Francesca Rescigno

Background/Objectives: VitroScreenORA^® (by VitroScreen srl) Dermo Papilla spheroids, based on two micro-physiological systems (non-vascularized DP and vascularized VASC-DP), were used to study the molecular mechanisms behind hair cycle regression. Methods: Dermal papilla cells (HFDPC) were cultured to develop both models. Hair cycle regression was induced by exposing DP spheroids to TGF-β1 for 72 h and/or FGF-18 for an additional 24 h. Catagen phase entrance was evaluated by modulating specific genes (FGF7, CCND1, and WNT5B). The VASC-DP model was obtained by sequentially co-culturing HFDPC and primary dermal microvascular endothelial cells (HMDEC), mimicking the surrounding capillary loop. The vascular system’s impact was assessed at 5 and 10 days using IF on CD31 (micro-vessels) and Fibronectin (FN). Nanostring nCounter^® technology was applied to investigate the transcriptional signature based on the WNT pathway. Extended culture time up to 11 days simulated natural hair cycle regression, monitored by versican and FN expression (IF). Minoxidil, Doxorubicin, and Retinol-based products were used to modify physiological aging over time. Results: Data shows that the vascular system improves tissue physiology by modulating the associated genes. Extended culture time confirms progressive DP structure degeneration that is partially recoverable with Retinol-based treatments. Conclusions: Both models provide a reliable platform to investigate the hair cycle, paving the way for new testing systems for personalized therapies.

Backgrounds/Objectives: Skin wound healing is a physiological process orchestrated by epithelial and mesenchymal cells able to restore tissue continuity by re-organizing themselves and the ECM. This research study aimed to develop an optimized in vitro experimental model of full-thickness skin, to address molecular and morphological modifications occurring in the re-epithelization and wound healing process. Methods: Wound healing starting events were investigated within an experimental window of 8 days at the molecular level by gene expression and immunofluorescence of key epidermal and dermal biomarkers. To mirror the behavior of infected wounds, the established wound healing model was then colonized with S. aureus, and the efficacy of a novel antibacterial agent, XZ.700, was investigated. Viable counts (CFU/tissue), IF, and ultrastructural analysis (SEM) were performed to evaluate S. aureus colonization inside and around the wound bed in an experimental window of 3 h of colonization and 24 h of treatment. Results: Endolysin showed an efficacy in counteracting bacterial growth and invasion within the wound bed, reducing the S. aureus load compared to its placebo, thanks to its selective antimicrobial activity interfering with biofilm formation. Conclusions: The preclinical in vitro infected wound model on FT-kin showed interesting applications to assess the repair efficacy of dermo-pharmaceutical and cosmetic formulations.

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Objective: Dermis spheroids from different donors (40 and 50 years old) were developed from primary fibroblasts to demonstrate their capacity to synthetize and organize the main dermal structural components when cultured in 3D microenvironment, forming endogenous de novo ECM according to their potential metabolic activity. Methods: Dermis spheroids were produced from primary human dermal fibroblasts at early passages in hanging drop culture system. Dermis models were characterized in terms of spheroid diameter, PICP release, collagen III and CD44 expression. Results: An increase of collagen III synthesis (101%) was found in the young donor compared to the old donor (23.5%) after seven days of culture by immunofluorescence. The progressive ECM assembly over the time and dermis maturation was showed by Masson’s trichrome staining and by immunofluorescence for collagen III and CD44; both molecules significantly accumulated in the dermal compartment from day seven to day 10 of culture with a global decrease for both spheroid models after 21 days of culture. Conclusion: Our results showed that specific culture conditions in the 3D scaffold-free microenvironment allowed the physiological and progressive ECM assembly of miniaturized dermis models reflecting phenotypic profile features of “young” and “old” native tissue from which cells were isolated with a potential application to personalized care approaches in dermatological research on aging processes and medicine.