An Experimental Study Of Ocular Surface Reconstruction With Cultured Limbal Corneal Epithelium In Vitro

Corneal disease including keratitis, traumatic cornea, thermal and chemicalburning, genetous abnormity, recurrent pterygium and tumor could cause injury ofocular surface tissue, descent of vision, blindness even. The corneal blind accountedfor a considerable proportion in the blind, there was little effective treatment on thecorneal blindness caused by the disease of ocular surface and the absence of corneallimbal stem cell, so the work proceeded at a glacial pace. The reconstructionoperation of ocular surface using mucous, membrane or conjunctiva had little clinicaleffect, because of different phenotype of the mucous membrane conjunctiva and theepithelium of cornea, the reconstruction of ocular surface using the mucousmembrane or conjunctiva remained elusive result, and resulted in neovasculization.The Allogeneic corneal transplantation was limited by the donor eyes, and thedevitalization of the frozen corneal limbal stem cell could not cause the effectivereconstruction of the normal ocular surface.It was realized that the corneal limbal stem cell was import to the ocular surface,and the deficiency or dysfunction of the corneal llimbal stem cell would causeepithelial healing obstacle and abnormal restoration, so the transplantation corneallimbal stem cell became hot. The human embryo stem cell had been successfullycultivated in vitro by American scientist, which reported on《science》magazine in1998. According to this research result, the stem cells can be transplanted to curesome diseases possibly. After one year, the American researcher discovered that the stem cell of mice myofiber tissue could be transversely differentiated into blood cell,which had been confirmed by other researcher. After that it was found so many stemcells had this kind of ability. All of this, the stem cell could be used to treat somekinds of disease. Tissue technology was born with the research of stem cell. It wasexcellent opportunity for oculist to take advantage of the tissue-engineered toreconstruct the ocular surface. The basic method of the tissue-engineered was to usea little limbal stem cell cultured in vitro. The germ of the corneal limbal stem cellwas planted in a good carrier which had good histocompatibility. This kind ofbiological tissue was transplanted in the damaged ocular surface. The advantage ofthis technology had: first, little material; second, little damage on the health eye;third, resolving the donor source and no immune rejection. The transplant of thecorneal stem cell could reconstruct the ocular surface because the HLA-DR antigenbecame more and more little in culture. So it resolved the stem cell source thatcorneal limbal stem cell was cultured in vitro, the allogeneic cultured corneal stemcell had been concentrated by most ophthalmologist. The transplantation of theallogeneic cultured stem cell might be the most suitable method to reconstruct thedamaged ocular surface.At present the ideal cultural system of corneal limbal stem cell was beingexplored, and it was the first-line factor that corneal limbai tissue Was acquired andcultured exactly. The research went along due to the lack of masculine sign ofcorneal limbal stem cell, and much important content hadn't been found out, forexample, the restricting factors of cell proloferation, cell differentiation and celldivision. A major breakthrough in the research would bring great social andeconomic benefits, and bring happiness to tens of thousands of corneal blinders.In summary, to attempt the corneal limbai stem cell was cultured in vitro, andthe tissue corneal limbal was be constructed successfully. To transplant the tissuecorneal limbal to animal models, ocular surface would be reconstructed, the smoothness and transparency of corneal surface was be restored, the normalanatomical structure and function of ocular surface was be restored, and thefoundations of theory and experimentation was be laid for further tissue ocularsurface reconstruction.The main points of the results from this study are summarized below:Ⅰ. To confirm the exact location of corneal precurcor epithelial cells or cornealstem cells (progenitor cells). Corneal of rabbits was assigned to 8 orientation zoneaccording to the basic reference of cornea and conjunctica limbus. Cornealspecimens were collected at 12, 6, 3 and 9 o'clock separately for the in vitro tissueculture. The morphology and area of the theca-form of corneal precursor epithelialcells in different limbus site were observed and measured daily for statisticalanalysis. It shew that: 1. Statistically significant differences were found in varioustissues at cells growth, theca-froming growth, theca-froming speed and theca-fromingshape. Close to the corneal conjunctiva limbus, the cells growth was the fastest, themembrane's growth was the earliest and best. 2. None of fibroblasts were seen insidethe corneal conjunctiva limbus. However, the fibroblasts were found during the culture.outside the corneal conjunctiva limbus and the numbers increased gradually outside. 3.No statistically significant difference was found in theca-forming shapeand rules ofcorneal precursor epithelium between horizontal level (3 and 9 o'clock) and verticallevel (12 and 6 o'clock). 4. The film with cultured in vitro was integrity,homogenate and clear. As the film was growing, it can resolve the fibroblasts oramniotic membrane epithelial cells. Tips: 1. Statistically significant differenceswere found in various tissues theca-froming physiological characteristic. Close to thecorneal conjunctiva limbus, the cells' growth was the fastest, the membrane's growthwas the earliest and best. 2. No a positive correlation is found between the richnessof palisade tissue and amount of corneal precursor epithelial cells 3: The film ofLimbal stem cells pesists the physiological characterigtic of the clear shape and the discrimination. 4. When the corneal precursor epithelial cells are cultured in vitro,they are better collcted closing to the corneal conjunctiva limbus.Ⅱ. To cultivate the limbal epithelial cells at different places of rabbit limbus in vitroso as to explore the physiological characteristic of Corneal stem cells. It shew that:1. When the similar films from tissue blocks emerged, films were compatible andlinked up, namely, neither film inside cells penetrated into the other, nor cellsdeposited or formed to block the other cells from entering barrier structure, andultimately resulting in trace between film connections free. 2. When the films fromthe limbal cells and the fibroblasts from the conjunctiva cells linked up, they competedeach other. 3. There are some limbal stem cells in limbal epithelial cells, filmsformed by limbal cells have physiological characteristics of keeping transparency andexclusiveness. Tips: 1. The competition between limbal cells and conjunctiva cellsis the test base of corneal conjunctiva from lack of corneal epithelial stem cells. 2.There are some limbal stem cells in limbal epithelial cells, it is important to maintaincornea's transparency and normal physiology.Ⅲ. To study and compare the physiological characteristic of intact and denudedamniotic membranes as a substrate for Corneal epithelial cells culture. Small biopsyspecimens of superficial cornea including epithelium were excised from the centraland. limbal, regions in rabbits. It shew that: 1. The theca-froming area andtheca-froming speed, of corneal epithelial cells with culture denuded amnioticmembrane was faster than intact amniotic membrane. The corneal epithelial cells'growing of denuded amniotic membrane was better. 2. During the process of filmgrowth, film forming cells would gradually dissolve amnion epithelial cells uponmeeting them, the film continued extending forward and maintained level, smooth andtransparent. Tips: 1. Different carriers, have some influences on the physiologicalcharacteristic of limbal stem cells. The corneal epithelial cells' growing of denudedamniotic membrane was better than intact amniotic membranes, it appears to be an excellent substrate for cultivation of Corneal epithelial cells, with a view totransplantation. 2. Corneal epithelial cells have physiological characteristics ofkeeping transparency and exclusiveness, it is important to maintain cornea'stransparency and normal physiology.Ⅳ. To reconstruct corneal epithelium in vitro by tissue engineering technique andprovide effective method and materials for clinical transplantation. As model of totallimbal deficiency had been built in right eyes of 20 rabbits by surgical removal oflimbal rim for 3 months. As model of total limbal deficiency had been built in righteyes of 16 rabbits, They were randomly divided into 2 groups: experimental andcontrol group. Small biopsy specimens from the limbal region of experimental groupwere taken and cultured on denuded amniotic membrane. When it was culturedabout 12 days, experimental group received grafts of amniotic membrane containingautologous cultured epithelial cells, whereas control group received grafts ofdenuded amniotic membrane alone. To observe for 3 months after'transplantation,the restoration of corneal epithelium was evaluated by phthology, and the cellsphenotype of corneal epithelium was examined by impression cytology. It shew that:1. Limbal epithelial cells can be cultured and proliferated on the amniotic membrane.2. Corneas of experimental group gradually epithelialized and progressive decreaseof vascularity and stromal infiltration in the limbal and peripheral zone. Theexamination of impression cytology shew that corneal epithelium beforetransplantation: PAS (+) , corneal epithelium'after transplantation: PAS (-). Thehistopathology shew 'that corneal epithelium before transplantation was deficiencemostly, corneal epithelium after transplantation was restored. Tips: Tissue engineeredcorneal epithelium can be reconstructed in vitro by culturing limbal epithelial cellson denuded amniotic membrane, corneal transplantation of cultivated limbalcornealepithelium on denuded amniotic membrane can repair the limbal deficiency.Ⅴ. To investigate the outcome of using human amniotic membrane as substrates for cultivating rabbit limbal corneal epithelial cells, and autologous or allogenoustransplanting them onto rabbits with total limbal deficiency. As model of total limbal.deficiency had been built in right eyes of 20 rabbits by surgical removal of limbalrim for 3 months. They were randomly divided into 2 groups of 10: Autologousgroup (5) and allogenous group (5). Small biopsy specimens from the limbal regionof 10 left eyes were taken and cultured on acellular amniotic membrane. When itwas cultured about 12 days, 5 of the right eyes then received grafts of amnioticmembrane containing Autologous cultured epithelial cells, 5 of the right eyes thenreceived grafts of amniotic membrane containing allogenous cultured epithelial cells.To observe for 3 months after transplantation, the restoration of corneal epitheliumwas evaluated by pathology, and the cells phenotype of corneal epithelium wasexamined by impression cytology. It shew that: Limbal epithelial cells can becultured and proliferated on the amniotic membrane, stratified layers of epitheliumwere formed after 12 days. Corneas of Autologous (5) or allogenous (3)transplanting gradually epithelialized and progressive decrease of vascularity andstromal infiltration in the limbal and peripheral zone. The examination of impressioncytology shew that corneal epithelium before transplantation: PAS (+), cornealepithelium after transplantation: PAS(-)The histopathology shew that cornealepithelium before transplantation was deficience mostly, corneal epithelium aftertransplantation was restored. However corneas of allogenous (2) transplanting hadhappened immunology rejection. Tips: Autologous transplantation of cultivatedlimbal corneal epithelium on amniotic membrane can reconstruct corneal epithelium.However, immunology rejection is main reason of limbal epithelial allogenoustransplantation.Ⅵ. To observe the effects of human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation for severesymblepharon resulting from eye burns. Two patients with severe symblepharon visited our hospital, to observe the effects of human limbal stem cells cultured onhuman amniotic membrane with themselves serum using allograft transplantation forsevere symblepharon resulting from eye burns. Eye movement, diplopia,development of corneal grafts and recoverment were observed. It shew that: aftertransplantation, two patients felt better and visual acuity improving a little, graftincluding amniotic membrane and stem cells firmly attached on limbus, cornealepithelium become smoothly, superficial neovasculization reduced significantly andblepharostenosis vanished. Tips: Human limbal stem cells cultured on humanamniotic membrane with themselves serum using allograft transplantation, is anideal treatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.To study the physiological characteristic of Corneal stem cells, it confirmed thatthe exact location of corneal stem cells located at limbus, it is important to maintaincornea's transparency and normal physiology. To study and compare thephysiological characteristic of intact and denuded amniotic membranes as a substratefor Corneal epithelial cells culture, it showed that denuded amniotic membraneappears to be an excellent substrate for cultivation of Corneal epithelial cells, with aview to transplantation. Corneal transplantation of cultivated limbal cornealepithelium on denuded amniotic membrane can repair the limbal deficiency, it can bereconstructed ocular surface. Human limbal stem cells cultured on human amnioticmembrane with themselves serum using allograft transplantation, is an idealtreatment for severe symblepharon resulting from eye burns. It supplies a newmethod for ocular surface reconstruction.