Study of proteoglycans in corneal stroma reconstructed by the self-assembly method
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1
CUO-Recherche, Centre de recherche du CHU de Québec, axe médecine régénératrice, Canada
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2
Centre de recherche en organogenèse expérimentale de l’Université Laval/LOEX, Université Laval, Canada
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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
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.