The effect of near-infrared photobiomodulation on in vitro cornea wound healing.

Abstract

Purpose : Photobiomodulation (PBM) is the use of non-ionizing light sources such as lasers and light-emitting diodes (LED) to alter cellular environments. While the effect of PBM on chronic wounds, skin defects, and second degree burns have been explored extensively, the potential of near infrared PBM in improving ocular wound healing and the cellular mechanisms associated with this treatment remains unclear. To gain a better understanding of the effects of near-infrared radiation (NIR) on the cornea, human corneal epithelial cells were exposed in vitro to varying levels of NIR. Cell viability, wound closure rates, mitochondria function, reactive oxygen species (ROS) and integrin expression were assessed.

Methods : HPV-immortalized human corneal epithelial cells (HCEC) were grown to confluence in tissue-culture treated plates or PET inserts. Cells were cultured using serum-free keratinocyte medium. The cells on PET inserts were then differentiated by exposing the monolayer to an air-liquid interface and feeding the basal side with 2% FBS in 1:1 DMEM/F12 (Postnikoff et al, 2014).
An in-house NIR in vitro testing LED platform controlled by an Arduino was used to expose cells to various NIR regimens. A scratch wound was created using a pipette tip and cells were treated daily with NIR for 10 minutes for up to 4 days. Monolayer cell viability was assessed using MTT and flow cytometry was used to characterize ROS, integrins α₃, α₅ and β₁. Images of the wound were captured daily by microscopy and wound dimensions were determined digitally.

Results : When compared to control cells (no NIR exposure), no difference in cell death and ROS were observed in cells exposed to NIR LED for energy levels up to 100 mW/cm². In both monolayer and stratified cultures, cell exposure to low energy levels of NIR resulted in a significant increase (p<0.05) in wound healing. HCEC exposed to NIR produced more ATP and significant changes in the level of expression of α₃, α₅ and β₁ (p<0.05) were also observed at 24 hours in cells exposed to NIR, highlighting the positive effects of NIR on HCEC.

Conclusions : Using monolayer and stratified cultures, our results indicate that near-infrared radiation promotes wound healing in human corneal epithelial cells in vitro and suggest that PBM treatment could be an effective therapeutic strategy for corneal wounds. Further in vivo studies are needed to demonstrate the safety and efficacy of NIR treatment.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.