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David Hui-Kang Ma, Yi-Jen Hsueh, Hung-Chi Chen, Jui-Yang Lai, Kathleen Sheng-Chuan Ma; ECM Protein Coating on Tilapia Fish Scales Improves HCEC Adhesion and Proliferation Through ILK/p63 Signaling. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1379. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of cultivated human corneal endothelial cells (HCECs) on a cell carrier has emerged as a potential therapeutic strategy. Because of characteristics such as transparency, intensity and biodegradability, processed Taiwan Tilapia fish scales were shown to be a better cell carrier than biomaterials like gelatin or chitosan. Therefore, we evaluate ECM components coating on the surface of fish scales for improvement of HCEC adhesion and proliferation.
The fish scales were harvested from whole tilapia fish skin and were treated with a decellularization, decalcification and sterilization process. The scales were coated with either fibronectin (FN), laminin (LN), collagen IV (Col IV) or FNC coating mix (FNC). B4G12 cells were cultured on the coated scales with human endothelial serum-free medium (Invitrogen) supplemented with 2%FBS and 10 ng/ml bFGF. Cell proliferation/adhesion was evaluated by MTT assay, BrdU labeling, immunoconfocal microscopy and SEM. Immunoblot and q-PCR were performed to observe the expression of intergrin-linked kinase (ILK), beta catenin, p63, cyclin D1, and p27.
Without coating, B4G12 cells adhered poorly to fish scales, forming only cell aggregates. LN, Col IV and FNC coatings facilitated B4G12 cells adhesion and proliferation, while FN only facilitated cell adhesion. In particular, cells cultured on LN- and FNC-coated fish scales were of greater density and showed a polygonal morphology. An immunoblotting assay confirmed that coating the scales with LN, Col IV or FNC up-regulated ILK phosphorylation and p63 expression in B4G12 cells, yet nuclear beta catenin level was not affected. In addition, FNC activated the cell cycle mediators cyclin D1 and p27.
This study indicates that with proper coating, processed fish scales may serve as an innovative cell carrier that may facilitate HCEC cell therapy. ECM coating increased the level of phospho-ILK (T173), p63, and cyclin D1 and decreased level of p27, but no significant difference in the nuclear translocation of beta catenin was found. This suggests that ILK may also regulate cyclin D1 via beta catenin-independent pathways and thus affects cell proliferation.
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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