A Novel Tissue Engineered Corneal Stromal Equivalent Based On Amniotic Membrane And Keratocytes

Purpose:To investigate a novel strategy in constructing tissue engineered corneal stromal equivalent based on amniotic membrane and keratocytesMethods:The ultra-thin amniotic membrane(UAM)was laminated,with human or rabbit corneal stromal cells(CSCs)distributed between the space of the layered UAMs.The multilayer UAM system was cultured in serum-free medium with FGF2,TGF-β3 and non-essential amino acid for 8 weeks.Calcein AM staining was used to evaluate cellular viability,morphology and arrangement.qRT-PCR and Western blot were performed to detect gene and protein expression in keratocytes.Optical coherence tomograph(OCT)visualized the cross sections and thickness of the UAM construction.The microstructure of the keratocyte-secreted extracellular matrix(ECM)was investigated by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).To evaluate the feasibility of the multilayer UAMs lamination for surgery,the tissue engineered stromal tissues were used to perform lamellar keratoplasty.Results:The expression of keratocyte-specific marker,such as CD34,ALDH3,CHST6,PTGDS and Keratocan,were upregulated on UAM surface.Lumican,one of the proteoglycan,and collagen I showed no significant difference between the cells grown on patterned UAM and culture dish.Fibronectin and a-SMA,the fibrotic markers,were significantly downregulated on UAM compared with the cells on cell culture dish.On UAM surface,CSCs were aligned in preferred orientation along linear groove of the patterned biomaterial,and maintained the unified growth manner even with ensuing culture.SEM showed dense,organized fibrils secreted by CSCs deposited on the aligned UAM substrate,and arranged in super-imposed,flat layers.The four-layer UAM was almost half thickness of human cornea(250±18μm),which showed promising optimal transparency.The average thickness of one-layer keratocyte-secreted ECM was about 30 μm.The TEM further displayed CSCs distributed in the construction,exhibited flat appearance,long processes into the developing ECM.When viewed at higher magnification,CSCs-generated ECM showed stratified,multilayered lamellae with orthogonal fibril arrangement,which was similar to the human cornea.To evaluate the efficacy of engineered corneal stroma-like tissue on corneal reconstruction,the 4 layered UAMs as a corneal stromal substitute was preformed in lamellar keratoplasty,4 weeks after operation,the implant was transparent,complete epithelialized and integrated well with the recipient cornea.The CSCs populated in the amniotic membrane,metabolically active,organized ECM deposited around the CSCs,which was composed of orthogonal collagen fiber with uniform and thin diameter.Conclusions:Our study established a novel 3D biomimetic corneal model to replicate the corneal stromal organization with multilayer UAM,which was capable of providing preferable environment for keratocyte flourishing and organized ECM deposition in vitro.Further the UAM functioned to promote ECM generation and remodeling after transplanted in vivo,establishing to be a new avenue for basic research and therapeutic potential.