Tripodi, Nicholas ORCID: 0000-0001-5062-0409 (2023) The molecular and cellular effects of polarized photobiomodulation on human fibroblasts in vitro. PhD thesis, Victoria University.

Abstract

Photobiomodulation (PBM) is a widely-used clinical therapy used to treat a myriad of different conditions, including in the treatment of wounds. Despite the utility of this therapy, the underpinning mechanisms of its biological effects remain unclear. The leading hypothesis is centred on mitochondrial cytochrome C oxidase (CcO). It posits that the photons emitted during PBM interact with CcO, in the process displacing Nitric Oxide, which allows oxygen to interact with CcO more readily, hence improving cellular metabolism. However, more contemporary research has shown that PBM can improve cellular biological functions in the absence of CcO. Beyond, the fundamental mechanisms of PBM there also remains debate concerning the optimum light exposure and treatment protocols of PBM. Variables such as wavelength, power, irradiation time, beam area, radiant energy, fluence, polarization state, pulse parameters and treatment cycles, are all factors which can influence the outcome of PBM. Of these, polarization—the property of light that specifies the direction of the oscillating electric field—is an intriguing variable to investigate. There is a small, but growing body of research that demonstrates that polarized PBM (P-PBM), when compared to otherwise matched non-polarized PBM (NP-PBM) may increase the biological efficacy of this therapy. Despite these promising results, more research is needed to elucidate the mechanistic changes that polarization can influence in the field of PBM. Therefore, this project aims to model the molecular and cellular effects of P-PBM in vitro, in a cell type known to be critical in the wound healing response, namely fibroblasts. Specifically, this project will compare the biological effects of P-PBM and NP-PBM on fibroblast cells in a model which represents the oxidative stress conditions found in chronic wounds. Firstly, in this thesis, a custom light source and stage is designed, constructed and profiled showing good intra-experiment reliability. From here the optimum light irradiation and cell culture parameters were determined through a series of pilot studies utilising multiple cellular viability, proliferation and apoptosis measurements. Using the aforementioned protocols, the effect of P-PBM compared to NP-PBM is profiled through cellular proliferation, migration, mitochondrial membrane potential and apoptosis studies. These results showed that largely, P-PBM exerts greater cell proliferative, metabolic and protective effects, when compared to NP-PBM and appropriate controls. Finally, the transcriptome of human dermal fibroblasts in response to PBM is profiled. This analysis demonstrated a number of differentially expressed genes related to both the mitochondria and extracellular matrix, as well as multiple significantly enriched ontological pathways. In sum, this project demonstrates that P-PBM in this setting, can exert a greater biological effect compared to otherwise matched NP-PBM and experimental controls, which has future applications in the treatment of wounds. Additionally, it demonstrates that PBM appears to influence multiple parts of the mitochondria, in addition to CcO, better shaping the fundamental underpinnings of PBM.

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