The penetration depth of blue light through epidermal tissues is reportedly limited to approximately 1 mm [29]. As the light passes through the epithelial layers to the underlying laminar propria, light is scattered and absorbed by photo-receptors within the resident keratinocytes and fibroblasts [29, 30], with potential to influence cellular activity. Oral mucosal tissues are approximately 0.3 mm thick, thinner than dermal tissues, and while the bulk of the oral epithelium presents as a keratinising protective barrier, non-keratinised epithelial cells of the sulcular and junctional epithelia surround the neck of the tooth [31, 32]. Consequently, any blue light treatment which has proven to be effective in reducing bacterial load in periodontal pockets [2,3,4], may also evoke cellular responses within surface facing keratinocytes, which may also penetrate through to the underlying fibroblasts populations that dominate these tissues. Within this study, we present novel data to indicate that blue light irradiation can increase the metabolic activity of pHGKs, with minimal effect for induction of apoptosis or ROS generation. Conversely, blue light irradiation is capable of significantly reducing the metabolic activity and increasing apoptosis of pHGFs, which our results would suggest is mediated by transient ROS generation.

The oral mucosa acts as a protective barrier and optimal wound healing is essential in the preservation of a functional healthy tissue. Keratinocytes play an important role in the constant re-epithelisation, supported by their high migratory and proliferative ability. Within the present study, irradiation of keratinocytes to blue light with fluence levels of 36 J/cm2 and above increased the metabolic activity, with statistical significance. These fluences represented a range of irradiance levels (300 or 100 mW/cm2) and duration (120–600 s), but responses were only dose-dependent upon the overall increasing level of fluence. Increases in metabolic activity detected by the reduction of Alamar Blue in the cytosolic environment directly correlate to an increase in the viable cell count, compared to untreated controls. Additionally, our results would indicate that following irradiation with blue light, pHGK do not experience significant oxidative stress, as near basal levels of ROS production were measured over the 30 min, following blue light treatment. Similarly, levels of LDH remained close to basal levels, suggesting blue light does not induce apoptosis. These observations align with previous studies that have determined the levels of enzymic antioxidants and products of oxidation within oral and dermal tissue biopsies [23]. Of note, immunodetection of SOD1, SOD2, SOD3 and catalase is considerably higher in the epidermal cells of both these tissues, compared with cells of the underlying laminar propria; a finding that correlates with the detection of significantly lower levels of oxidative stress-induced, biomolecular damage within epithelial tissues [23]. To the authors knowledge at time of publication, the results from the present study represent the first observation of the effect of blue light on the cellular behaviour on primary human gingival epithelial cells, but our results do align with similar studies which identified negligible effects of blue light on dermal keratinocytes, potentially reflecting genotypic and phenotypic differences between oral mucosal and dermal epithelial cells [18, 23, 33]. Collectively, these findings would suggest that epithelial cells of the oral mucosa possess good intracellular mechanisms to withstand blue light-derived, oxidative stress. However, this conclusion should be balanced against the observations that blue light can induce DNA damage in human dermal epithelial cells, which were reparable through base excision repair and nucleotide repair pathways [34]. Within dermal tissues, the generation of DNA damage has been linked with premature cell senescence and skin aging [34]. The nature of DNA damage caused by blue light in gingival epithelial cells is yet to be determined.

Within the oral mucosa, fibroblasts play important roles within wound healing; providing paracrine signalling between the different cell types, including the epithelial and immune cells and orchestrating angiogenesis, collagen synthesis and ECM formation. Moreover, fibroblasts of the oral mucosa are genotypically and phenotypically “younger” than dermal fibroblasts, which has been attributed to their preferential wound healing and minimal scarring properties influenced by differences in matrix metalloproteinase-mediated ECM remodelling and lower TGFβ1 secretion that limit myofibroblast differentiation, collagen deposition and fibrosis [24, 26, 35,36,37]. Noting different response to those observed by pHGKs, the irradiation of pHGFs resulted in a small dose dependent decrease in the metabolic activity. Although this decrease was only statistically different for cells irradiated with fluence of 30 J/cm2, a corresponding increase in apoptosis is also witnessed, which may account for the decrease in viable cells. The observed differences in cellular responses of gingival epithelial and fibroblasts cells is also reported in dermal tissues, with fibroblasts more susceptible to blue light resulting in decreased proliferation and induced cytotoxicity [14, 18]. Opsins are intracellular light sensitive G protein-coupled receptors and following light activation opsins can activate a myriad of cell signalling pathways, including wound healing responses in skin [38, 39]. All four opsins have been reported to be present in the epidermal and dermal layers of the skin [38], with cells of the two layers likely to express different opsin profiles, resulting in light of variable wavelengths triggering different cell signalling pathways. However, it is notably that OPN3 (also known as encephalopsin/panopsin and sensitive to light within the 460–470 nm blue light range) are highly expressed in fibroblasts [38]. Such difference may partially account for the higher cytotoxic influences of blue light on fibroblasts cell populations. Differences in opsin profiles, may also partially account for the higher cytotoxic effects of 418 nm violet irradiation on both the pHGFs and pHGKs used in this study, with OPN1 activated by light with an approximate 425 nm wavelength [38, 39]. Violet light has been shown to cause higher levels of oxidative stress, lysosomal and mitochondrial damage which has the capacity to act as a stressor of cellular homeostasis through the accumulation of lipofuscin, a granular pigment product arising from oxidized lipids, proteins, and metal ions [40].

Cells irradiated in the presence of the ROS scavenger NAC, saw significant reductions in the generation of ROS, most evident in pHGFs at the higher dose of 60 J/cm2. The presence of NAC during blue light treatment also reduced the generation of ROS in pHGKs, although observed differences between NAC treated and untreated cells did not reach statistical significance. NAC ameliorates oxidative challenges through the provision of sulfhydryl groups, which serve as a precursor to replenish GSH levels, thus driving GSH/GSSG ratios in favour of a reducing environment [41, 42]. The concentration of NAC during blue light treatment, therefore, dictates the amount of ROS scavenged. Nonetheless, our results suggest that blue light treatments do promote ROS production.

Blue light treatment can also disrupt intrinsic molecular function triggered by oxidative stress induced by ROS generation, most notably superoxide radical species (O2.−) generated via mitochondrial electron transport chain activity [43]. In alleviating the resultant oxidative stress, intracellular generation of ROS is also known to mediate a variety of cellular responses, such as NRF2 signalling that regulates antioxidant gene transcription [44]. In observing the detrimental effects of blue light in oral fibroblasts, this study continued to investigate changes in the gene transcription of a wide range of mitochondrial antioxidant candidates in pHGFs and pHGKs, identifying only minimal changes. Of note, small increases (approximately 2-fold) were observed in mRNA levels for NQO1, GSR and GSS in pHGKs, which were slightly more pronounce in pHGFs. However, further analyses suggested that these increases did not yield a consequential change in the protein levels in the fibroblast populations examined within this study. Antioxidant levels for SOD1, SOD2 and catalase remained relatively unchanged. Immunohistochemical analysis for oral mucosal tissues have demonstrated the constitutive presence of these antioxidants associated with the epithelial cells and fibroblasts of the lamina propria [23], and these levels may have been appropriate to neutralise any transient production of ROS generated by blue light treatment. Similar justification may be true in considering no changes were noted for glutathione peroxidase 1 and 4.

All the above intracellular antioxidants are themselves regulated at a gene level by Nuclear Factor Erythroid 2-related Factor 2 (NRF2) [44, 45], and it is noted that blue light likewise did not induce any significant changes in the gene expression of this key regulatory transcription factor. However, the activity of NRF2 is tightly regulated by KEAP1. Whist no significant increases in KEAP1 were observed, under oxidative stress conditions, KEAP1 is known to be oxidised at reactive cysteine residues resulting in KEAP1 inactivation. This event subsequently results in the release of tethered cytosolic NRF2 which translocates to the nucleus, where it binds with antioxidant response elements to initiate gene transcription of genes, including NQO1 and GSS [45]. Within this study, observed by immuno-localisation of the protein NRF2 in the cytosol of pHGFs and pHGKs, we have obtained preliminary evidence to suggest that blue light treatment is associated with the movement of NRF2 to align with the cytoskeleton and engage with the nucleus (results not included), presumably in the activation of antioxidant response element genes.