## 4 Discussion
Concomitantly, cells irradiated with violet-blue and NIR light exhibited augmented expression of focal adhesion proteins namely vinculin, which appeared mainly aggregated at the end of stress fiber-like structures, and p-FAK which, among other functions, is required for stress fiber formation in fibroblasts [62]. This may be an interesting finding when considering that the formation of mature adhesion complexes associated to robust actin stress fibers results in force transmission from myofiblabats to the surrounding ECM, essential for the contraction and closure of healing wounds [63]. Of note, we found that the cells exposed to red PBM also showed an up-regulation of the expression levels of vinculin. This is not surprising when considering that, beside its structural and adhesive role in focal adhesion contacts for cell/matrix connection, vinculin may also act as signaling protein in cell mechanotransduction and in the modulation of several molecular signaling, involved in differentcellular processes such as survival, growth/proliferation (e.g., ERK) and differentiation in many types of cells, including those of mesenchymal origin [64; 65; 66; 67]. In this study we also highlighted the capability of violet-blue and NIR light to stimulate the secretive ability of the cells in terms of type-1 collagen synthesis, differently from red light. Interestingly, the different collagen expression in the cells subjected to the different PBM, well correlated with cell morphology and cytoskeleton organization we observed. Indeed, it has been reported that fibroblasts exhibiting a reduced assembly of actin cytoskeleton and with rounded or elongated aspect, showed a decline of type-1 collagen production [68; 69]. Of interest, FAK-dependent collagen synthesis by fibroblasts has been reported, which may contribute to further support our conclusions concerning the effect of violet-blue and NIR light in stimulating the secretive ability of fibroblasts [70]. We also found that the cells treated with violet-blue and even more NIR PBM showed the cytoplasmic expression of \(\alpha\)-sma. Even if this protein does not appear organized along the stress fiber-like structures as occurring in mature myofibals, our findings further suggest the potential acquisition of a proto-myofiboblastic phenotype after violet-blue and NIR PBM. However, the effects of violet-blue or NIR PBM on myofiblast differentiation and function deserve to be investigated deeper in our cell model. By contrast, red PBM treated cells did not exhibit \(\alpha\)-sma expression according with previous reports showing the capability of red PBM to inhibit in vitro TGF-\(\beta\)1/Smad3-mediated transition of fibroblasts towards myofibals or to reduce in vivo myofiblast population and collagen deposition [4; 71]. Finally, we found that red PBM was able to induce an increase of MMPs/TIMPs ratio and promote MMPs' functionality, differently from violet-blue and NIR PBM treatments that elicited an opposite effect. Besides the role of these enzymes as secreted proteins on the ECM, involved in the maintenance of a fine balance between ECM synthesis and degradation, essential for proper wound healing (e.g., necessary for favoring cell migration and provisional scar remodeling and removal), it appears worth mentioning that a positive correlation between MMPs' expression/function and proliferation of different cells has been proposed [35; 72; 73]. Thus, the possibility that tested PBM treatments influence fibroblast proliferation via MMPs' modulation may not be excluded. In this study, we only tested an energy density of 0.4 J/cm\({}^{2}\) for red, violet-blue and NIR light essentially to compare only the effects of different wavelengths on fibroblast responses, eliminating the potential puzzling effect of energy density. This energy density value has been demonstrated to be an optimal one, safe and able to exert biostimulatory effects on the cells in different experimental in vitro conditions [4; 5; 34; 74].