CN1061812C - Method for preparing skin grafts - Google Patents

Abstract

Tissue obtained from a human or animal donor. The tissue may be fixed by a fixation method. The cell components that cause rejection can be removed from the tissue by chemical treatment, thereby leaving the extracellular matrix (ECM) with its original shape, biological structure, ultrastructure, orientation and durability. Viable cells are seeded on the resulting ECM as a cell frame to form an animate graft for transplantation or allograft.

Description

Translated fromChinese

制备皮肤移植体的方法Method for preparing skin grafts

本发明涉及从人或动物组织所得到的组织制备生物细胞外基质的方法。The present invention relates to a method for the preparation of biological extracellular matrices from tissues obtained from human or animal tissues.

器官或组织的损伤或缺失是人类健康中最经常发生、最具灾难性且花费最昂贵的问题之一。因此，组织工程学作为一个新的领域便应运而生，其是利用生物学和工程学原理来开发损伤组织的功能替代物。Damage or loss of organs or tissues is one of the most frequent, catastrophic and costly problems in human health. As a result, tissue engineering has emerged as a new field that utilizes principles of biology and engineering to develop functional substitutes for damaged tissue.

组织工程是一个应用工程和生命科学原理研究正常和病理状态哺乳组织间结构一功能关系并进而研制能恢复，保持或改善组织功能的领域。它包括将活细胞与天然或人工的细胞外基质一起用于研制用于恢复或替代组织功能的移植体。Tissue engineering is a field that applies the principles of engineering and life sciences to study the structure-function relationship between normal and pathological mammalian tissues, and then to develop tissues that can restore, maintain or improve tissue functions. It involves the use of living cells together with natural or artificial extracellular matrices to develop grafts that restore or replace tissue function.

对于制备新的组织替代物所采取的三种基本构思之一是将细胞置于基质上或之中。该基质来自天然材料或合成的生物降解的聚合物纤维框架，其被作为ECM。One of the three basic concepts adopted for the preparation of new tissue substitutes is to place cells on or in a matrix. The matrix is derived from a natural material or a synthetic biodegradable polymer fiber framework, which serves as the ECM.

尽管已开发出许多人工或合成的移植物，但这些组织替代物仍不能恰当地取代天然高度复杂的结构。今天在组织移植中所面临的挑战是克服机体的免疫系统并尽力改善移植物的功能。尽管在本领域已取得了很大进展，但目前的组织移植仍存有许多问题，这包括炎症，降解，瘢痕，感染，钙化，咬合和排斥。因此，目前广泛深入的医学生物材料研究是面向改善移植组织移植物的工程，但理想的人工移植物还未研制出来。Although many artificial or synthetic grafts have been developed, these tissue substitutes still cannot adequately replace natural highly complex structures. The challenge in tissue transplantation today is to overcome the body's immune system and try to improve the function of the graft. Although great progress has been made in this field, there are still many problems with current tissue transplantation, including inflammation, degradation, scarring, infection, calcification, occlusion and rejection. Therefore, the current extensive and in-depth research on medical biomaterials is aimed at improving the engineering of transplanted tissue grafts, but ideal artificial grafts have not yet been developed.

美国专利4，801，299记载了一种通过用洗涤剂除去组织中细胞成份从而得到供移植用的细胞外基质的方法。但如该组织预先用固定剂固定的话，则该洗涤剂不能用于该组织的制备。U.S. Patent No. 4,801,299 describes a method for obtaining an extracellular matrix for transplantation by removing cellular components of tissue with detergents. However, if the tissue is previously fixed with a fixative, the detergent cannot be used in the preparation of the tissue.

本发明人在研究中出人意料地发现通过将来自人或动物供体的组织先用固定液固定，然后用作为消化液的碱性溶液除去固定组织中细胞得到细胞外基质，该细胞外基质具有优良的组织完整性和作为移植物所需具备的其它性质，并使供体的来源更广，从而为供体提供了广泛来源。其中所述动物可以是猪、牛或羊。本发明基于上述发现得以完成。The present inventors unexpectedly found in the research that the extracellular matrix obtained by fixing the tissues from human or animal donors with a fixative, and then using an alkaline solution as a digestive solution to remove cells in the fixed tissues has excellent The integrity of the tissue and other properties required as a graft, and the source of the donor is wider, thus providing a wide range of sources for the donor. Wherein said animal can be pig, cow or sheep. The present invention has been accomplished based on the above findings.

本发明的目的是提供一种加工来自人或动物的生物组织的方法，其是通过化学处理来制备脱细胞化的以胶原蛋白为主的ECM，所得到的生物组织用于人类或动物的移植。本发明提供了制备改进的ECM的有效方法，该ECM一般具有该组织的原有形状且同时保留了所需要的生物结构。该组织可由已知方法得到，该组织可是宿主的或从尸体或供体得到的。该组织在固定后进行脱细胞。The object of the present invention is to provide a method for processing biological tissues from humans or animals, which is to prepare decellularized collagen-based ECM by chemical treatment, and the obtained biological tissues are used for human or animal transplantation . The present invention provides an efficient method of producing an improved ECM that generally has the original shape of the tissue while retaining the desired biological structure. The tissue may be obtained by known methods, and the tissue may be of the host or obtained from a cadaver or donor. The tissue was decellularized after fixation.

得到该组织后，对其进行固定，然后进行脱细胞，其中脱细胞用的消化液选自碱金属氢氧化物或其碳酸盐或碳酸氢盐，氢氧化铵，碱金属醇化物，有机胺或它们的混合物。可采用氧氢氧化钠或氢氧化钾溶液。然后将所得到的ECM洗涤，孵育并保存。After the tissue is obtained, it is fixed and then decellularized, wherein the digestive solution for decellularization is selected from alkali metal hydroxide or its carbonate or bicarbonate, ammonium hydroxide, alkali metal alcoholate, organic amine or their mixtures. Sodium oxyhydroxide or potassium hydroxide solution can be used. The resulting ECM was then washed, incubated and stored.

在移植前，作为细胞框架的ECM可用来自宿主(自身细胞)或其它人的细胞种植。该机体组织的EGM也可作为基质材料用于组织替代。Before transplantation, the ECM as a cellular framework can be seeded with cells from the host (own cells) or from other people. The EGM of body tissue can also be used as a matrix material for tissue replacement.

本发明包括通过固定处理，除去细胞，灭菌和细胞重建步骤获得生物组织，从而提供用于移植的生物替代组织。The present invention involves obtaining biological tissues through the steps of fixation, decellularization, sterilization and cellular reconstitution, thereby providing biological replacement tissues for transplantation.

在优选实施方案中，所获得的组织优选是与随后进行植入的移植物相同的组织。例如皮肤用于代替皮肤，血管用于代替血管，软骨用于代替软骨等。In a preferred embodiment, the tissue obtained is preferably the same tissue as the graft that is subsequently implanted. For example, skin is used instead of skin, blood vessels are used instead of blood vessels, cartilage is used instead of cartilage, etc.

用人的尸体或动物供体得到的生物组织具有1cm³体积或更大，其获取后马上置于带有固定剂的固定液中，如戊二醛或甲醛。该固定液可包含于缓冲液如磷酸钠缓冲液中(PBS)的2％甲醛和1.5％戊二醛。该组织在固定液中最少保留时间约2小时，或直到该组织完全被固定。组织固定后，用适宜材料对其洗涤几次，如用蒸馏水。Biological tissues obtained from human cadavers or animal donors having a volume of 1 cm³ or more are immediately placed in a fixative solution with a fixative such as glutaraldehyde or formaldehyde. The fixative may comprise 2% formaldehyde and 1.5% glutaraldehyde in a buffer such as sodium phosphate buffered saline (PBS). The tissue is left in the fixative for a minimum of about 2 hours, or until the tissue is completely fixed. After the tissue is fixed, it is washed several times with a suitable material, such as distilled water.

在优选实施方案中，该组织然后被放在NaOH或KOH溶液或任何其它碱性溶液中以进行该组织的孵育，从而除去固定组织中细胞。该组织于室温在该溶液中的保留时间为约12小时-5天。溶液优选含1.5-2.0N NaOH或KOH的碱性溶液。浓度范围为0.1N-5N，温度范围为约4℃-60℃，这取决于所加工供体组织的类型和获得该脱细胞化ECM所需的时间长度。更高浓度，更高的温度或更长的消化将损害该结果。实践中，在室温用NaOH或KOH处理更长时间会导致弹性纤维的消化。所得ECM可通过显微镜和扫描电子显微镜观察到。In a preferred embodiment, the tissue is then placed in NaOH or KOH solution or any other alkaline solution for incubation of the tissue to remove cells from the fixed tissue. The retention time of the tissue in the solution at room temperature is about 12 hours to 5 days. The solution is preferably an alkaline solution containing 1.5-2.0N NaOH or KOH. Concentrations range from 0.1 N to 5 N and temperatures range from about 4°C to 60°C, depending on the type of donor tissue being processed and the length of time required to obtain the decellularized ECM. Higher concentrations, higher temperatures or longer digestions will compromise this result. In practice, longer treatment with NaOH or KOH at room temperature leads to digestion of elastic fibers. The resulting ECM can be visualized by microscopy and scanning electron microscopy.

组织脱细胞化后，进行组织的洗涤。洗涤组织的溶液可以是蒸馏水和缓冲液，如磷酸钠。After the tissue is decellularized, the tissue is washed. The solution for washing the tissue can be distilled water and a buffer such as sodium phosphate.

在该步中，可变化洗涤溶液，优选为约每30分钟，且持续洗涤直到ECM呈半透明状。洗涤ECM后，将ECM于氨基酸如天门冬氨酸，谷氨酸或甘氨酸溶液中孵育以消除残余的游离固定剂并降低pH。具体的氨基酸可是0.5％天门冬氨酸或0.1M甘氨酸溶液。缓冲液中ECM孵育最好将洗涤液pH带到约7.4。加工后，可以在移植前不损害ECM结构的任何方式放置并贮存该脱细胞的ECM。During this step, the wash solution may be changed, preferably about every 30 minutes, and the wash continued until the ECM is translucent. After washing the ECM, the ECM is incubated in a solution of amino acids such as aspartic acid, glutamic acid or glycine to eliminate residual free fixative and lower the pH. Specific amino acids may be 0.5% aspartic acid or 0.1M glycine solution. Incubation with ECM in buffer best brings the pH of the wash solution to about 7.4. After processing, the decellularized ECM can be placed and stored in any manner that does not damage the structure of the ECM prior to transplantation.

该ECM结构提供了用于细胞粘着，生长和分化的适宜有效的生物底物。该ECM可在移植前通过细胞培养技术在其上种植活细胞作为细胞框架而成为替代组织，或通过各种医学步骤直接作为体内或体外植入的模板。This ECM structure provides a suitable and efficient biological substrate for cell adhesion, growth and differentiation. The ECM can be used as a replacement tissue by planting living cells on it as a cell framework through cell culture technology before transplantation, or directly as a template for in vivo or in vitro implantation through various medical procedures.

本发明提供了一种组织产品，其包括良好防腐的ECM。该ECM调节与其接触的细胞行为上起着活性的并且综合的作用。The present invention provides a tissue product comprising well preserved ECM. The ECM plays an active and integrative role in modulating the behavior of cells in contact with it.

本发明就是制备一个完好保留的细胞外基质，目前大量的研究已经证明了细胞外基质对与其相接触的细胞的各种行为起着非常重要的调节作用，围绕在细胞外的基质是一种相对很稳定的结构物质，但是只认为围绕在细胞外的基质对于细胞仅仅起着惰性的支持和框架的作用，这种看法已成为了过去，构成细胞外基质的每一种成份对于与它相接触的细胞的生长，分化都有着特殊的功能，由它们结合而构成的具有三维立体空间的细胞外基质，对于细胞的生长、分化、代谢、调节都起着不可替代的作用，反过来由于这些细胞外基质是由细胞所产生，所以细胞对于围绕在其周围的细胞外基质的形成起着这样或那样的作用和影响，在细胞的表面有着与ECM相连结的特殊的受体，这样细胞基质的组成成份和空间结构对于细胞的代谢、形态生长分化有着密切相关的作用。The present invention is to prepare a well-retained extracellular matrix. At present, a large number of studies have proved that the extracellular matrix plays a very important role in regulating the various behaviors of cells in contact with it. The matrix surrounding the extracellular matrix is a relatively It is a very stable structural substance, but it is only considered that the matrix surrounding the extracellular matrix only acts as an inert support and framework for the cells. This view has become a thing of the past. The growth and differentiation of human cells have special functions. The three-dimensional extracellular matrix formed by their combination plays an irreplaceable role in the growth, differentiation, metabolism and regulation of cells. In turn, these cells The extracellular matrix is produced by the cells, so the cells play one or another role and influence on the formation of the extracellular matrix around them. There are special receptors connected to the ECM on the surface of the cells, so that the cell matrix Composition and spatial structure are closely related to cell metabolism, morphological growth and differentiation.

因此，对于通过该技术特殊处理过的组织，由于所有能够被宿主识别出对宿主表达组织免疫抗原性的细胞物质被去除，而完整保留下来的ECM其形态，组织结构，超微结构，定位和耐受性等都与原有组织的基质一样。Therefore, for tissues specially treated by this technique, since all cellular substances that can be recognized by the host and express tissue immune antigenicity to the host are removed, the morphology, tissue structure, ultrastructure, location and Tolerance and so on are the same as the matrix of the original tissue.

当与目前市场的移植物比较时，本发明所制备的ECM具有难以模仿的天然生物构造并在应用中具有优越性能。该ECM与由ECM成份如胶原或纤维蛋白涂覆的化学聚合物底物组成的移植体比较，其绝对无毒，不会引起不良过敏活性且可促进细胞繁殖。当本发明与美国专利4，801，299的ECM和方法比较时，本发明通过更简易，低花费的方法在更短的时间内制备ECM，同时保留了所需的天然结构，且供体的来源更广泛，方便。The prepared ECM of the present invention has an inimitable natural biological structure and superior performance in application when compared with the grafts currently on the market. The ECM is absolutely non-toxic, does not cause adverse allergic activity and promotes cell proliferation in contrast to implants composed of ECM components such as collagen or fibrin-coated chemical polymer substrates. When the present invention is compared with the ECM and method of U.S. Patent No. 4,801,299, the present invention prepares ECM in a shorter time by a simpler, less expensive method, while retaining the desired native structure, and the donor's The sources are wider and more convenient.

Claims (15)

1. one kind by having removed the cell composition, keeps extracellular matrix and prepare the method for dermatoplasty body, and it may further comprise the steps:

A. obtain skin histology from the human or animal,

B. the skin of gained is fixed with fixative,

C. will be selected from alkali metal hydroxide or its carbonate or bicarbonate, ammonium hydroxide, alkali alcoholate, the alkaline solution of organic amine or their mixture is used for described fixing skin histology, removing the cell composition in the skin histology,

D. with buffer solution the Skin Cell epimatrix is washed.Wherein the Skin Cell epimatrix is meant the skin histology of being handled by above-mentioned steps c except the cell composition,

E. the Skin Cell epimatrix that is obtained is hatched in hatching solution so that eliminate residual fixer composition and reduce the pH value of Skin Cell epimatrix.

2. the described method of claim 1, Incubating Solution wherein is an asparatate.

3. the described method of claim 1, Incubating Solution wherein is a glutamic acid.

4. the described method of claim 1, wherein incubating clothing liquid is glycine.

5. the described method of claim 1, alkaline solution wherein is a sodium hydroxide solution.

6. the described method of claim 2, wherein alkaline solution is a sodium hydroxide solution.

7. the described method of claim 3, wherein alkaline solution is a sodium hydroxide solution.

8. the described method of claim 3, wherein alkaline solution is a sodium hydroxide solution.

9. the described method of claim 1, alkaline solution wherein is a potassium hydroxide solution.

10. the described method of claim 2, wherein alkaline solution is a potassium hydroxide solution.

11. the described method of claim 3, wherein alkaline solution is a potassium hydroxide solution.

12. the described method of claim 4, wherein alkaline solution is a potassium hydroxide solution.

13. the arbitrary desired method of claim 1-12, the template that wherein prepared ECM can be used as the value of moving body is directly used in human or animal's transplanting.

14. the arbitrary desired method of claim 1-12, wherein prepared ECM can be gone up from host or other human or animal's living cells by plantation and the transplant of transplanting as the human or animal.

15. the desired method of claim 1, wherein said animal can be pig, ox, sheep.