Corneal Cell Matrix-Conditioned Amniotic Membrane with Improved Biochemical Properties and Corneal Wound Healing Potential: An In Vitro and In Vivo Study

Several attempts have been made to engineer the amniotic membrane (AM) to improve its biomechanical and biological characteristics close to corneal tissue for corneal wound dressing applications. In this study, a decellularized AM (dAM) is conditioned with limbal epithelial stem cells (LESCs) by freeze-thawing (cAM), and fully characterized in vitro for mechanical behaviors, degradation, and cytobiocompatibility, and in vivo implantation in comparison with dAM. The LESCs-conditioning improved the LESCs adhesion property of cAM compared with dAM with no cytotoxicity against LESCs in both dAM and cAM. Complete epithelial coverage with improved corneal wound healing and collagen synthesis, and also no scarring is observed in the wounds treated with dAM and cAM. Notably, in the cAM group, epithelial layers are well delaminated and collagen fibers are arranged in a basket weave pattern. A significant up-regulation in limbal/epithelial genes (ABCG2 and Ck3) expression is observed in the wounds treated with cAM, compared with other groups. Both dAM and cAM significantly down-regulated the pro-inflammatory genes (IL6 and TNF alpha), compared with the control wound. In conclusion, this study reveals that LESCs-conditioning is a viable strategy for the development of corneal wound dressing and now promises to proceed with clinical investigations.