Development and Characterization of Decellularized Human Corneal Stroma as a Scaffold for Tissue Engineering

Abstract

Purpose: To evaluate the potential use of decellularized human corneas as a scaffold for cultivating human corneal endothelial cells.

Methods: Human corneal tissues (N=20) not suitable for transplantation were used. Corneal endothelial cells were isolated by explant culture method. For preparation of the scaffold, corneal epithelium and endothelium were removed mechanically and remaining corneal stroma was decellularized using enzymatic method.The resulting acellular matrices were then subjected to Haematoxylin-Eosin (H&E) staining to visualize cellular remnants; quantitative analysis to determine the DNA content; Scanning Electron Microscopy (SEM) for collagen fibril morphology; Alcian blue staining to analyse extracellular matrix (ECM); Immunohistochemistry for structural proteins collagen type I, II, IV, fibronectin; and cytotoxicity assay of decellularized cornea using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide. Tensile strength of corneal tissues &Light transmission ratios was estimated using uniaxial load testing equipment &UV-visible spectrophotometer. Native cornea served as control for all the experiments. 10,000 human corneal endothelial cells were cultured on decellularized scaffold for 14 days and then analyzed using SEM, histology and Immunocytochemistry for ZO-1, and Na+ /K+-ATPase.

Results: H&E staining demonstrated efficient elimination of cellular components. Alcian blue confirmed good preservation of the extracellular matrix and major structural proteins collagen type I, II IV & fibronectin were retained. The amount of DNA in decellularized cornea was 32+ 7.27ng/mg whereas in native cornea it was 133+8.3ng/mg(p<0.05).The tensile strain at break was 38.8% & 42.4%;Young’s Modulus was 0.10 MPa& 0.14MPa and light transmittance was 24.5% &22.7% in decelluarized and native corneas respectively. In vitro cytotoxicity assays excluded the presence of soluble toxins. Corneal endothelial cells could be efficiently cultured and expanded on the acellular matrix. The SEM & Alizarin red staining of cultured cells over decelluarized stroma showed surface covered with a uniform monolayer of cells and they also expressed functional markers Na+ /K+-ATPase & ZO-1.

Conclusions: Decellularized corneal stroma retains biomechanical and functional properties to support endothelial cell proliferation and expansion.

Keywords: 481 cornea: endothelium • 484 cornea: stroma and keratocytes