Morphological and kinetic study of oral keratinocytes assembly on reconstituted basement membrane: Effect of TEGDMA

Abstract

Objective: Open wounds of oral cavity require rapid healing. The cytotoxic monomer, triethylene glycol dimethacrylate (TEGDMA) can leach out from dental restoratives, reach the oral epithelial barrier and trigger an immune response. It is speculated that low and moderate concentrations of TEGDMA (0.5 and 1.5 mmol/L, respectively) influence the assembly kinetics and morphology of the keratinocyte layers overlying the extracellular matrix (ECM) in vivo. A three-dimensional cell system composed of immortalized oral keratinocytes (iMOK) cultured on reconstituted basement membrane (ECM) was used to investigate the development of epithelial layers upon exposure to TEGDMA.

Methods: Adherence and opposing movement of adjacent keratinocytes using actin protrusions (lamellipodia and filopodia) to create spheroids, and their fusion capacity to establish subsequent layers were tested at different time points. Fluorescent, confocal, differential interference contrast microscopy and image processing were employed to quantify the morphological modifications over time.

Results: Increasing concentrations of TEGDMA decreased the number of viable cells that utilized the actin protrusions and led to a delay in the communication/interaction among cells. Consequently, cells assembly was affected and the formation of more than a single layer prevented. Areas of basal-like proliferating cells were replaced with the increasing areas of non-replicating large cell population and extended gaps.

Conclusions: These findings suggest that TEGDMA may prevent rapid sealing of open wounds by keratinocytes and suppress the establishment of a resistant and impermeable barrier against pathogen internalization. The iMOK-ECM-based platform facilitated the validation and quantification of solubilized dental materials impact on the reconstitution of epithelial layer.

Keywords: 3D cell culture; Actin protrusions; Assembly kinetics; Cytotoxicity; Dental restoration; Oral keratinocytes; Triethylene glycol dimethacrylate.