Study of proteoglycans in corneal stroma reconstructed by the self-assembly method

Abstract

Event Abstract Back to Event Study of proteoglycans in corneal stroma reconstructed by the self-assembly method Gaëtan Le-Bel1, Jean-Michel Bourget1, Sébastien Larochelle2, Sylvain Guérin1, 3, Véronique Moulin2 and Lucie Germain1, 2, 3 1 CUO-Recherche, Centre de recherche du CHU de Québec, axe médecine régénératrice, Canada 2 Centre de recherche en organogenèse expérimentale de l’Université Laval/LOEX, Université Laval, Canada 3 Département d’ophtalmologie et d’ORL, Faculté de médecine, Université Laval, Canada Context and Objectives: Corneal transparency is due to a particular collagen fibril architecture defined by a constant diameter and a regular spacing of collagen fibrils within lamellae. Proteoglycans (PGs) are glycoproteins located between collagen fibrils involved in the determination of these two parameters. The objective of this study was to compare the expression profile of PGs of corneal and dermal tissue-engineered stromal models. Materials and Methods: Human corneal fibroblasts (HCFs) were isolated from donor cornea obtained at our local eye bank (n=2). Isolated human dermal fibroblasts (HDFs) were used as control (n=2). Cells were cultured in the presence of ascorbic acid to allow formation of extracellular matrix sheets. Gene expression of various PGs (lumican, keratocan, decorin, biglycan, mimecan, syndecan) was evaluated by PCR at different culture times. After 12 days, the mRNA was isolated for further transcriptome analysis by DNA microarray. The role of lumican was evaluated by knockdown using small interfering RNAs delivered by a retroviral vector to produce stable populations of HCFs (4 independent targets). Efficiency of the knockdown was evaluated by Western blot and immunofluorescence. Results: Expression of the majority of PGs analyzed was maximal at day 11 of culture with ascorbic acid. We found a similar gene expression profile for matrix proteins and PGs for all the populations, except for TNC, COL15A1 and COL5A3, which were higher in HDFs compared to HCFs. Decorin and lumican were highly expressed in cultured HCFs and HDFs while keratocan was not detected. HCF knockdowns for lumican were obtained successfully and the populations that were most inhibited were identified. Conclusion: These results demonstrated that HCFs present a dermal rather than a corneal stromal phenotype since expression of keratocan was not detected. Since the expression profiles of matrix proteins were similar between the HDFs and HCFs, differences in transparency of the reconstructed tissues are potentially due to differences in matrix organization rather than composition of these tissues. Next steps of the project will be to evaluate the diameter and spacing of collagen fibrils as well as transparency of the tissue-engineered corneal stroma with HCFs knockdown for lumican. These studies will allow to better define the role of lumican in the formation of a transparent corneal stroma. Keywords: Extracellular Matrix, Tissue Engineering, microstructure, complex tissue orgnization Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Le-Bel G, Bourget J, Larochelle S, Guérin S, Moulin V and Germain L (2016). Study of proteoglycans in corneal stroma reconstructed by the self-assembly method. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00343 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Google Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Google Scholar Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain PubMed Gaëtan Le-Bel Jean-Michel Bourget Sébastien Larochelle Sylvain Guérin Véronique Moulin Lucie Germain Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.