Minimally-invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has been so far the most commonly interrogated body fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Minimally-invasive technologies have emerged as a method to sample this fluid in a pain-free manner and often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate-peptide nucleic acid hybrid material for sequence-specific sampling, isolation and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (~6.5μL in 2 min), this platform technology also enables for the first time the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cellfree nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression.

The dermal papilla is a cluster of mesenchymal cells located at the base of the hair follicle which have a number of important roles in the regulation of hair growth. As a consequence, in vitro models of these cells are widely used to study the molecular mechanisms which underlie hair follicle induction, growth and maintenance. While dermal papilla from rodent hair follicles can be digested prior to cell isolation, the unique extracellular matrix composition found in human dermal papilla renders enzymes such as trypsin and collagenase insufficient for digestion of the dermal papilla into a single cell suspension. As such, to grow human dermal papilla cells in vitro, the papilla has to first be isolated via a micro‐dissection approach from the follicle. In this article we describe the micro‐dissection and culture methods, which we use within our laboratory, for the study of human dermal papilla cells.

SummaryBackgroundWhile several commercial dermoepidermal scaffolds can promote wound healing of the skin, the achievement of complete skin regeneration still represents a major challenge.ObjectivesTo perform biological characterization of self‐assembled extracellular matrices (ECMs) from three different subpopulations of fibroblasts found in human skin: papillary fibroblasts (Pfi), reticular fibroblasts (Rfi) and dermal papilla fibroblasts (DPfi).MethodsFibroblast subpopulations were cultured with ascorbic acid to promote cell‐assembled matrix production for 10 days. Subsequently, cells were removed and the remaining matrices characterized. Additionally, in another experiment, keratinocytes were seeded on the top of cell‐depleted ECMs to generate epidermal‐only skin constructs.ResultsWe found that the ECM self‐assembled by Pfi exhibited randomly oriented fibres associated with the highest interfibrillar space, reflecting ECM characteristics that are physiologically present within the papillary dermis. Mass spectrometry followed by validation with immunofluorescence analysis showed that thrombospondin 1 is preferentially expressed within the DPfi‐derived matrix. Moreover, we observed that epidermal constructs grown on DPfi or Pfi matrices exhibited normal basement membrane formation, whereas Rfi matrices were unable to support membrane formation.ConclusionsWe argue that inspiration can be taken from these different ECMs, to improve the design of therapeutic biomaterials in skin engineering applications.

In skin homeostasis, dermal fibroblasts are responsible for coordinating the migration and differentiation of overlying epithelial keratinocytes. As hairy skin heals faster than nonhairy skin, we took bio-inspiration from the follicle and hypothesized that follicular fibroblasts would accelerate skin re-epithelialization after injury faster than interfollicular fibroblasts. Using both in vitro and ex vivo models of human skin wound closure, we found that hair follicle dermal papilla fibroblasts could accelerate closure of in vitro scratch wounds by 1.8-fold and epithelial growth capacity by 1.5-fold compared with controls (P < 0.05). We used a cytokine array to determine how the dermal papilla fibroblasts were eliciting this effect and identified two cytokines, sAXL and CCL19, that are released at significantly higher levels by follicular fibroblasts than by interfollicular subtypes. Using sAXL and CCL19 individually, we found that they could also increase closure of epithelial cells in a scratch wound by 1.2-and 1.5-fold, respectively, compared with controls (P < 0.05). We performed an unbiased transcriptional analysis, combined with pathway analysis, and postulate that sAXL accelerates wound closure by promoting migration and inhibiting epithelial differentiation of skin keratinocytes. Long term, we believe these results can be exploited to accelerate wound closure of human skin in vivo.

A major problem of diabetic foot ulcers is ischemia contributing to delayed wound closure. The gap junction protein Connexin43 (Cx43) is differentially remodelled during 'normal' and 'chronic' wound healing events. The mechanisms by which this protein is involved remains unresolved. This work investigated the expression and post translational modification of Cx43 and Pannexin 1 (Panx1) in skin biopsies from patients undergoing arterial reconstruction or major limb amputation for critical limb ischaemia and in keratinocytes cultured in normoxic (N) and hypoxic (H) (1% oxygen, 5% carbon dioxide and 94 % nitrogen) conditions. Skin tissue biopsies (proximal and distal to the point of venous bypass surgery) from diabetic and non-diabetic patients were processed for immunohistochemistry and stained with antibodies specific to Cx43 and Cx43Ser 368. To model the events in vitro HaCaT cells were grown in 12 well plates, scrape wounded and cultured in N or H environments for up to 48 hours (h). Cell movement into the denuded area was recorded. Cells were fixed or protein harvested and expression of hypoxic inducible factor 1-a (HIF-1a), Cx43, Cx43Ser 368 , Panx1 and Ki67 determined by immunocytochemistry and Western blot analysis. Cx43 and Cx43Ser 368 expression was significantly increased in the epidermis of skin biopsies isolated from the distal position (i.e. locations of ischaemia) in both diabetic and non-diabetic tissue. Scratch wound assay determined delayed wound closure in hypoxic conditions, that was confirmed by induction of HIF-1a expression. Cx43 and Cx43 Ser368 staining showed translocation of the protein to the nucleus. No changes in Panx1 were observed. In conclusion, post translational phosphorylation of Cx43 occurs in chronically wounded diabetic and ischemic skin. Under H conditions, scrape wound closure is reduced and immunofluorescence suggests that Cx43 and Cx43Ser 368 translocate to the nucleus after 24 h hypoxia. Thus, hypoxia impacts on Cx expression but not Panx1 expression in the epidermis.

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