Purchase this article with an account.
Jian Wu, Yiqin Du, William R. Wagner, James L. Funderburgh; Bioengineering Human Corneal Stroma Using Adult Stem Cells on an Aligned Nanofibrous Substrate: Effects of Growth Factors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):319.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
This study focused on bioengineering corneal stromal tissue using human corneal stromal stem cells (hCSSCs). We recently demonstrated that an aligned nanofibrous substrate provides topographic cues to initiate and guide organization of a stroma-like extracellular matrix (ECM) by hCSSCs (Wu et al., Biomaterials, PMID 22078813). The current study examined the effect of soluble factors, fibroblast growth factor 2 (FGF2) and transforming growth factor-beta 3 (TGFβ3) in effecting matrix deposition and organization in this system.
Aligned nanofibrous substrates were prepared by electrospinning biodegradable poly(ester urethane) urea (PEUU) onto a high-speed rotating mandrel. hCSSCs were seeded onto the aligned nanofibrous substrate and cultured in serum-free medium supplemented with ascorbate-2-phosphate, insulin and FGF2, 10 ng/mL, TGFβ3, 0.1 ng/mL, or both factors. After 6 wks, secreted ECM was evaluated by transmission electron microscopy, wholemount immunostaining, and immunoblotting of culture media. Gene expression was examined by qPCR.
Cultures supplemented with FGF2-only secreted collagen fibrils strongly aligned with the nanofibrous substrate; however, TGFβ3 induced distinct orthogonal collagenous layers. Genes of stromal ECM secretion, KERA, B3GnT7, and CHST6 were upregulated in all cultures. Stromal ECM components keratan sulfate, dermatan sulfate, lumican, decorin, and keratocan were detected in secreted ECM and in culture media. The combination of FGF2 and TGFβ3 produced significant synergetic effect in construct thickness, in packing of collagen fibrils, and in expression of cornea-specific ECM components: keratan sulfate, lumican, and keratocan.
On aligned PEUU nanofibrous substrates, spatial self-organization of collagen-based ECM by hCSSCs exhibits characteristic responses to specific growth factors. Presence of both FGF2 and TGFβ3 produced a significant synergetic effect, leading to stratified multilayered lamellae with orthogonally-oriented collagen fibrils, mimicking that of human corneal stroma. This study provides a new tool for bioengineering of a well-organized, collagen-based construct with appropriate nanoscale structure for corneal repair and regeneration.
This PDF is available to Subscribers Only