CN1473551A - Artificial tissue engineeing biological cornea - Google Patents
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- CN1473551A CN1473551A CNA031401139A CN03140113A CN1473551A CN 1473551 A CN1473551 A CN 1473551A CN A031401139 A CNA031401139 A CN A031401139A CN 03140113 A CN03140113 A CN 03140113A CN 1473551 A CN1473551 A CN 1473551A
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Abstract
The present invention discloses a kind of artificial tissue engineering biological cornea. The present invention utilizes homogeneous and xenogenous or heterogeneous cornea substrate as carrier, which may be preprocessed, and plants and cultures cells on it to constitute engineering biological cornea. The cornea constituted with the carrier has very low immunogenicity, unchanged thickness and diopter after transplantation, grown autogenous nerve, mostly restored cornea aesthesia, firmly adhered epithelium and normal connection between the cells and between the cell and the substrate membrane.
Description
Technical field
The present invention relates to a kind of carrier construction that is used to make up engineered biological cornea.
Background technology
Corneal blindness occupies the first place of global blinding oculopathy, and fortunately, corneal blindness is that curability is blind, successful corneal transplantation can help the corneal blindness patient to recover vision, but the immunological rejection after donor material source scarcity and the transplanting has seriously restricted extensively carrying out and curative effect of this operation.
Tissue engineering is the principle and the method for application engineering and life sciences, it makes cell combine with carrier by different tissue construction modes, constitute and have specific three dimensional structure and bioactive artificial organ or organ, be used for substituting or repairing the disease damage organ and the tissue of human body, reach the purpose of recovery organization form and function.Indicate that medical science will surmount the old model of tissue and organ transplantation, enter the epoch of artificial manufacturing tissue and organ, make up the biological cornea of artificial organ through engineering approaches by the organizational project means and will solve or alleviate this imbalance between supply and demand.The essential raw material that makes up tissue by organizational project is carrier construction and seed cell.
The bottleneck of the engineered biological cornea research of present restriction is a carrier construction, because the physiology and the optical characteristics of cornea uniqueness require to be used to make up biological corneal stroma and must have (1) suitable cell growth and adherent microenvironment; (2) stable corneal thickness and corneal curvature, stable to keep diopter; (3) high grade of transparency; (4) moderately intensity and elasticity; (5) good innervation.Otherwise, with the clinical practice and the effect of the biological cornea of influence.
But make up cornea at present or all can not satisfy above-mentioned requirements with the carrier macromolecular material and the collagen of the similar engineered tissue of cornea:
1) be that the cornea of vector construction is not only transparent poor with the macromolecular material, and can not set up the corneal nerve domination, as: the three-dimensional Cornu Bovis seu Bubali film that Mimami etc. (1993) make up with artificial synthetic substrate, be exactly because the defective of artificial substratum (opaque weak) with stretching resistance, can not be used for transplanting (Mimami Y, et al.Reconstruction of cornea in three-dimensionalcollagen gel matrix culture.Invest Ophthalmol Vis Sci.1993; 34 (7): 2316.).The transparency and the poor stability of materials such as polyglycolic acid/polylactic acid (PGA/PLA) class also found in research, require further improvement, require further improvement (Shang Qingxin, Hu Xiaojie, Cao Yilin: the experimentation of tissue engineering technique structure corneal stroma if be used to make up engineered biological cornea, Chinese Journal of Ophthalmology, 2001,37:252).
2) the cornea intensity of collagen synthetic substrate vector construction is poorer.Griffith etc. (1999) utilize transgenic technology to set up " immortalization " cell line at laboratory successfully recombinated function, the structure biological cornea similar to normal cornea, the maximum of being described as in human corneal bioengineered tissue and the tissue transplantation by " science " magazine breaks through and milestone (Griffith M, et al.Functional human corneal equivalents constructed from cell lines.Science 1999; 286 (5447): 2169-72.), also be because insufficient strength fails to carry out zoopery, differ just farther from requirements for clinical application, in addition since collagen easily by collagenase digesting, therefore unstable, and then influence the optical property of cornea and the long-term surviving of transplanted cells.
Normal corneal stroma is that keratocyte normally adheres to the basis with long-term surviving, healthy limbus of corneae substrate has formed stable cell " tabernacle ", supported limbal stem cell normal differentiation and propagation, this is the not available advantage of macromolecular material and collagen carrier.Lange etc. (1993) are that carrier arrives New Zealand white rabbit with corneal endothelial transplantation with cattle Descemet ' s film, all are planted sheet and keep transparent 12-17 days (Lange-TM, et al.Corneal endothelial celltransplantation using Descemet ' s membrane as a carrier.J-Cataract-Refract-Surg.1993; 19 (2): 232-5.), Bohnke etc. (1998,1999) be carrier with Descemet ' s film, also successfully with people and porcine cornea transplanted endothelial cell to human eye (Bohnke-M, et al.Detection of neurone-specific enolase in long-termcultures of human corneal endothelium.Graefes Arch Clin ExpOphthalmol.1998; 236 (7): 522-6. and Bohnke-M, et al.Transplantation ofcultured adult human or porcine corneal endothelial cells onto humanrecipients in vitro.Part II:Evaluation in the scanning electron microscope.Cornea.1999; 18 (2): 207-13.).From beginning in 1994, we are to the hetero stroma of cornea immunogenicity, transplant back cornea nerve recovery, the plant bed histology changes, transplant back different times corneal transparence, series of studies has been carried out in aspect such as diopter and varied in thickness, found that: the immunogenicity of corneal stroma is very low, the thickness of corneal stroma after the xenotransplantation, diopter remains unchanged, autologous nerve can be grown into, big recovery of corneal sensation, epithelial cell adheres to firmly, iuntercellular asks that with cell and substrate physiology is connected normally, the monkey corneal stroma is implanted people's inter-lamination of cornea, kept transparent nearly 10 years, and shown that utilizing hetero stroma of cornea was present optimal structure cornea carrier.
The constructing technology aspect, the main method that makes up engineered biological cornea at present both at home and abroad is that liquid-vapor interface and 3 D stereo are cultivated, after the three-dimensional corneal transplantation of Zieske etc. (1994) report reorganization, cell has hemi desmosome, anchored fiber and continuous basement membrane form, the distribution of α enolase and K3 and normal cornea edge identical (Zieske JD, et al.Basement membraneassembly and differentiation of cultured corneal cells:importance ofculture environment and endothelial cell interaction.Exp Cell Res.1994; 214:621-33.).Engelmann etc. (1999) obtain pig respectively and human endothelial cell reaches 3,450/mm
2With 1,850/mm
2Maximum whole density (Engelmann K, et al.Transplantation of adult human or porcine comeal endothelial cells ontohuman recipients in vitro.Part I:Cell culturing and transplantationprocedure.Cornea.1999; 18:199-206.).
Summary of the invention
The invention provides a kind of biological cornea of artificial organ through engineering approaches, to solve the problem that above-mentioned artificial cornea exists with good carrier.
The biological cornea of artificial organ through engineering approaches of the present invention, preparation in accordance with the following methods: get fresh, take off that cell is handled or preserve after hetero stroma of cornea, after removing endothelial cell and corneal epithelial cell, plant corresponding cell respectively on its surface, be configured to the biological cornea of the artificial organ through engineering approaches with normal cornea structure.In addition, use micro-lamellar cornea cutting edge, miniature cornea lathe or laser cutting to process above-mentioned hetero stroma of cornea and can make to become and have different dioptric corneal stroma sheets, as the carrier that makes up biological cornea.
Main inventive point of the present invention is with the corneal stroma to be carrier, the biological cornea of the artificial organ through engineering approaches that makes, immunogenicity is very low, can be used for xenotransplantation, thickness, the diopter of transplanting the back corneal stroma remain unchanged, and autologous nerve can be grown into, big recovery of corneal sensation, epithelial cell attaches firmly, and physiology is connected normally between iuntercellular and cell and basement membrane.
The specific embodiment
The inventor is to the hetero stroma of cornea immunogenicity and transplant back cornea nerve recovery, the plant bed histology changes, transplant back different times corneal transparence, systematic study has been carried out in aspect such as diopter and varied in thickness, wherein, people → monkey, monkey → people, pig → rabbit, people → rabbit has all carried out the transplantation experiments of preservation and fresh corneal stroma, found that: the immunogenicity of corneal stroma is very low, can be used for xenotransplantation, transplant the thickness of back corneal stroma, diopter remains unchanged, and autologous nerve can be grown into, big recovery of corneal sensation, epithelial cell attaches firmly, and physiology is connected normally between iuntercellular and cell and basement membrane.The inventor thinks that hetero stroma of cornea has the cultured cell of becoming carrier, is used for the potential of synthetic artificial bio-membrane's cornea.
Utilize fresh, take off that cell is handled or preserve after hetero stroma of cornea, corneal stroma as animal classes such as pig, sheep, cat, monkey, cattle, horse, donkey or Fish, after removing endothelial cell and corneal epithelial cell, plant corresponding cell respectively on its surface, be configured to the biological cornea of the artificial organ through engineering approaches with normal cornea structure.Process above-mentioned hetero stroma of cornea, make it have different external shape (according to the processing of the cornea shape of clinical needs) and obtain different diopters (diopter can from-40D～+ 40D) after, can be used for making up artificial organ through engineering approaches biology cornea.Be most preferred embodiment of patent of the present invention below:
Examples of implementation 1: get fresh heterogenic cornea, utilize Dispase II digestion method to remove the epithelial cell and the endotheliocyte of its front and rear surfaces, with this hetero stroma of cornea sheet is carrier, with allogeneic limbus of corneae cell and " immortalization " endotheliocyte is seed cell, utilize microgravity associating gas-liquid interface method to make seed cell and hetero stroma of cornea carrier close attachment, forming front surface is 5～7 layers of epithelial cell, the rear surface is the biological cornea of monolayer endothelial cell, carry out zoopery, according to transplanting the back immunity, the histology recovers, optometry changes, the research observed result of aspects such as operation method improves and improves making up and implantation method.
Examples of implementation 2: the heterogenic cornea of getting dry or freezing preservation, remove the epithelial cell and the endotheliocyte of heterogenic cornea front and rear surfaces with laser ablation, use micro-lamellar cornea cutting edge, laser or miniature cornea machined into then, make it have certain diopter, with this hetero stroma of cornea sheet is carrier, with embryo stem cell source corneal epithelial cell, the endothelial cell of inducing differentiation is seed cell, utilizes microgravity and gas-liquid interface method to make up biological cornea.
The density of the biological endothelial cell that newly constructs reaches 2000 cell/mm
2More than, corneal epithelial cell reaches 5 ~ 7 layers.Have that immunogenicity is low, cellularity and function be normal, transplants back thickness, diopter remains unchanged, autologous nerve can be grown into, big recovery of corneal sensation, epithelial cell attaches firmly, physiology is connected normally between iuntercellular and cell and basement membrane.
Examples of implementation 3: epithelial cell and the endotheliocyte of removing the heterogenic cornea front and rear surfaces with laser ablation, use micro-lamellar cornea cutting edge, laser or miniature cornea machined into, make it have certain diopter, simultaneously or merely at its front and rear surfaces coating water proof material (as crosslinked silicon compound etc., require material and the corneal stroma surface adhesive is good, coating transparency height, particularly have and the compatibility of eyeball favorable tissue and stability), make the water proof material surface-crosslinked, be used for viable transplantation at corneal stroma.
Claims (7)
1, a kind of biological cornea of artificial organ through engineering approaches, preparation in accordance with the following methods: get fresh, take off that cell is handled or preserve after allogeneic or hetero stroma of cornea, remove behind endothelial cell and the corneal epithelial cell as carrier, plant corresponding cell respectively on its surface, be configured to the biological cornea of the artificial organ through engineering approaches with normal cornea structure.
2, according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: described allogeneic or heterogenic cornea are handled through enzyme digestion or laser ablation method, remove endothelial cell and corneal epithelial cell, become the corneal stroma carrier that contains micro-collagen flaggy merely.
3, according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: the cornea of manufacturing the corneal stroma carrier can be the fresh cornea that contains stromal cell, also can be the cornea of handling through preserving that does not contain keratocyte.
4, according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: corneal stroma is made to become with miniature cornea machined into through micro-lamellar cornea cutting edge has different dioptric corneal stroma eyeglasses, as the carrier that makes up biological cornea.
5, by the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: the cell in the carrier surface plantation is allogeneic limbus of corneae cell, endothelial cell and the embryo stem cell source keratocyte of inducing differentiation.
6, according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: the method for cell seeding adopts microgravity, gas-liquid interface method, three little structure methods or unites the structure method.
7, according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: can be coated with the water proof material in the corneal stroma carrier front and rear surfaces of manufacturing simultaneously or merely.
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| CNB031401139A CN1326502C (en) | 2003-08-07 | 2003-08-07 | Artificial tissue engineeing biological cornea |
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| CNB031401139A CN1326502C (en) | 2003-08-07 | 2003-08-07 | Artificial tissue engineeing biological cornea |
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| CN1473551A true CN1473551A (en) | 2004-02-11 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007071169A1 (en) * | 2005-12-20 | 2007-06-28 | Zhiguang (Gz) Biotech Co., Ltd | Biological artificial cornea and method of making |
| CN100333702C (en) * | 2004-04-28 | 2007-08-29 | 浙江大学医学院附属邵逸夫医院 | Exogenous cornea substrate without cells and its preparation method and use |
| CN100386061C (en) * | 2005-05-17 | 2008-05-07 | 浙江大学医学院附属邵逸夫医院 | A frozen heterogeneous corneal stroma with low antigen content and its preparation method |
| WO2008131639A1 (en) * | 2007-04-25 | 2008-11-06 | Zhongshan Ophthalmic Center, Sun Yat-Sen University | A cell-removing cornea substrate and a method of preparation thereof |
| US7674289B2 (en) | 2005-07-29 | 2010-03-09 | Grandhope Biotech Co., Ltd. | Biological artificial ligament and method of making |
| WO2011094965A1 (en) * | 2010-02-05 | 2011-08-11 | 陕西瑞盛生物科技有限公司 | Tissue engineering cornea producing method |
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| CN105445476A (en) * | 2015-12-17 | 2016-03-30 | 中山大学中山眼科中心 | Tissue scaffold based sodium ion channel detection method of three dimensional optic cup originated neuron-like cells |
| CN105688282A (en) * | 2016-03-11 | 2016-06-22 | 广州宏畅生物科技有限公司 | Novel biological artificial cornea capable of realizing cellularization through in-vivo induction as well as realizing quick transparency |
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| Publication number | Publication date |
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| CN1326502C (en) | 2007-07-18 |
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