Cellular response of limbal epithelial cells on electrospun poly-ε-caprolactone nanofibrous scaffolds for ocular surface bioengineering: a preliminary in vitro study.

Purpose: The aim of this study was to develop a synthetic stromal substrate for limbal epithelial cell (LEC) expansion that can serve as a potential alternative substrate to replace human amniotic membrane (HAM).

Methods: Nanofibers were fabricated using 10% poly-ε-caprolactone (PCL) solution dissolved in trifluoroethanol (TFE) via an electrospinning process. Nanofibers were characterized for surface morphology, wetting ability, pore size, mechanical strength, and optical transparency using scanning electron microscopy (SEM), contact angle measurement, microtensile tester, and UV-Vis spectrophotometer, respectively. The human corneal epithelial (HCE-T) cell line was used to evaluate the biocompatibility of nanofibers based on their phenotypic profile, viability, proliferation, and attachment ability. Subsequently, human LECs were cultivated on biocompatible nanofibers for two weeks and their proliferation capability analyzed using MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole)) proliferation assay. Immunofluorescent (IF) staining and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to check the molecular marker expression; SEM was used to study the morphology.

Results: The average fiber diameter of PCL was 132±42 nm. Pore size varied from 0.2 to 4 microns with a porosity of 85%. The tensile strength of the PCL membrane was 1.74±0.18 MPa (Mega Pascal); strain was 30.08±2.66%. The water contact angle was 90°. Biocompatibility results indicated that the polymer surface was highly biocompatible, as HCE-T cells could favorably attach and proliferate on the polymer surface. SEM figures showed that the corneal epithelium was firmly anchored to the polymer surface via a continuous cell sheet and was able to retain a normal corneal phenotype. MTT assay confirmed that cells were metabolically active on nanofibers (p<0.05) and gradually increased in their number for up to two weeks. IF and RT-PCR results revealed no change in the expression profile of LECs grown on nanofibers when compared to those grown on glass coverslips and human amniotic membrane (HAM). Confocal microscopy illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional (3D) corneal epithelium, which was viable for two weeks.

Conclusions: Electrospun nanofibers provide not only a milieu supporting LEC expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to HAM.