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Kristan Sorenson Worthington, Emily Kaalberg, Robert F Mullins, Edwin M Stone, Budd Tucker; Fabrication and characterization of tissue-engineered multilayer outer retinal grafts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3172.
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© ARVO (1962-2015); The Authors (2016-present)
As AMD associated photoreceptor cell death is typically preceded by loss of cells of the underlying retinal pigment epithelium (RPE) and choroidal vasculature, an effective restorative treatment strategy will likely require transplantation of multi layered cellular grafts. Recent data suggests that autologous induced pluripotent stem cells may be the optimal cell source for such application. However, current bolus cell delivery methods typically result in significant cell loss (as much as 99%) and limited cellular integration following transplantation. The purpose of this study is to overcome these limitations by combining state of the art stem cell and biodegradable tissue engineering technologies to fabricate multi-layered outer retinal grafts.
Cross-linked methacrylate-functionalized tropoelastin and chitosan served as biomaterial substrates for the delivery of RPE and photoreceptor precursor cells, respectively. Chemical reactions were confirmed by NMR. The mechanical properties of the constructs were measured using dynamic mechanical analysis while cellular survival, identity, morphology and directionality were characterized using rt-PCR, Western blot, and immunocytochemical approaches.
Tropoelastin and glycol chitosan were both successfully modified with methacrylate groups by reaction with methacrylic anhydride and AOHPMA, respectively. Tropoelastin based Bruch’s Membrane mimics were fabricated using photo-crosslinking with 365nm light, while chitosan hydrogel based outer nuclear layer mimics were fabricated with 650nm light. The mechanical properties of materials were tuned to match the compressive modulus of native retinal tissue by adjusting the polymer cross-linking density. RPE and photoreceptor precursor cells survived on their respective materials and exhibited lineage-specific markers.
These results lay the foundation for testing of autologous cell/polymer-based therapeutic modalities in vivo in animal models of retinal degenerative diseases. The constructs developed can be used as transplantation grafts or as closer in vitro approximations to the in vivo retinal environment than those offered by traditional cell culture methods. Further, the inclusion of additional layers in future studies, such as a 3D-printed choroid-like structure, could result in a powerful regenerative graft for a variety of retinal degenerative diseases of the outer retina.
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