The Study On Human Amniotic Epithelial Cell Sheet Engineering Of Fibrin Gels And Its Re-establishment Of Corneal Epithelium

Objective:(1)To research the biologic characteristics of human amniotic epithelial cells(HAECs) and to investigate the method of culturing HAECs in vitro,and to lay a foundation for tissue engineering of corneal epithelium.(2)To study the status of HAECs growing on fibrin gels(FG) and the effect of using fibrin gels and aprotinin on the proliferation of HAECs;and to investigate the feasibility of FG for HAECs sheet engineering.(3)To explor the possibility of corneal epithelium reconstruction by using HAECs sheet engineering of fibrin gels.Methods:(1) The HAECs were cultured respectively by using tissue digesting method.The cells were subcultured after they became confluent and were investigated morphologically by inverted microscope,Hematoxylin-eosin staining,and identified by cytokeratin immunocytochemistry.(2)The HAECs were seeded in 96-well microplates, the absorbance(A) of HAECs were detected by the methods of MTT after 48 hours on the effect of various concentration of aprotinin.The HAECs were seeded on FG and cultureplates without FG respectively,and compared the cloning efficiency of cells.When the HAECs seeded on FG were confluent,the HAECs sheets were detached from the plate and examed by inverted microscope,Hematoxylin-eosin staining,and scanning electron microscopy(SEM).(3)Stem cell deficiency(SCD)models were created in one eye of adult albino rabbits by a ring lamellar limbal dissection combineed with perilimbal conjunctiva dissection..Corneal epithelium reconstruction procedures were performed in 2 months.During the surgery process,conjunctivalized tissues and neovascularization membrane were excised,then the cultivated HAECs sheets engineering of fibrin gels were placed onto the corneal surface and covered by soft contact lens instead of suturing.Then blepharons were sutured up.After postoperative 2-3 days,opened the stylolitic blepharons,removed the soft contact lens,and observed the survival of HAECs sheets and the stability of ocular surface.After postoperative 1 month,histological and immunohistochemical studies were performed to examine the source of corneal epithelium.Results:(1) The HAECs adhere and grow within 24 hours and form a confluent monolayer in 6-8 days.Most of the cultured HAECs are polygon and are of typical slabstone,and the passage cells still maintained the characteristics of the primary cells.The cells grow very well and could be cultured for a long time.Cytokeratin monoclonal antibody staining is positive.(2)The results of MTT showed:aprotinin could inhibit the proliferation of HAECs in vitro notablely,when the concentration of aprotinin exceed 4×10~5KIU/L.The prepared sheets of FG were smoth and transparent. With cell growth,FG could degrade partly,and obtain cell sheet engineeting with a small amount of FG.The cloning efficiency of HAECs growing on the FG have no significant difference with the HAECs growing on the cultureplates without FG.HAECs could grow well on the FG in vitrio and most of the cultured cells were round or polygon and typical slabstone-like appearance.Many microvilli were observed on cell surfaces by SEM.HAECs had stratificated growth tendency and HAECs sheets engineering of fibrin gels are transparent.(3) After the surgery of corneal limbal dissection,one rabbit was failed model for postoperative serious symblepharon,and seven others developed corneal conjunctivalization,vascularization,and stromal opacity.After ocular surface transplantation procedure,three transplanted epithelial sheets survived well with negative fluorescence staining and improved corneal transparency.Neovascularization developed below the epithelial sheet,but it was limitied to the peripheral cornea region.Two rabbits got keratoleukoma due to the sheets of FG had not degraded.One rabbit was condemned for the HAECs sheet slough on day 3 after the first transplantation procedure.After 1 month,cross-sections of transplanted sheets showed compact an'angement of epithelial cell and appeared similar to normal corneal epithelium,immunohistochemical studies proved weakly positive expression of keretin-12.Conclusions:(1) The HAECs could be cultured for a long time.(2) HAEC sheets were transparent and successfully cultured by using a biodegradable fibrin sealant.(3) A commercially available fibrin sealant was an effective me,am of tissue engineering to create a carrier-free,transpantable HAEC sheets.And the sheets were potential graf to re-establish corneal epithelial layer.