Age-Related Changes in the Fibroblastic Differon of the Dermis: Role in Skin Aging.

Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. The processes developing in the skin during aging are based on fundamental molecular mechanisms associated with fibroblasts, the main cellular population of the dermis. It has been revealed that the amount of fibroblasts decreases markedly with age and their functional activity is also reduced. This inevitably leads to a decrease in the regenerative abilities of the skin and the progression of its aging. In this review we consider the mechanisms underlying these processes, mainly the changes observed with age in the stem/progenitor cells that constitute the fibroblastic differon of the dermis and form their microenvironment (niches). These changes lead to the depletion of stem cells, which, in turn, leads to a decrease in the number of differentiated (mature) dermal fibroblasts responsible for the production of the dermal extracellular matrix and its remodeling. We also describe in detail DNA damages, their cellular and systemic consequences, molecular mechanisms of DNA damage response, and also the role of fibroblast senescence in skin aging.

Deciphering Mesenchymal Drivers of Human Dupuytren's Disease at Single-Cell Level.

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.

Inhibition of class I HDACs preserves hair follicle inductivity in postnatal dermal cells.

Induction of new hair follicles (HFs) may be an ultimate treatment goal for alopecia; however, functional cells with HF inductivity must be expanded in bulk for clinical use. In vitro culture conditions are completely different from the in vivo microenvironment. Although fetal and postnatal dermal cells (DCs) have the potential to induce HFs, they rapidly lose this HF inductivity during culture, accompanied by a drastic change in gene expression. This suggests that epigenetic regulation may be involved. Of the various histone deacetylases (HDACs), Class I HDACs are noteworthy because they are ubiquitously expressed and have the strongest deacetylase activity. This study revealed that DCs from postnatal mice rapidly lose HF inductivity and that this reduction is accompanied by a significant decrease in histone H3 acetylation. However, MS-275, an inhibitor of class I HDACs, preserves HF inductivity in DCs during culture, increasing alkaline phosphatase activity and upregulating HF inductive genes such as BMP4, HEY1, and WIF1. In addition, the inhibition of class I HDACs activates the Wnt signaling pathway, the most well-described molecular pathway in HF development, via increased histone H3 acetylation within the promoter region of the Wnt transcription factor LEF1. Our results suggest that class I HDACs could be a potential target for the neogenesis of HFs.

Engineered extracellular vesicle mimetics from macrophage promotes hair growth in mice and promotes human hair follicle growth.

Recent studies clearly show that cell-derived extracellular vesicles (EVs, including exosomes) can promote hair growth. However, large-scale production of EVs remains a big hurdle. Recently, extracellular vesicle mimetics (EMs) engineered by extrusion through various membranes are emerging as a complementary approach for large-scale production. In this study, to investigate their ability to induce hair growth, we generated macrophage-engineered EMs (MAC-EMs) that activated the human dermal papilla (DP) cells in vitro. MAC-EMs intradermally injected into the skin of C57BL/6 mice were retained for up to 72 h. Microscopy imaging revealed that MAC-EMs were predominately internalized into hair follicles. The MAC-EMs treatment induced hair regrowth in mice and hair shaft elongation in a human hair follicle, suggesting the potential of MAC-EMs as an alternative to EVs to overcome clinical limitation.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Cyclic Adenosine Monophosphate Eliminates Sex Differences in Estradiol-Induced Elastin Production from Engineered Dermal Substitutes.

Lack of adult cells' ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.

Tailoring the growth and proliferation of human dermal fibroblasts by DNA-based polymer films for skin regeneration.

The use of DNA as a functional biomaterial for therapeutic, diagnostic, and drug delivery applications has been prominent in recent years, but its use as a scaffold for tissue regeneration is still limited. This study aimed to evaluate the biocompatibility and interaction of DNA-based polymeric films (DNA-PFs) with primary human fibroblasts (PHF) for regenerative medicine and wound healing purposes. The morphological characterization of the films was performed by scanning electron microscopy, SEM-energy-dispersive X-ray spectroscopy, and atomic force microscopy analysis. Cell viability, cell cycle kinetics, oxidative stress, and migration studies were carried out at 48 and 72 hr of incubation and compared to control cells. Cell adhesion was impaired in the first 24 hr, DNA-PFs with higher concentrations of DNA (1.0 and 2.0 g/L) this effect was not seen in DNA-PFs (0.5 g/L), explained by the difference in topography and roughness of DNA-PFs, but it was overcome after 48 hr of incubation. PHF seeded on DNA films showed higher proliferation and migration rates than the control after 48 hr of incubation, with the maintenance of cell morphology and lower cytotoxicity and oxidative stress during the evaluation time. Therefore, these results indicate that DNA-PFs are highly biocompatible and provide a suitable microenvironment for dermal fibroblasts to maintain their activity, helping build new and more complex biomaterials suitable for future tissue repair applications.

Transmembrane collagens-Unexplored mediators of epidermal-dermal communication and tissue homeostasis.

Tissue homeostasis is maintained through constant, dynamic and heterogeneous communication between cells and their microenvironment. Proteins that are at the same time active at the intracellular, cell periphery and deeper extracellular levels possess the ability to, on the individual molecular level, influence the cells and their microenvironment in a bidirectional manner. The transmembrane collagens are a family of such proteins, which are of notable interest for tissue development and homeostasis. In skin, expression of all transmembrane collagens has been reported and deficiency of transmembrane collagen XVII manifests with distinct skin phenotypes. Nevertheless, transmembrane collagens in skin remain understudied despite the association of them with epidermal wound healing and dermal fibrotic processes. Here, we present an overview of transmembrane collagens and put a spotlight on them as regulators of epidermal-dermal communication and as potential players in fibrinogenesis.

A developmental basis for the anatomical diversity of dermis in homeostasis and wound repair.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re-enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

An on-chip wound healing assay fabricated by xurography for evaluation of dermal fibroblast cell migration and wound closure.

Dermal fibroblast cell migration is a key process in a physiological wound healing. Therefore, the analysis of cell migration is crucial for wound healing research. In this study, lab-on-a-chip technology was used to investigate the effects of basic fibroblast growth factor (bFGF), mitomycin C (MMC), MEK1/2 inhibitor (U0126) and fetal calf serum (FCS) on human dermal fibroblast cell migration. The microdevice was fabricated consisting of microchannels, pneumatic lines and pneumatically-activated actuators by xurographic rapid prototyping. In contrast to current approaches in in vitro wound healing such as scratch assays and silicone inserts in wellplate format, which show high variability and poor reproducibility, the current system aims to automate the wounding procedure at high precision and reproducibility using lab-on-a-chip. Traumatic wounding was simulated on-chip on fibroblast cell monolayers by applying air pressure on the flexible circular membrane actuator. Wound closure was monitored using light microscopy and cell migration was evaluated using image analysis. The pneumatically controlled system generates highly reproducible wound sizes compared to the conventional wound healing assay. As proof-of-principle study wound healing was investigated in the presence of several stimulatory and inhibitory substances and culture including bFGF, MMC, U0126 MEK1/2 inhibitor as well as serum starvation to demonstrate the broad applicability of the proposed miniaturized culture microsystem.

Evaluation of the Effect of Plasma from Patients with Trophic Ulcers on the Function of Dermal Fibroblasts, Mesenchymal Stem Cells, and Endothelial Cells.

We studied the effect of platelet lysate and platelet-poor plasma from patients with trophic ulcers with and without type 2 diabetes mellitus on proliferation, migration, and apoptosis of human dermal fibroblast, mesenchymal stem cells, and endothelial cells. It is shown that plasma obtained from patients with type 2 diabetes mellitus produced inhibitory effects.

Pelnac® Artificial Dermis Assisted by VSD for Treatment of Complex Wound with Bone/Tendon Exposed at the Foot and Ankle, A Prospective Study.

Purpose: This study aims to assess the efficacy and safety of Pelnac dermal regeneration template assisted by vacuum sealing drainage (VSD) and a split-thickness skin graft to cover the large skin and soft-tissue defects at foot and ankle. Methods: This study began from March 2013, up to February 2017. A total of 16 patients met the inclusion and exclusion criteria and were included. For every patient, 2 separate operations were performed, the first being thorough debridement of necrotic tissues immediate coverage of VSD at continuous negative pressure suction, and the second being the autologous split-thickness skin graft. At each follow-up, relevant data were documented. Results: The average follow-up was 16.5 months (range, 12 to 42 months). No infections, hematoma, or seroma were observed. 13 out of 16 patients had a complete skin graft "take" (100%). Patients' satisfaction of esthetic appearance was 76.5 ± 5.2/100. The VSS value was 2.2 ± 2.1, representing a good result. Regarding the sensory recovery, the response "normal or near normal" could be obtained in 14/16 patients. Mean AROM for extension/flexion of the ankle was 48.5 ± 4.8° (range 35-62°), and 93.7% (15/16) of patient could obtain a satisfying functional result. Conclusions: Our report indicated Pelnac provided an effective method for management of complex wounds with underlying bone or tendons exposed.

[Short peptides: regulation of skin function during aging.]

Short peptides are applied for supporting skin function during ageing, because they can permeate the intact stratum corneum of the epidermis and affect the cells of the dermis. Short peptides are part of natural metabolism of cells and many of them have geroprotective properties. In the review we are considering the base sorts of peptides that are used for normalized skin fibroblasts function: matrikines, carnosine, collagen peptides, cytokine and growth factor analogs, defensins, immunoprotective peptides and polyfunctional peptides. Polyfunctional peptides (AcSDKP, KED, AEDG, AED) have geroprotective properties, slow apoptosis and stimulate skin cell proliferation, also increase functional activity of skin fibroblasts, normalize intracellular matrix hemostasis. Polyfunctional peptides are the antioxidants and immunoprotectors and can activate microcirculation in dermis. Peptide regulation of skin function during ageing are the fast-developing and prospective area in molecular gerontology.

Comparative transcriptome analysis of transcultured human skin-derived precursors (tSKPs) from adherent monolayer culture system and tSKPs-derived fibroblasts (tFBs) by RNA-Seq.

Transcultured human skin derived precursors (tSKPs) from adherent monolayer culture system have similar characteristics as traditional skin derived precursors (SKPs), making tSKPs a suitable candidate for regenerative medicine. tSKPs can differentiate into fibroblasts. However, little is known about the molecular mechanism of the transition from tSKPs to fibroblasts. Here, we compared the transcriptional profiles of human tSKPs and tSKPs-derived fibroblasts (tFBs) by RNA-Sequence aiming to determine the candidate genes and pathways involving in the differentiation process. A total of 1042 differentially expressed genes (DEGs) were identified between tSKPs and tFBs, with 490 genes up-regulated and 552 genes down-regulated. Our study showed that these DEGs were significantly enriched in tumor necrosis factor signaling pathway, focal adhesion, extracellular matrix-receptor interaction and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway. A further transcription factors (TFs) analysis of DEGs revealed the significantly down-expressed TFs (p21, Foxo1and Foxc1) in tFBs were mostly the downstream nodes of PI3K-Akt signaling pathway, which suggested PI3K-Akt signaling pathway might play an important role in tSKPs differentiation. The results of our study are useful for investigating the molecular mechanisms in tSKPs differentiation into tFBs, making it possible to take advantage of their potential application in regenerative medicine.

Molecular Mechanisms of Hair Growth and Regeneration: Current Understanding and Novel Paradigms.

Hair is a defining feature of mammals and has critical functions, including protection, production of sebum, apocrine sweat and pheromones, social and sexual interactions, thermoregulation, and provision of stem cells for skin homeostasis, regeneration, and repair. The hair follicle (HF) is considered a "mini-organ," consisting of intricate and well-organized structures which originate from HF stem and progenitor cells. Dermal papilla cells are the main components of the mesenchymal compartments in the hair bulb and are instrumental in generating signals to regulate the behavior of neighboring epithelial cells during the hair cycle. Mesenchymal-epithelial interactions within the dermal papilla niche drive HF embryonic development as well as the postnatal hair growth and regeneration cycle. This review summarizes the current understanding of HF development, repair, and regeneration, with special focus on cell signaling pathways governing these processes. In particular, we discuss emerging paradigms of molecular signaling governing the dermal papilla-epithelial cellular interactions during hair growth and maintenance and the recent progress made towards tissue engineering of human hair follicles.

The aging skin microenvironment dictates stem cell behavior.

Aging manifests with architectural alteration and functional decline of multiple organs throughout an organism. In mammals, aged skin is accompanied by a marked reduction in hair cycling and appearance of bald patches, leading researchers to propose that hair follicle stem cells (HFSCs) are either lost, differentiate, or change to an epidermal fate during aging. Here, we employed single-cell RNA-sequencing to interrogate aging-related changes in the HFSCs. Surprisingly, although numbers declined, aging HFSCs were present, maintained their identity, and showed no overt signs of shifting to an epidermal fate. However, they did exhibit prevalent transcriptional changes particularly in extracellular matrix genes, and this was accompanied by profound structural perturbations in the aging SC niche. Moreover, marked age-related changes occurred in many nonepithelial cell types, including resident immune cells, sensory neurons, and arrector pili muscles. Each of these SC niche components has been shown to influence HF regeneration. When we performed skin injuries that are known to mobilize young HFSCs to exit their niche and regenerate HFs, we discovered that aged skin is defective at doing so. Interestingly, however, in transplantation assays in vivo, aged HFSCs regenerated HFs when supported with young dermis, while young HFSCs failed to regenerate HFs when combined with aged dermis. Together, our findings highlight the importance of SC:niche interactions and favor a model where youthfulness of the niche microenvironment plays a dominant role in dictating the properties of its SCs and tissue health and fitness.

Peel Test to Assess the Adhesion Strength of the Dermal-Epidermal Junction in Tissue-Engineered Skin.

Innovative therapies combining gene-corrected stem cells and the production of bioengineered tissues to treat epidermolysis bullosa are emerging. However, quantitative tests to measure the adhesion forces between two highly viscoelastic substrates such as those found in bilayered bioengineered skin are needed and are still lacking. The objective of this study was to develop a mechanical test to measure the dermal-epidermal adhesion strength of our bilayered tissue-engineered skin substitute (TES) produced with the self-assembly method. We developed a peel test, which allows the displacement of both skin layers in a T configuration, based on the ASTM International standard. A MATLAB program was written to process and analyze raw data. The experimental setup was tested by measuring the dermal-epidermal adhesion strength in TESs produced with normal or collagen VII-deficient cells. Our peel testing method allowed us to detect the impact of the absence of collagen VII in the dermal-epidermal adhesion strength of TESs and also to examine the progression of the dermal-epidermal adhesion strength in relation to culture time in normal TES. Impact statement This study describes a method for assessing the adhesion strength at the dermal-epidermal junction of individual tissue-engineered skin substitute (TES). An ASTM standardized protocol of peel testing was designed to measure this important mechanical property. Our innovative approach will serve as a quality control in the production, improvement, and application of TESs for the treatment of pathologies affecting the dermal-epidermal adhesion such as epidermolysis bullosa. Data presented contribute to research on the interfaces between biological substrates and provide a reference factor for the characterization of products derived from tissue engineering.

Extracellular Vesicles Derived from Senescent Fibroblasts Attenuate the Dermal Effect on Keratinocyte Differentiation.

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated ß-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.