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Jaganmohan Reddy Reddy, Maxim Gerasimov, May Griffith; Functional modification of Collagen like peptides for cellular specificity and function. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4135. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our most recent works show that small collagen like peptide (CLP) with functional properties similar to collagen when conjugated with PEG can serve as collagen analog. Further CLP-PEG implants, like full-length recombinant human collagen implants, promoted stable, functional regeneration of neo-corneas that were optically transparent among Gottingen pigs. However, CLP-PEG implants, like collagen implants, does not show the biomechanical properties of elasticity and viscoelasticity similar to human donor corneas. Moreover, CLP-PEG implants does not show cell type or tissue type specificity to tailor the concerning needs during pathogenesis of the respective tissue. Our goal in this study is to optimize the implants for pathological specificity to grow specific cells such as epithelial cells, keratocytes, and endothelial cells.
Preparation of Hydrogels: CLP-PEG conjugated lyophilized product obtained from UAB Ferentis (Lituania) is made into a 12% (W/W) solution and crosslinked with EDC-NHS before molding into flat sheet. Similarly, Recombinant Human Collagen III (RHCIII) (Fibrogen Inc., USA) and PEG-DGEA sheets are made from an initial solution of 18% (W/W).Immuno Cytochemistry: HCEC were plated on to the respective hydrogels in a 96 well plate and cultured in the KSFM media with supplements. After 48 hrs, the cells were fixed with 4% PFA, permeabilized with methanol for 10 min and stained with corresponding primary and secondary antibodies.
HCEC cultured on PEG hydrogels and PEG-DGEA hydrogels showed cell clustering while the separation of the cell adhesion motif (DGEA or RGDS) with CLP showed more dispersed cells similar to RHCIII hydrogels. Even though cells showed staining for proliferation marker Ki67 in all the hydrogels, the expression of cell adhesion interacting molecules integrin β 1 showed similar expression with a DGEA concentration of 250 mg/ml as that of RHCIII and Gelatin. EGFR, expression also correlated as the Integrin β 1 expression among the collagen based hydrogels. Similar to our animal studies, we observed increased expression of Collagen I in the CLP-PEG implants and PDMS than the Gelatin and RHCIII hydrogels.
In this study, we show that punctuation of the cell adhesion motifs among the synthethic ECM mimetic proteins enables the mimicking of such proteins as the native ECM and thus providing a better matrices for modeling tissue specific regeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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