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Jian Wu, Sayan Basu, Martha L Funderburgh, William R Wagner, James L Funderburgh; Aligned Fibrous poly(lactide-co-glycolide) (PLGA) Substrates for Corneal Stromal Regeneration by human corneal stromal stem cells (hCSSCs). Invest. Ophthalmol. Vis. Sci. 2014;55(13):4619.
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© ARVO (1962-2015); The Authors (2016-present)
Bioengineering corneal stromal tissue is a critical step in the development of a functional corneal bioequivalent. We recently demonstrated that topographic cue provided by aligned nanofibrous surface in concert with FGF2 and TGFβ3 (Wu et al., Biomaterials, PMID 22078813, Tissue Engineering Part A, PMID: 23557404) induced hCSSCs to generate a well-organized stroma-like extracellular matrix (ECM). In this study, we further investigated the influences of scaffold composition and degradation rate on tissue deposition by hCSSCs.
An aligned fibrous scaffold was prepared by electro-spinning using biodegradable poly(lactide-co-glycolide) (PLGA) with LA/GA ratios of 100/0 compared to 50/50. hCSSCs were seeded on the aligned fibrous PLGA scaffold, and cultured in a serum-free keratocyte differentiation medium. ECM secreted by hCSSCs on the substrate was characterized by electron microscopy, two-photon fluorescent microscopy, and rheometry, and gene expression was examined by q-PCR.
During 4 months of culture, the PLGA (50/50) scaffold gradually degraded and almost disappeared, while the PLGA (100/0) scaffold remained essentially intact. Electron microscopy and two-photon fluorescent microscopy demonstrated that collagenous ECM deposited by hCSSCs on the PLGA (50/50) substrate was much more abundant than that on PLGA (100/0). As the substrate degraded, the ECM secreted by hCSSCs on PLGA (50/50)-based scaffold gradually became transparent, although not optically clear, and exhibited shear modulus of ~50 kPa as shown by rheological characterization. Gene expression profiles showed that PLGA (50/50) fibrous substrate induced up-regulation of mRNA for several tissue-specific markers of keratocytes (KERA, ALDH, B3GNT7, CHST6, and COL-I, V, VI) much more than in hCSSCs cultured on PLGA (100/0) substrate.
These observations demonstrated that the composition and the degradation rate of biodegradable PLGA scaffold plays an important role the corneal stromal tissue generation by hCSSCs. When the degradation rate of the PLGA approximated the rate of tissue deposition by hCSSCs, the construct volume and quality was enhanced. Production of stromal ECM tissue in vitro with appropriate optical and mechanical properties is crucial in the development of a functional corneal bioequivalent, and offers the potential as use in lamellar keratoplasty.
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