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Fatima N Syed-Picard, Yiqin Du, Rachelle Palchesko, Martha L Funderburgh, Andrew Hertsenberg, Adam W Feinberg, James L Funderburgh; Scaffold-free engineering of stromal lamellar tissue. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3456.
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Blinding corneal scarring is usually treated with allogeneic graft tissue, however, there is a worldwide shortage in donor tissue. The goal of the study is to engineer tissue replacements that emulate the structure of native corneal stroma for regenerative therapy. Novel scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can easily be transplantated in vivo without introducing the additional variables associated with exogenous scaffolding.
Corneal stromal stem cells (CSSC) were isolated from human corneal rims. Scaffold-free corneal tissues were engineered by culturing CSSC on fibronectin coated polydimethylsiloxane substrates containing 10 µm wide, straight grooves spaced 10 μm apart to direct CSSC organization and the generation of a parallel-organized matrix. Keratocyte differentiation was induced using a growth factor-supplemented serum-free keratocyte differentiation medium (PMID: 17962455). After 10 days in culture, CSSC generated a tissue sheet that could be separated with forceps from the substrate. Scaffold-free corneal engineered tissues were implanted in vivo into mouse corneal stromal pockets. Results are representative of experiments replicated at least 3 times.
Scaffold-free engineered corneal stromal constructs were approximately 4 μm thick, cellular and collagenous tissue sheets. Similar to native corneal stroma, engineered corneal tissues contained long collagen fibrils organized in parallel with approximately uniform diameter as seen by transmission electron microscopy. Immunostaining revealed the matrix comprised type I collagen and keratocan. After in vivo implantation, scaffold-free engineered corneal stromal tissue remained in the mouse corneal stroma for up to 8 weeks, and the expression of human corneal stromal matrix molecules was detected. Furthermore, light scatter measurements detected with optical coherence tomography indicated that scaffold-free corneal stromal tissues became transparent in the mouse corneal stroma.
Scaffold-free engineered constructs emulated stromal lamellae of native corneal stromal tissues. These constructs were easily manipulated and became transparent after transplantation into the mouse corneal stroma. We believe that multi-lamellar assemblies of these sheets will provide full thickness stromal tissue suitable for grafting as an alternative to allogeneic donor tissue.
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