Scratch assay is a consolidated practice in our laboratory and TLVM helps not only to empirically follow biological phenomena but also to quantify some of those. In this way, in a single multiwell, different events may be analyzed and diverse treatments compared contemporary on the same human cells (same passage, same incubation conditions). For the first time, Q-switched 1064 nm Nd:YAG laser treatments were compared to control in scratched HaCaT monolayer. Preliminary viability evaluation with MTT test showed that there is an initial stress caused by laser treatment that in fact reduced metabolic activity of cells treated respect to the ones added with hyaluronan or control. This finding is in agreement with the slower reparation rate found in wound healing assay, at least in the first 18–24 h.
Thus in this experimental model, laser treatment proved to fasten reparation. However, when we included HA-based treatments, these were the most powerful in increasing the repair rate, in fact, overall the wound closure occurred earlier. In agreement with previous studies, we confirmed that HCC is the best performing gel in keratinocytes monolayer regeneration. However, restoring the epidermis thickness and barrier integrity in wound healing require a strong integration of signaling molecules such as cytokines, growth factors, integrin, ECM, and metalloproteases. To better highlight the possibly occurring synergy between laser treatments and HA in skin repair, biochemical key biomarkers were investigated. Scratched keratinocytes, treated with laser, showed IL-1 alpha, IL-1 beta, and TNF-alpha downregulated gene expression mostly after 48 h compared to untreated controls. All HA-treated samples also reduced the cytokines tested. Finally, coupled treatments succeeded in further reduction, especially laser + HCC treatments, that for IL-1 alpha and IL-1 beta at 48 h showed the lowest expression level. TNF-alpha was reduced already at 24 h both for laser and laser + HCC treatments, specifically the latter showed a maximal 1.8-fold reduction respect to control, and an improvement of 30% respect to the sole laser treatment (Fig.
3). Proinflammatory cytokines favor the inflammatory phase of wound healing, prompting the clearance of microorganisms and stimulating the expression of growth factors. Among those, we chose to evaluate the effect of laser on TGF-beta expression, which represents the most important ligand, in keratinocyte migration, during re-epithelialization [
19]. TGF-beta as anti-inflammatory cytokine reduces the persistent inflammatory state, associated at chronic wound, that alters the normal wound healing. It is a growth and differentiation factor; it acts in remodeling by stimulating the synthesis of collagen and fibronectin [
20] and thus the deposition of the extracellular matrix, important for re-epithelization. In laser-treated keratinocytes, there is an increase in the gene expression of TGF beta after 24 h; HHA-treated samples after laser treatment do not show a higher expression, while laser + HCC proved slightly superior both at 24 and 48 h (Fig.
3). At these times, we found activation of αV and β3 integrins, whose expression increased until 48 h, especially for HCC in combination with laser treatment. These data were confirmed at protein level. Cytokines promote the expression of many classes of adhesion molecules. These adhesion molecules are determining factor for the diapedesis of neutrophils, including selectins and integrins which interact with those already present on the membrane surface of endothelial cells. The expression of αV and β3 during re-epithelization favors binding to fibronectin, vitronectin, and collagen to initiate epithelial migration on a provisional matrix. Integrins play a central role in cell adhesion contacts function as signaling centers, and the linkages between ECM and actin cytoskeleton allow adhesion sites to serve as traction sites for cell movement [
21]. Another interesting aspect of this study concerns the modulation of AQP3 expression in scratched keratinocytes monolayers treated with laser. Besides, this specific biomarker was often investigated in the characterization of biological response of HA-based fillers [
22]. The skin of deficient mice in AQP3 shows reduced hydration, reduced elasticity, and delayed barrier formation as a result of damage [
23]. In our previous study about effects of laser on markers of rejuvenation [
24], we have demonstrated for the first time that laser increases AQP3 gene expression in human keratinocytes, favoring moisturizing and barrier function of skin. Among its different functions, AQP3 promotes the migration and proliferation of keratinocytes during healing [
25,
26]. In fact, wound closure was delayed in AQP3-deficient keratinocytes. Hara and collaborators have, furthermore, showed that glycerol administration in the diet corrects healing delay in AQP3-null mice, favoring not only migration but also proliferation of keratinocytes [
26]. We investigated AQP3 expression that was significantly increased by laser treatments compared to untreated controls; further increment was found by coupling HCC treatments to laser. The increase proved time dependent and the maximum expression was observed at 48 h. AQP3 upregulation suggests that laser effects are expressed not only on accelerated healing of the wound, but also on hydration, elasticity, and restoration of the barrier during skin care, as supported also by protein expression level. Q-switched laser on scratched HaCat and fibroblast monolayers was used in previous studies [
27]. However, this is the first time that time lapse videomicroscopy is used to quantify healing rate, that laser treatment is combined to HA formulation, and that a whole array of biomarkers is assessed in these experimental conditions both with RT-PCR and western blotting. In our opinion, the most interesting part of this research resides into the potential synergic effect of the laser treatments with HCC. The latter are used in dermatology and esthetic medicine; however, being a recently proposed formulation and having showed peculiar and interesting behavior on fibroblast and stem cells are good candidates to new approaches and combined clinical treatments.
In fact, our results suggest that these treatments may contribute to enhance tissue repair, to reduce chronic inflammation in the wound, as previously reported [
27,
28]. Besides, laser treatment was already reported to favor hydration and to enhance antimicrobial molecules expression (HBD-2), also HCC proved efficient in this respect [
27,
28], and in prompting migration [
29]. Due to these results and the supportive previous scientific literature, complications due to cells overgrowth as hypertrophic or keloid scarring can be solved with supportive therapies, such as the use of Q-switched Nd:YAG laser eventually coupled to HA and above all HCC treatments. Excessive scarring can dramatically influence the patient’s quality of life both physically and psychologically.
These results may support clinical dermatologist to assess new protocols conjugating laser treatments with the application of topical or injective HA-based gels to improve bioremodelling in scars/skin defects/keloids/fibrotic scars. Resolving the excessive inflammation, the laser treatments activate stop signals that block further formation of fibrosis or hypertrophic scar.