An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering

Abstract

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Mimicking corneal stroma using keratocyte‐loaded photopolymerizable methacrylated gelatin hydrogels

“…To date, several tissue‐engineered products were developed using synthetic and/or natural materials. Products mimicking only one layer (epithelium, stroma, or endothelium; Teichmann et al, ; Wu et al, ; Zhang et al, ), two layers (hemi‐cornea epithelium and stroma; Wang et al, ; Zorlutuna, Tezcaner, Kiyat, et al, ), or all three layers of cornea (Vrana et al, ) were studied. These were in various forms including foams (Acun & Hasirci, ; Vrana et al, ), meshes (Kador & Ahearne, ), films (Kilic, Girotti, Rodriguez‐Cabello, & Hasirci, ; Zorlutuna et al, ), and decellularized corneal tissues (Ponce Márquez et al, ).…”

Natural Biomaterials for Corneal Tissue Engineering, Repair, and Regeneration

“…When compared to denuded unmodified AM, the resulting engineered epithelium was thicker and more transparent when ultrathin AM was used as the scaffold. Further, the increased transparency and stratification of the epithelium on the ultrathin AM were maintained after implantation into a rabbit model . This strategy, while improving the transparency, did not address the lack of mechanical strength; however, that may not be necessary if it is used to engineer a full epithelium and glued as opposed to sutured into place, as was done in this study.…”

“…The resulting construct was ≈30 µm in thickness with improved transparency and was used as a substrate for cultured epithelial cells. ( Figure ) . When compared to denuded unmodified AM, the resulting engineered epithelium was thicker and more transparent when ultrathin AM was used as the scaffold.…”

“…In another approach, researchers investigated an alternative to chemical crosslinking to increase the transparency of the AM by creating ultrathin pieces by digestion of the stromal portion with collagenase IV . The resulting construct was ≈30 µm in thickness with improved transparency and was used as a substrate for cultured epithelial cells.…”

“…D) Hoechst whole mount staining of IAM, DAM, and UAM (Bar: 100 µm). Reproduced under the terms of the CC‐BY license . Copyright 2016, The Authors, published by Springer Nature.…”