CN1326502C - Artificial tissue engineeing biological cornea - Google Patents
Artificial tissue engineeing biological cornea Download PDFInfo
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- CN1326502C CN1326502C CNB031401139A CN03140113A CN1326502C CN 1326502 C CN1326502 C CN 1326502C CN B031401139 A CNB031401139 A CN B031401139A CN 03140113 A CN03140113 A CN 03140113A CN 1326502 C CN1326502 C CN 1326502C
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Abstract
The present invention discloses an artificial tissue engineering biologic cornea. A homogeneous variant or heterogeneous corneal stroma is used by the present invention as a carrier, and the tissue engineering biologic cornea is constructed after cells are planted and cultivated on the surface of the carrier. The carrier can be used without processing, or can be used after being preprocessed. The cornea constructed by using the carrier has the characteristics of very low immunogenicity, unchanged thickness and diopter after transplantation, the inward growth of self nerves, the most recovery of corneal sensation, the firm attachment of epithelial cells, and normal physiological connection between cells and between the cells and a basal 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 comeal 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. the biological cornea of an 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 seed 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 is the fresh cornea that contains stromal cell, or through preserving the cornea of handling 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. according to the biological cornea of the described artificial organ through engineering approaches of claim 1, it is characterized in that: the seed cell in the carrier surface plantation is allogeneic limbus of corneae cell and endothelial cell, or induces embryo stem cell source corneal epithelial cell, the endothelial cell of 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: be coated with the water proof material in the corneal stroma carrier front and rear surfaces of manufacturing simultaneously.
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| CNB031401139A CN1326502C (en) | 2003-08-07 | 2003-08-07 | Artificial tissue engineeing biological cornea |
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Cited By (1)
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| CN101433478B (en) * | 2007-11-14 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
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| 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 |
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| WO2016049345A1 (en) * | 2014-09-24 | 2016-03-31 | The Regents Of The University Of California | Three-dimensional bioprinted artificial cornea |
| CN104971384B (en) * | 2015-07-29 | 2017-12-08 | 东莞博与再生医学有限公司 | A kind of preparation method of tissue engineering comea |
| CN105445476B (en) * | 2015-12-17 | 2018-03-23 | 中山大学中山眼科中心 | A kind of sodium-ion channel detection method of the three-dimensional optic cup Derived Nerve like cell based on organization bracket |
| 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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| CN101433478B (en) * | 2007-11-14 | 2012-05-23 | 上海交通大学医学院附属第九人民医院 | Whole layer biological cornea as well as construction method and use thereof |
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