Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.
The treatment of chronic wounds still challenges modern medicine because of these wounds’ heterogenic pathophysiology. Processes such as inflammation, ischemia and bacterial infection play major roles in the progression of a chronic wound. In recent years, preclinical wound models have been used to understand the underlying processes of chronic wound formation. However, the wound models used to investigate chronic wounds often lack translatability from preclinical models to patients, and often do not take exaggerated inflammation into consideration. Therefore, we aimed to investigate prolonged inflammation in a porcine wound model by using resiquimod, a TLR7 and TLR8 agonist. Pigs received full thickness excisional wounds, where resiquimod was applied daily for 6 days, and untreated wounds served as controls. Dressing change, visual documentation and wound scoring were performed daily. Biopsies were collected for histological as well as gene expression analysis. Resiquimod application on full thickness wounds induced a visible inflammation of wounds, resulting in delayed wound healing compared to non-treated control wounds. Gene expression analysis revealed high levels of IL6, MMP1 and CD68 expression after resiquimod application, and histological analysis showed increased immune cell infiltration. By using resiquimod, we were able to show that prolonged inflammation delayed wound healing, which is often observed in chronic wounds in patients. The model we used shows the importance of inflammation in wound healing and gives an insight into the progression of chronic wounds.
Burn injuries initiate numerous processes such as heat shock response, inflammation and tissue regeneration. Reliable burn models are needed to elucidate the exact sequence of local events to be able to better predict when local inflammation triggers systemic inflammatory processes. In contrast to other ex vivo skin culture approaches, we used fresh abdominal skin explants to introduce contact burn injuries. Histological and ultrastructural analyses confirmed a partial-thickness burn pathology. Gene expression patterns and cytokine production profiles of key mediators of the local inflammation, heat shock response, and tissue regeneration were analyzed for 24 h after burn injury. We found significantly increased expression of factors involved in tissue regeneration and inflammation soon after burn injury. To investigate purely inflammation-mediated reactions we injected lipopolysaccharide into the dermis. In comparison to burn injury, lipopolysaccharide injection initiated an inflammatory response while expression patterns of heat shock and tissue regeneration genes were unaffected for the duration of the experiment. This novel ex vivo human skin model is suitable to study the local, early responses to skin injuries such as burns while maintaining an intact overall tissue structure and it gives valuable insights into local mechanisms at the very beginning of the wound healing process after burn injuries.
Purpose Dermal open flow microperfusion (dOFM) has previously demonstrated its utility to assess the bioequivalence (BE) of topical drug products in a clinical study. We aimed to characterize the sources of variability in the dermal pharmacokinetic data from that study. Methods Exploratory statistical analyses were performed with multivariate data from a clinical dOFM-study in 20 healthy adults evaluating the BE, or lack thereof, of Austrian test (T) and U.S. reference (R) acyclovir cream, 5% products. Results The overall variability of logAUC values (CV: 39% for R and 45% for T) was dominated by inter-subject variability (R: 82%, T: 91%) which correlated best with the subject’s skin conductance. Intra-subject variability was 18% (R) and 9% (T) of the overall variability; skin treatment sites or methodological factors did not significantly contribute to that variability. Conclusions Inter-subject variability was the major component of overall variability for acyclovir, and treatment site location did not significantly influence intra-subject variability. These results support a dOFM BE study design with T and R products assessed simultaneously on the same subject, where T and R treatment sites do not necessarily need to be next to each other. Localized variation in skin microstructure may be primarily responsible for intra-subject variability.
Skin permeability can be assessed by measuring the passive electrical properties of the skin, which enables correction of skin permeability variations. This allows reduction of experiment numbers in future pharmacokinetic and pharmacodynamic studies with human skin ex vivo and in vivo and leads to diminished study costs.
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