Abstract
Purpose :
To investigate the effect of GAGs on the ECM expression and deposition of hTM cells cultured on 3D natural biopolymer scaffolds and the role of Dex on its remodeling under static and dynamic conditions.
Methods :
Anisotropic porous scaffolds were fabricated from natural biopolymers present in the TM extracellular matrix, specifically collagen and a variety of glycosaminoglycans (GAGs), to allow the study of hTM cells’ response to extracellular environment composition. 100,000 hTM cells were seeded on the scaffolds and cultured for up to 2 weeks in the presence and absence of 100 nM dexamethasone (Dex). Dynamic 3D cell culture was performed on scaffolds housed in a perfusion chamber connected to a peristaltic pump. mRNA expression levels of elastin, laminin, and MMP-2 were quantified using qPCR. ECM protein deposition was visualized using confocal microscopy. Pressure changes were measured using pressure transducers connected to the dynamic cell culture constructs.
Results :
hTM cells cultured on the collagen-only (CO) scaffolds expressed significantly higher amounts of mRNA for the studied ECM proteins compared to hTM cells seeded on tissue culture dishes. The expression of these ECM proteins was also impacted by the composition of those scaffolds. In particular, the addition of chondroitin sulfate (CS) increased the expression of these proteins by up to 8-fold when compared to CO. Dex increased the expression of elastin and decreased expression of laminin and MMP-2 after two weeks of culture in scaffolds. The morphology of elastin and laminin proteins was also impacted as a result of dexamethasone addition. Pressure measured across the scaffolds was higher in cells cultured in the presence of Dex as compared to non-treated controls.
Conclusions :
Extracellular matrix remodeling has been shown to play an important role in the IOP homeostasis, increasing the aqueous humor outflow resistance of the TM. Our results show collagen and GAGs are important in providing signals to the cells for the expression of important ECM proteins. CS can potentially be responsible for the promotion of elastin fiber formation. Our results suggest that the presence of GAGs decreases the expression of laminin likely via the integrin pathway when cells are cultured in the presence of Dex. Increased pressure across scaffolds is a result of decreased MMP-2 expression and its effect on ECM protein degradation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.