Q-switched Nd-YAG laser alone and in combination with innovative hyaluronic acid gels improve keratinocytes wound healing in vitro

Anna De Filippis,Anna Virginia Adriana Pirozzi,Maria Antonietta Tufano,Chiara Schiraldi,Adone Baroni,Antonella D’Agostino

doi:10.1007/s10103-020-03145-5

Anna De Filippis, Anna Virginia Adriana Pirozzi + Show 4 more

Open Access

https://doi.org/10.1007/s10103-020-03145-5

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Abstract

During the last years, several attempts have been accomplished to improve the wound healing. Device application aimed at enhancing skin ability to reconstruct its damaged sites through a proper dermal regenerative process. In particular, Q-switched Nd-YAG laser (Medlite C6 laser, Conbio, USA) applied with a fluence of 8 J/cm2, a pulse width of 5 ns, and a spot size of 4 mm exerts a photo-mechanical action that improve skin repair. Besides, hyaluronan hybrid cooperative complexes (HCC) widely exploited in dermoesthetic applications proved specific actions on keratinocytes and fibroblasts monolayer repair. We evaluated this specific laser treatment in vitro on a wound healing model based on human keratinocytes (HaCaT) alone and in combination with HCC. In addition, we evaluated key biomarkers of dermal repair. Scratched HaCaT monolayers were treated with laser and successively with HA-based formulations (HHA and HCC). For each treatment and the control samples, at least 3 different wells were analyzed. Wound closure was quantified, measuring five view filed for each well at increasing incubation time, exploiting time lapse videomicroscopy and image analysis, permitting to compare the different healing rate of treatments respect to control. By real-time PCR and western blotting, we evaluated biomarkers of wound regeneration, such as integrins, aquaporin three (AQP3), and proinflammatory cytokines. The ANOVA test was used to assess statistical significance of the results obtained. Laser-treated cells achieved wound closure in about 37 h, faster than the control, while when coupled to HCC, the complete reparation was obtained in 24 h. Integrin αV was upregulated by treatments, with in particular about four-fold increase respect to the control when HCC + laser was used. In addition, integrin β3 was upregulated by all treatments especially with the combination of laser and HCC proved more efficient than others (~ 14-folds). A slighter but significant increase of AQP3 gene expression of 61% was found for laser treatment while the latter combined with HCC determined an upregulation of 72%. By coupling laser treatment and HCC, further healing improvement and consistent biomarker modulation was observed. Our results may support clinical implementation of new dermatology protocols conjugating laser treatments with topical or injective HA formulations as a valid tool in treatments to repair scars or other skin defects.

Highlights

Wound healing is a physiological dynamic complex response to tissue damage, which consists of three overlapping phases: inflammation, tissue formation, and tissue remodeling [1]
We found activation of αV and β3 integrins, whose expression increased until 48 h, especially for hybrid cooperative complexes (HCC) in combination with laser treatment
In our previous study about effects of laser on markers of rejuvenation [24], we have demonstrated for the first time that laser increases aquaporin 3 (AQP3) gene expression in human keratinocytes, favoring moisturizing and barrier function of skin

Summary

Introduction

Wound healing is a physiological dynamic complex response to tissue damage, which consists of three overlapping phases: inflammation, tissue formation, and tissue remodeling [1]. It is well established that a pivotal role is played by a superbly orchestrated cross-talk among blood cells, parenchymal cells, soluble mediators, and extracellular matrix, which lead together to proper wound repair and tissue regeneration, restoring the skin barrier [1]. Aquaporins (AQPs) are intrinsic membrane proteins involved in water transport, among them aquaporin 3 (AQP3) is involved in glycerol transport reported as “aquaglyceroporin,” localized in the skin and to the basal epidermis layer [3] and sebaceous glands [4]; AQP3 promotes the migration and proliferation of keratinocytes during healing [3]. Most of them may act as “start signals” able to trigger relatively sedentary cell lineages at the wound margin, to fill the wound site, prompting proliferation and synthesis of new extracellular matrix [2]

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