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Xin Che, Changkai Jia, Han Wu, Shangkun Ou, Xin He, Wei Li; A novel tissue engineered corneal stromal equivalent. Invest. Ophthalmol. Vis. Sci. 2018;59(9):532. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate a novel strategy in constructing tissue engineered corneal stromal equivalent based on amniotic membrane and keratocytes.
The ultra-thin amniotic membrane (UAM) was laminated, with human corneal stromal cells (hCSCs) 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).
On UAM surface, hCSCs were aligned in preferred orientation along linear groove of the patterned biomaterial, and maintained the unified growth manner even with ensuing culture. The expression of keratocan, lumican and collagen 1 showed no significant difference between the cells grown on patterned UAM and culture dish.α-SMA, the fibrotic marker, was significantly downregulated on UAM compared with the cells on culture dish. The four-layer UAM was almost half thickness of human cornea (250±18 μm), which showed promising optimal transparency. The average thickness of one-layer keratocyte-secreted ECM was about 30 μm. SEM showed dense, organized fibrils secreted by hCSCs deposited on the aligned UAM substrate, and arranged in super-imposed, flat layers. The TEM further displayed hCSCs distributed in the construction, exhibited flat appearance, long processes into the developing ECM. When viewed at higher magnification, hCSCs-generated ECM showed stratified, multilayered lamellae with orthogonal fibril arrangement, which was similar to the human cornea.
Our study established a novel 3D biomimetic corneal model to replicate the human cornea stromal organization with multilayer UAM, which was capable of providing preferable environment for keratocyte culture and organized ECM deposition. The 3D UAM laminations can be considered a tissue engineered corneal stromal equivalent with potential of future clinic application.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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