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Purpose: We examined whether amniotic membrane transplantation (AMT) is useful in preparing the perilimbal stroma to enhance the success of allograft limbal transplantation (ALT)., Methods: Forty-seven eyes of 42 consecutive patients with cytologically proven limbal deficiency (LD) were included in this prospective study. Based on the severity of LD, group A (mild) with 18 eyes received AMT alone, group B (moderate) with 13 eyes received AMT and ALT, and group C (severe) with 16 eyes received AMT, ALT and penetrating keratoplasty. All except for group A received continuous systemic cyclosporin A., Results: Except for the two eyes with atopy, all amniotic membrane-covered surfaces showed rapid epithelialization in 2-4 weeks, reduced inflammation, vascularization and scarring, and became smooth. For the mean follow-up period of 23 months, 38 eyes (82.6%) showed visual improvement, consisting of > or = 6 lines (15 eyes), 4-5 lines (10 eyes), 1-3 lines (13 eyes). Visual improvement was noted in 16/18 eyes (88.9%) in group A, in 10/13 eyes (77%) in group B, and in 12/16 eyes (75%) in group C. In group C corneal graft rejection occurred in 12 of 16 eyes (75%). In group B and C, early reversible limbal allograft rejection was noted in 3 of 29 eyes (10.3%) and a recurrent limbal deficiency was observed in 8 of 29 eyes (27.6%)., Conclusion: For partial LD with superficial involvement, AMT alone is sufficient and hence superior to ALT because of no need for using cyclosporin A. For total LD, additional ALT is needed and AMT helps reconstruct the perilimbal stroma with reduced inflammation and vascularization, which collectively may enhance ALT success.

The ocular surface epithelia with the tear film as a unit provide clear vision and comfort and serve as the first line the defense. It is important to understand how ocular surface health is maintained and how ocular surface failure occurs. Furthermore, it is timely to summarize new information concerning action mechanisms and clinical uses of amniotic membrane transplantation for ocular surface reconstruction. When appropriately processed and preserved, amniotic membrane as a native matrix can be used as a graft to restore conjunctival surfaces following removal of lesions such as pterygium, tumor, scar, symblepharon, and conjunctivochalasis. It can also be used as a graft to restore corneal surfaces suffering from limbal stem-cell deficiency. For partial limbal deficiency, amniotic membrane alone is frequently sufficient, while for total limbal deficiency it needs to be combined with limbal stem-cell transplantation with or without corneal transplantation. When used as a graft or patch, amniotic membrane can facilitate healing of persistent corneal ulcers and recurrent corneal erosion and reduce corneal haze following keratectomy. Reported data indicate that amniotic membrane transplantation facilitates rapid healing with recovery of a normal epithelial phenotype in the epithelium and reduces inflammation, vascularization, and scarring in the stroma.

The role of the various isoforms of transforming growth factor-beta (TGF-beta) concerning the regulation of proliferation, differentiation and wound healing of the corneal epithelium is controversial. We investigated various concentrations of TGF-beta 1 and 2 in a serum-free, defined clonal growth assay concerning the proliferation of limbal and peripheral corneal epithelium. Concentrations between 0.05 and 5.0 ng/ml of either TGF-beta 1 or 2 caused a significant, dose-dependent decrease of both colony formation and colony proliferation in limbal and peripheral corneal epithelial cultures. These results suggest a physiological role for TGF-beta as a negative growth regulator targeting primarily the transient amplifying cells of the limbal and corneal epithelium.

The basal limbal epithelium contains corneal stem cells, which are responsible for the maintenance of the corneal epithelial mass. In our study of the regulation of limbal progenitor cells, we investigated the proliferation of limbal and corneal epithelial cells in a defined single-cell clonal culture system. In a serum-free defined medium the limbal epithelium generated significantly fewer colonies than the cornea, which also proliferated more slowly. The addition of fetal calf serum resulted in the stimulation of a unique subpopulation of progenitor cells at the limbus. In contrast, proliferation of the peripheral corneal epithelium was inhibited by serum. These results further support the concept of a limbal location of corneal stem cells and suggest that limbal progenitor cells might be regulated preferentially by serum factors.

The regulation of proliferation of transient amplifying cells from the corneal epithelium by all-trans-retinoic acid was investigated by means of a serum-free clonal growth assay. While retinoic acid did not alter cell attachment, a dose-dependent and significant inhibition of both colony formation and clonal proliferation was observed. The data indicate that retinoic acid inhibits the proliferation of corneal transient amplifying cells. Therefore, retinoic acid might also play a role in regulating the proliferation of the corneal epithelium in vitro.

The limbal basal epithelium contains the stem cells of the corneal epithelium. In order to study the regulation of these cells we developed models allowing their investigation. While an explant model based on n-heptanol debridement did not allow outgrowth of corneal epithelium onto culture dishes, a serum-free single-cell clonal growth supported colony growth in limbal cells. MCDB 151 medium was supplemented with insulin, transferrin, selenium, hydrocortisone, phospho-/ethanolamine and calcium. Single cells (5,000) were seeded onto 60 mm plastic dishes following combined digestion with dispase and trypsin/EDTA. Increasing CFE was noted until day 6, allowing increasing recruitment into proliferation. This model will serve as a tool for the further investigation of factors that might govern the regulation of the differentiation and proliferation of limbal epithelial cells.