Effect of Helichrysum italicum in Promoting Collagen Deposition and Skin Regeneration in a New Dynamic Model of Skin Wound Healing

Abstract

Natural products have many healing effects on the skin with minimal or no adverse effects. In this study, we analyzed the regenerative properties of a waste product (hydrolate) derived from Helichrysum italicum (HH) on scratch-tested skin cell populations seeded on a fluidic culture system. Helichrysum italicum has always been recognized in the traditional medicine of Mediterranean countries for its wide pharmacological activities. We recreated skin physiology with a bioreactor that mimics skin stem cell (SSCs) and fibroblast (HFF1) communication as in vivo skin layers. Dynamic culture models represent an essential instrument for recreating and preserving the complex multicellular organization and interactions of the cellular microenvironment. Both cell types were exposed to two different concentrations of HH after the scratch assay and were compared to untreated control cells. Collagen is the constituent of many wound care products that act directly on the damaged wound environment. We analyzed the role played by HH in stimulating collagen production during tissue repair, both in static and dynamic culture conditions, by a confocal microscopic analysis. In addition, we performed a gene expression analysis that revealed the activation of a molecular program of stemness in treated skin stem cells. Altogether, our results indicate a future translational application of this natural extract to support skin regeneration and define a new protocol to recreate a dynamic process of healing.

Keywords: Helichrysum italicum; bioreactor; dynamic cultures; fibroblasts; molecular mechanisms; stem cells; tissue regeneration; wound healing.

Figure 1

Analysis of collagen deposition in SSCs during wound healing. Immunohistochemical analysis of the expression of collagen type I (red) was assessed in SSCs after scratch assay and treatment with two different concentrations of HH (30% and 20%). Control cells (CTRLs) were maintained in basic growing medium after scratch assay. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars: 40 µm, magnification 40×. The figures are representative of different independent experiments.

Figure 2

Analysis of collagen deposition in HFF1s during wound healing. Immunohistochemical analysis of the expression of collagen type I (red) was assessed in HFF1s after scratch assay and treatment with two different concentrations of HH (30% and 20%). Control cells (CTRLs) were maintained in basic growing medium after scratch. Nuclei are labelled with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars: 40 µm, magnification 40×. The figures are representative of different independent experiments.

Figure 3

Gene expression analysis of Sox2 (a), Oct-4 (b), and NANOG (c) in SSCs cultured in the presence of the different concentrations of HH after the scratch assay. The expression of each gene was normalized to GAPDH and plotted as the fold change (2^−∆∆Ct) relative to the mRNA expression of the untreated control (CTRL); * p value ≤ 0.05.

Figure 4

Gene expression analysis of p16 (a), Bmi1 (b), and TERT (c) in SSCs cultured in the presence of the different concentrations of HH after the scratch assay. The expression of each gene was normalized to GAPDH and plotted as the fold change (2^−∆∆Ct) relative to the mRNA expression of the untreated control (CTRL); * p value ≤ 0.05.

Figure 5

Scheme represents the chamber (Live Box2) and the cells cultured: SSCs (1) and HFF1s (2). The chamber connects to a reservoir of culture medium (R) by a peristaltic pump (PP) (blue lines and arrows in the scheme).