matrix materials", a comprehensive analysis discussion on feasibility, safety and application of acellular matrix is carried out, and it is considered that the natural acellular biological scaffold material is obtained by mainly utilizing homologous or heterologous organs/tissues through acellular and antigen removal treatment. The acellular matrix material mainly comprises collagen and other antigenic proteins, and the interaction between the implanted body and the implanted product is affected by the method of acellular treatment, residual cell components and the like, so that it is difficult to completely remove cell membranes and nuclear materials. The safety evaluation of the acellular matrix also included residual DNA of scaffold after decellularization. More extracellular matrix materials are porcine tissue materials, contain a small amount of galactose antigenic determinants, and the galactose antigens existing on the surface of vascular endothelial cells are the main reasons of the rejection of the xenogeneic organ transplantation. Galactose antigens present in the porcine extracellular matrix were found to cause elevated levels of antibodies in serum, indicating that such extracellular matrix materials are immunogenic.
In conclusion, in the preparation process of the acellular matrix, the alpha-galactosyl antigen is effectively removed, the biocompatibility of the acellular matrix is improved, and the antigenicity of the acellular matrix is reduced, which is a problem to be solved for preparing high-performance acellular matrix materials.
3. Summary of the invention
At present, most research institutions and production enterprises adopt a technical method combining an enzymatic method and a chemical method to prepare an acellular matrix, and the acellular matrix is characterized by short tissue treatment time and large capacity, and has the problems that metabolic substances generated after enzyme treatment are difficult to remove, the purification to a natural tissue component environment is difficult, the DNA removal rate is low, alpha-galactosyl which can also generate antigen tissue immune reaction is not removed, and tissue rejection reaction occurs in products.
Through the intensive research on the acellular biotechnology, the invention adopts the special acellular biotechnology of the acellular alpha-galactosyl antigen to carry out the tissue acellular preparation of the acellular matrix.
By using the special biological technology for removing the alpha-galactosyl antigen cells, an enzyme treatment method is not adopted, the generated enzyme metabolites are effectively avoided, the alpha-galactosyl for generating antigen reaction in the tissues is removed, and the DNA removal rate is the highest in the existing various cell removal methods (the verification experiment of DNA determination is carried out), so that the tissue immune reaction can be avoided, and the better degradation and absorption can be realized.
The basic technical route is as follows: collecting specific tissues of fresh and alive pigs qualified for quarantine → pretreatment of tissues (removing tissue impurities and removing blood interstitial fluid) → degreasing by a proprietary method → removing cell DNA (proprietary technology) → removing virus (chemical virus inactivation) → removing alpha-galactosyl antigen (proprietary technology) → preparing a required dosage form and specification according to clinical application.
4. Description of the drawings
FIG. 1 is a photograph of a prepared acellular matrix slice, which is stained by HE (high intensity electrophoresis) by using pig skin which is a special technology for removing alpha-galactosyl antigen as a raw material, wherein the material is in a loose net structure, cells are not seen, a large amount of light red collagen fibers still keep a porous structure, and the collagen fibers are not damaged in the acellular process, so that the acellular effect is good. FIG. 2 is a graph of galactose standard showing a linear relationship is satisfactory.
5. Detailed description of the preferred embodiments
The preparation method comprises removing impurities such as attachments, blood, fat, etc. from tissues of pig such as fresh dermis, small intestine submucosa, bladder submucosa, blood vessel, heart valve, omentum majorana, cornea, etc., treating with sodium hydroxide of certain concentration, and treating with low-concentration iodized salt, NaH and phosphate buffer solution2PO4And sequentially treating the boron-containing salt solution to finally obtain the acellular matrix biomedical material, and through related detection, the results of removing DNA and α -galactosyl antigen are good.
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