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In clinical practice to treat complex injuries, the application of electrical stimulation (ES) directly to the skin complicates the wound. In this work, the effect of a conductive hydrogel mediated electric field on skin regeneration is investigated. Polypyrrole incorporated matrices of gelatin and silk fibroin were prepared by two-step interfacial polymerization. The maximum electrical conductivity of 10 -4 S cm -1 was achieved when 200 mM polypyrrole was loaded. Mechanically stable and cytocompatible hydrogels were evidenced to have antioxidant and blood compatible characteristics. Human dermal fibroblast cells responded to pulsed stimulation of 100 or 300 mV mm -1 as observed from the increased expressions of TGFβ1, αSMA, and COLIAI genes. Further, the increase in the αSMA protein expression with the magnitude of electrical stimulation also suggested transdifferentiation of the fibroblast to myofibroblast. Moreover, Raman spectroscopy identified two fingerprint regions (collagen and lipid) to differentiate ES treated and nontreated samples. Therefore, the combination of hydrogels and electrical stimulation has potential therapeutic effects for accelerating the rate of skin regeneration.

Background: In vitro culture of fibroblasts is a technique based on cell isolation, physiological characterization, and cryopreservation. This technique has not been described for Galea spixii, therefore, it can be used to learn about its cellular biology and genetic diversity., Objective: We established fibroblast lines of six G. spixii individuals from several passages (second, fifth, eighth, and tenth) and cryopreserved them., Methods: Fibroblasts recovered from skin biopsies were identified based on morphology, immunocytochemistry, and karyotyping. The cells were analyzed for morphology, ultrastructure, viability, proliferation, metabolism, oxidative stress, bioenergetic potential, and apoptosis before and after cryopreservation., Results: After the eighth passage, the fibroblasts showed morphological and karyotypic changes, although their viability, metabolism, and proliferation did not change. An increase in oxidative stress and bioenergetic potential from the fifth to the eighth passages were also observed. Post cryopreservation, cell damage with respect to the ultrastructure, viability, proliferative rate, apoptotic levels, oxidative stress, and bioenergetic potential were verified., Conclusion: Fibroblasts up to the tenth passage could be cultured in vitro. However, cells at the fifth passage were of better quality to be used for reproductive techniques. Additionally, optimization of the cryopreservation protocol is essential to improve the physiological parameters of these cells., Competing Interests: Declaration of Competing Interest The authors declare having no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Numerous studies have demonstrated the efficacy of extremely low frequency-pulsed electromagnetic fields (ELF-PEMF) in accelerating the wound healing process in vitro and in vivo. Our study focuses specifically on ELF-PEMF applied with the Magnomega® device and aims to assess their effect during the main stages of the proliferative phase of dermal wound closure, in vitro. Thus, after the characterization of the EMFs delivered by the Magnomega® unit, primary culture of human dermal fibroblasts (HDFs) were exposed, or not for the control culture, to 10-12 and 100 Hz ELF-PEMF. These parameters are used in clinical practice by physiotherapists in order to enhance healing of dermal lesions in patients. HDFs proliferation was first assessed and revealed an increase in the expression of one of the two genetic markers of cell proliferation tested (PCNA and MKI67), after initial exposure of the cells to 10-12 Hz PEMF. Next, migration of HDFs was investigated by performing scratch assays on HDF layers. The observed wound closure kinetics corroborate the early organization of actin stress fibers that was revealed in the cytoplasm of HDFs exposed to 100 Hz ELF-PEMF. Also, maturation of HDFs into myofibroblasts was significantly increased in cells exposed to 10-12 or to 100 Hz PEMF. The present study is the first to demonstrate, in vitro, an early stimulation of HDFs, after their exposure to ELF-PEMF delivered by the Magnomega® device, which could contribute to an acceleration of the wound healing process., (© 2024 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society.)

During the physiological healing of skin wounds, fibroblasts recruited from the uninjured adjacent dermis and deeper subcutaneous fascia layers are transiently activated into myofibroblasts to first secrete and then contract collagen-rich extracellular matrix into a mechanically resistant scar. Scar tissue restores skin integrity after damage but comes at the expense of poor esthetics and loss of tissue function. Stiff scar matrix also mechanically activates various precursor cells into myofibroblasts in a positive feedback loop. Persistent myofibroblast activation results in pathologic accumulation of fibrous collagen and hypertrophic scarring, called fibrosis. Consequently, the mechanisms of fibroblast-to-myofibroblast activation and persistence are studied to develop antifibrotic and prohealing treatments. Mechanistic understanding often starts in a plastic cell culture dish. This can be problematic because contact of fibroblasts with tissue culture plastic or glass surfaces invariably generates myofibroblast phenotypes in standard culture. We describe a straight-forward method to produce soft cell culture surfaces for fibroblast isolation and continued culture and highlight key advantages and limitations of the approach. Adding a layer of elastic silicone polymer tunable to the softness of normal skin and the stiffness of pathologic scars allows to control mechanical fibroblast activation while preserving the simplicity of conventional 2-dimensional cell culture., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Platelet-rich plasma (PRP) has become an important regenerative therapy. However, the preparation method of PRP has not been standardized, and the optimal platelet concentration for PRP used in skin wound repair is unclear, leading to inconsistent clinical efficacy of PRP. Therefore, the development of standardized preparation methods for PRP and the investigation of the dose-response relationship between PRP with different platelet concentrations and tissue regeneration plays an important role in the development and clinical application of PRP technology. This study has developed an integrated blood collection device from blood drawing to centrifugation. Response surface methodology was employed to optimize the preparation conditions, ultimately achieving a platelet recovery rate as high as 95.74% for PRP (with optimal parameters: centrifugation force 1730× g , centrifugation time 10 min, and serum separation gel dosage 1.4 g). Both in vitro and in vivo experimental results indicate that PRP with a (2×) enrichment ratio is the most effective in promoting fibroblast proliferation and skin wound healing, with a cell proliferation rate of over 150% and a wound healing rate of 78% on day 7.

Background: In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion., Methods: Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses., Results: Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group., Conclusions: The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps., (© 2024. The Author(s).)

Various strategies have extensively explored enhancing the physical and biological properties of chitosan and cellulose scaffolds for skin tissue engineering. This study presents a straightforward method involving the addition of glycerol into highly porous structures of two polysaccharide complexes: chitosan/carboxymethyl cellulose (Chit/CMC) and chitosan/oxidized cellulose (Chit/OC); during a one-step freeze-drying process. Adding glycerol, especially to Chit/CMC, significantly increased stability, prevented degradation, and improved mechanical strength by nearly 50%. Importantly, after 21 days of incubation in enzymatic medium Chit/CMC scaffold has almost completely decomposed, while foams reinforced with glycerol exhibited only 40% mass loss. It is possible due to differences in multivalent cations and polymer chain contraction, resulting in varied hydrogen bonding and, consequently, distinct physicochemical outcomes. Additionally, the scaffolds with glycerol improved the cellular activities resulting in over 40% higher proliferation of fibroblast after 21 days of incubation. It was achieved by imparting water resistance to the highly absorbent material and aiding in achieving a balance between hydrophilic and hydrophobic properties. This study clearly indicates the possible elimination of additional crosslinkers and multiple fabrication steps that can reduce the cost of scaffold production for skin tissue engineering applications while tailoring mechanical strength and degradation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The skin, the outermost organ of the human body, is vital for sensing and responding to stimuli through mechanotransduction. It is constantly exposed to mechanical stress. Consequently, various mechanical therapies, including compression, massage, and microneedling, have become routine practices for skin healing and regeneration. However, these traditional methods require direct skin contact, restricting their applicability. To address this constraint, we developed shear wave stimulation (SWS), a contactless mechanical stimulation technique. The effectiveness of SWS was compared with that of a commercial compression bioreactor used on reconstructed skin at various stages of maturity. Despite the distinct stimulus conditions applied by the two methods, SWS yielded remarkable outcomes, similar to the effects of the compression bioreactor. It significantly increased the shear modulus of tissue-engineered skin, heightened the density of collagen and elastin fibers, and resulted in an augmentation of fibroblasts in terms of their number and length. Notably, SWS exhibited diverse effects in the low- and high-frequency modes, highlighting the importance of fine-tuning the stimulus intensity. These results unequivocally demonstrated the capability of SWS to enhance the mechanical functions of the skin in vitro, making it a promising option for addressing wound healing and stretch mark recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The central nervous system (CNS) is surrounded by three membranes called meninges. Specialised fibroblasts, originating from the mesoderm and neural crest, primarily populate the meninges and serve as a binding agent. Our goal was to compare fibroblasts from meninges and skin obtained from the same human-aged donors, exploring their molecular and cellular characteristics related to CNS functions. We isolated meningeal fibroblasts (MFs) from brain donors and skin fibroblasts (SFs) from the same subjects. A functional analysis was performed measuring cell appearance, metabolic activity, and cellular orientation. We examined fibronectin, serpin H1, β-III-tubulin, and nestin through qPCR and immunofluorescence. A whole transcriptome analysis was also performed to characterise the gene expression of MFs and SFs. MFs appeared more rapidly in the post-tissue processing, while SFs showed an elevated cellular metabolism and a well-defined cellular orientation. The four markers were mostly similar between the MFs and SFs, except for nestin, more expressed in MFs. Transcriptome analysis reveals significant differences, particularly in cyclic adenosine monophosphate (cAMP) metabolism and response to forskolin, both of which are upregulated in MFs. This study highlights MFs' unique characteristics, including the timing of appearance, metabolic activity, and gene expression patterns, particularly in cAMP metabolism and response to forskolin. These findings contribute to a deeper understanding of non-neuronal cells' involvement in CNS activities and potentially open avenues for therapeutic exploration., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Stem cells are recognized as an important target and tool in regenerative engineering. In this study, we explored the feasibility of engineering amniotic fluid-derived mesenchymal stem cell-secreted molecules (afMSC-SMs) as a versatile bioactive material for skin regenerative medicine applications in a time- and cost-efficient and straightforward manner. afMSC-SMs, obtained in powder form through ethanol precipitation, effectively contributed to preserving the self-renewal capacity and differentiation potential of primary human keratinocytes (pKCs) in a xeno-free environment, offering a potential alternative to traditional culture methods for their long-term in vitro expansion, and allowed them to reconstitute a fully stratified epithelium sheet on human dermal fibroblasts. Furthermore, we demonstrated the flexibility of afMSC-SMs in wound healing and hair regrowth through injectable hydrogel and nanogel-mediated transdermal delivery systems, respectively, expanding the pool of regenerative applications. This cell-free approach may offer several potential advantages, including streamlined manufacturing processes, scalability, controlled formulation, longer shelf lives, and mitigation of risks associated with living cell transplantation. Accordingly, afMSC-SMs could serve as a promising therapeutic toolbox for advancing cell-free regenerative medicine, simplifying their broad applicability in various clinical settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Physiologically-relevant in vitro skin models hold the utmost importance for efficacy assessments of pharmaceutical and cosmeceutical formulations, offering valuable alternatives to animal testing. Here, an advanced immunocompetent 3D bioprinted human skin model is presented to assess skin sensitization. Initially, a photopolymerizable bioink is formulated using silk fibroin methacrylate, gelatin methacrylate, and photoactivated human platelet releasate. The developed bioink shows desirable physicochemical and rheological attributes for microextrusion bioprinting. The tunable physical and mechanical properties of bioink are modulated through variable photocuring time for optimization. Thereafter, the bioink is utilized to 3D bioprint "sandwich type" skin construct where an artificial basement membrane supports a biomimetic epidermal layer on one side and a printed pre-vascularized dermal layer on the other side within a transwell system. The printed construct is further cultured in the air-liquid interface for maturation. Immunofluorescence staining demonstrated a differentiated keratinocyte layer and dermal extracellular matrix (ECM)-remodeling by fibroblasts and endothelial cells. The biochemical estimations and gene-expression analysis validate the maturation of the printed model. The incorporation of macrophages further enhances the physiological relevance of the model. This model effectively classifies skin irritative and non-irritative substances, thus establishing itself as a suitable pre-clinical screening platform for sensitization tests., (© 2024 Wiley‐VCH GmbH.)

Delayed re-epithelization and weakened skin contractions are the two primary factors that hinder wound closure in large-scale acute or chronic wounds. However, effective strategies for targeting these two aspects concurrently are still lacking. Herein, an antioxidative active-shrinkage hydrogel (AHF@AS Gel) is constructed that can integratedly promote re-epithelization and skin constriction to accelerate large-scale acute and diabetic chronic wound closure. The AHF@AS Gel is encapsulated by antioxidative amino- and hydroxyl-modified C 70 fullerene (AHF) and a thermosensitive active shrinkage hydrogel (AS Gel). Specifically, AHF relieves overactivated inflammation, prevents cellular apoptosis, and promotes fibroblast migration in vitro by reducing excessive reactive oxygen species (ROS). Notably, the AHF@AS Gel achieved ≈2.7-fold and ≈1.7-fold better re-epithelization in acute wounds and chronic diabetic wounds, respectively, significantly contributing to the promotion of wound closure. Using proteomic profiling and mechanistic studies, it is identified that the AHF@AS Gel efficiently promoted the transition of the inflammatory and proliferative phases to the remodeling phase. Notably, it is demonstrated that AS Gel alone activates the mechanosensitive epidermal growth factor receptor/Akt (EGFR/Akt) pathway and promotes cell proliferation. The antioxidative active shrinkage hydrogel offers a comprehensive strategy for acute wound and diabetic chronic wound closure via biochemistry regulation integrating with mechanical forces stimulation., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Skin is constantly exposed to injuries that are repaired with different outcomes, either regeneration or scarring. Scars result from fibrotic processes modulated by cellular physical forces transmitted by integrins. Fibronectin (FN) is a major component in the provisional matrix assembled to repair skin wounds. FN enables cell adhesion binding of α5β1/αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5/αIIb is the synergy site located in close proximity to the RGD motif. The mutation to impair the FN synergy region ( Fn1 syn/syn ) demonstrated that its absence permits complete development. However, only with the additional engagement to the FN synergy site do cells efficiently resist physical forces. To test how the synergy site-mediated adhesion affects the course of wound healing fibrosis, we used a mouse model of skin injury and in-vitro migration studies with keratinocytes and fibroblasts on FN syn . The loss of FN synergy site led to normal re-epithelialization caused by two opposing migratory defects of activated keratinocytes and, in the dermis, induced reduced fibrotic responses, with lower contents of myofibroblasts and FN deposition and diminished TGF-β1-mediated cell signalling. We demonstrate that weakened α5β1-mediated traction forces on FN syn cause reduced TGF-β1 release from its latent complex.

Background: Xylosyltransferases-I and II (XT-I and XT-II) catalyze the initial and rate limiting step of the proteoglycan (PG) biosynthesis and therefore have an import impact on the homeostasis of the extracellular matrix (ECM). The reason for the occurrence of two XT-isoforms in all higher organisms remains unknown and targeted genome-editing strategies could shed light on this issue., Methods: XT-I deficient neonatal normal human dermal fibroblasts were generated by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated proteins (Cas) 9 system. We analyzed if a reduced XT-I activity leads to abnormalities regarding ECM-composition, myofibroblast differentiation, cellular senescence and skeletal and cartilage tissue homeostasis., Results: We successfully introduced compound heterozygous deletions within exon 9 of the XYLT1 gene. Beside XYLT1 , we detected altered gene-expression levels of further, inter alia ECM-related, genes. Our data further reveal a dramatically reduced XT-I protein activity. Abnormal myofibroblast-differentiation was demonstrated by elevated alpha-smooth muscle actin expression on both, mRNA- and protein level. In addition, wound-healing capability was slightly delayed. Furthermore, we observed an increased cellular-senescence of knockout cells and an altered expression of target genes knowing to be involved in skeletonization., Conclusion: Our data show the tremendous relevance of the XT-I isoform concerning myofibroblast-differentiation and ECM-homeostasis as well as the pathophysiology of skeletal disorders.

Background: Skin innervation is crucial for normal wound healing. However, the relationship between nerve receptors and wound healing and the intrinsic mechanism remains to be further identified. In this study, we investigated the role of a calcitonin gene-related peptide (CGRP) receptor component, receptor activity-modifying protein 1 (RAMP1), in mouse skin fibroblast (MSF) proliferation., Methods: In vivo, Western blotting and immunohistochemical (IHC) staining of mouse skin wounds tissue was used to detect changes in RAMP1 expression. In vitro, RAMP1 was overexpressed in MSF cell lines by infection with Tet-On-Flag-RAMP1 lentivirus and doxycycline (DOX) induction. An IncuCyte S3 Live-Cell Analysis System was used to assess and compare the proliferation rate differences between different treatment groups. Total protein and subcellular extraction Western blot analysis, quantitative real-time-polymerase chain reaction (qPCR) analysis, and immunofluorescence (IF) staining analysis were conducted to detect signalling molecule expression and/or distribution. The CUT & RUN assay and dual-luciferase reporter assay were applied to measure protein-DNA interactions., Results: RAMP1 expression levels were altered during skin wound healing in mice. RAMP1 overexpression promoted MSF proliferation. Mechanistically, total Yes-associated protein (YAP) and nuclear YAP protein expression was increased in RAMP1-overexpressing MSFs. RAMP1 overexpression increased inhibitory guanine nucleotide-binding protein (G protein) α subunit 3 (Gαi3) expression and activated downstream protein kinase A (PKA), and both elevated the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activated it, promoting the transcription of YAP, elevating the total YAP level and promoting MSF proliferation., Conclusions: Based on these data, we report, for the first time, that changes in the total RAMP1 levels during wound healing and RAMP1 overexpression alone can promote MSF proliferation via the Gαi3-PKA-CREB-YAP axis, a finding critical for understanding RAMP1 function, suggesting that this pathway is an attractive and accurate nerve target for skin wound treatment. Video Abstract., (© 2022. The Author(s).)

Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced into the coaxial polycaprolactone/gelatin (PCL/GEL) nanofiber mat with CIP loaded into the PCL (core layer) and TH loaded into the GEL (shell layer), developing antibacterial wound dressing with the co-delivering of the two antibiotics (PCL-CIP/GEL-TH). The nanostructure, physical properties, drug release, antibacterial property, and in vitro cytotoxicity were investigated accordingly. The results revealed that the CIP shows a long-lasting release of five days, reaching the releasing rate of 80.71%, while the cumulative drug release of TH reached 83.51% with a rapid release behavior of 12 h. The in vitro antibacterial activity demonstrated that the coaxial nanofiber mesh possesses strong antibacterial activity against E. coli and S. aureus . In addition, the coaxial mats showed superior biocompatibility toward human skin fibroblast cells (hSFCs). This study indicates that the developed PCL-CIP/GEL-TH nanofiber membranes hold enormous potential as wound dressing materials.

Countless biophysical studies have sought distinct markers in the cellular mechanical response that could be linked to morphogenesis, homeostasis, and disease. Here, an iterative-fitting methodology visualizes the time-dependent viscoelastic behavior of human skin cells under physiologically relevant conditions. Past investigations often involved parameterizing elastic relationships and assuming purely Hertzian contact mechanics, which fails to properly account for the rich temporal information available. We demonstrate the performance superiority of the proposed iterative viscoelastic characterization method over standard open-search approaches. Our viscoelastic measurements revealed that 2D adherent metastatic melanoma cells exhibit reduced elasticity compared to their normal counterparts-melanocytes and fibroblasts, and are significantly less viscous than fibroblasts over timescales spanning three orders of magnitude. The measured loss angle indicates clear differential viscoelastic responses across multiple timescales between the measured cells. This method provides insight into the complex viscoelastic behavior of metastatic melanoma cells relevant to better understanding cancer metastasis and aggression., (© 2022. The Author(s).)

The skin is subject to both intrinsic aging caused by metabolic processes in the body and extrinsic aging caused by exposure to environmental factors. Intrinsic aging is an important obstacle to in vitro experimentation as its long-term progression is difficult to replicate. Here, we accelerated aging of a full-thickness skin equivalent by applying periodic mechanical stimulation, replicating the circadian rhythm for 28 days. This aging skin model was developed by culturing a full-thickness, three-dimensional skin equivalent with human fibroblasts and keratinocytes to produce flexible skin-on-a-chip. Accelerated aging associated with periodic compressive stress was evidenced by reductions in the epidermal layer thickness, contraction rate, and secretion of Myb. Increases in β-galactosidase gene expression and secretion of reactive oxygen species and transforming growth factor-β1 were also observed. This in vitro aging skin model is expected to greatly accelerate drug development for skin diseases and cosmetics that cannot be tested on animals.

As a source of growth factors for expediting wound healing and tissue regeneration, plasma-rich plasma (PRP) has been extensively applied in diverse fields including orthopaedics, ophthalmology, oral and maxillofacial surgery, dentistry, and gynaecology. However, the function of PRP in metabolic regulations remains enigmatic. A standardized method was devised herein to enrich growth factors and to lyophilize it as enhanced PRP (ePRP) powder, which could become ubiquitously available without mechanical centrifugation in clinical practice. To identify metabolic reprogramming in human dermal fibroblasts under ePRP treatment, putative metabolic targets were identified by transcriptome profiling and validated for their metabolic effects and mechanism. ePRP does not only promote wound healing but re-aligns energy metabolism by shifting to glycolysis through stimulation of glycolytic enzyme activity in fibroblasts. On the contrary, oxygen consumption rates and several mitochondrial respiration activities were attenuated in ePRP-treated fibroblasts. Furthermore, ePRP treatment drives the mitochondrial resetting by hindering the mitochondrial biogenesis-related genes and results in a dampened mitochondrial mass. Antioxidant production was further increased by ePRP treatment to prevent reactive oxygen species formation. Besides, ePRP also halts the senescence progression of fibroblasts by activating SIRT1 expression. Importantly, the glycolytic inhibitor 2-DG can completely reverse the ePRP-enhanced wound healing capacity, whereas the mitochondrial inhibitor oligomycin cannot. This is the first study to utilize PRP for comprehensively investigating its effects on the metabolic reprogramming of fibroblasts. These findings indicate that PRP's primary metabolic regulation is to promote metabolic reprogramming toward glycolytic energy metabolism in fibroblasts, preserving redox equilibrium and allowing anabolic pathways necessary for the healing and anti-ageing process.

Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility 1 . The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (T H 2) cells within a distinct microanatomical niche. T H 2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as T H 2-interacting fascial fibroblasts (TIFFs), which expand in response to T H 2 cytokines to form subcutaneous fibrous bands. Activation of the T H 2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified T H 2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Biofabrication of skin tissue equivalents using 3D bioprinting technology has gained much attention in recent times due to the simplicity, the versatility of the technology and its ability in bioengineering biomimetic tissue histology. The key component being the bioink, several groups are actively working on the development of various bioink formulations for optimal skin tissue construction., Methods: Here, we present alginate (ALG), gelatin (GEL) and diethylaminoethyl cellulose (DCEL) based bioink formulation and its application in bioprinting and biofabrication of skin tissue equivalents. Briefly, DEAE cellulose powder was dispersed in alginate solution with constant stirring at 60 °C to obtain a uniform distribution of cellulose fibers; this was then mixed with GEL solution to prepare the bioink. The formulation was systematically characterized for its morphological, physical, chemical, rheological, biodegradation and biocompatibility properties. The printability, shape fidelity and cell-laden printing were assessed using the CellInk bioprinter., Results: The bioink proved to be a good printable, non-cytotoxic and stable hydrogel formulation. The primary human fibroblast and keratinocyte-loaded 3D bioprinted constructs showed excellent cell viability, collagen synthesis, skin-specific marker and biomimetic tissue histology., Conclusion: The results demonstrated the successful formulation of ALG-GEL-DCEL bioink and its application in the development of human skin tissue equivalents with distinct epidermal-dermal histological features., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

Exposure of cells or biological tissues to high-power pulses of terahertz (THz) radiation leads to changes in a variety of intracellular processes. However, the role of heating effects due to strong absorption of THz radiation by water molecules still stays unclear. In this study, we performed numerical modelling in order to estimate the thermal impact on water of a single THz pulse as well as a series of THz pulses. A finite-element (FE) model that provides numerical solutions for the heat conduction equation is employed to compute the temperature increase. A simple expression for temperature estimation in the center of the spot of THz radiation is presented for given frequency and fluence of the THz pulse. It has been demonstrated that thermal effect is determined by either the average power of radiation or by the fluence of a single THz pulse depending on pulse repetition rate. Human dermal fibroblasts have been exposed to THz pulses (with an energy of [Formula: see text] and repetition rate of 100 Hz) to estimate the thermal effect. Analysis of heat shock proteins expression has demonstrated no statistically significant difference ([Formula: see text]) between control and experimental groups after 3 h of irradiation., (© 2021. The Author(s).)

Tissue-engineered skin equivalents are reconstructed the functions of human skin and can be used as an alternative to animal experiments in basic study or as cultured skin for regenerative medicine. Recent studies confirmed that epidermal tight junctions (TJs), which are complex intercellular junctions formed in the stratum granulosum of human skin, play an important part in the formation of the skin barrier function. In well-formed reconstructed human skin models, there are several reports on the expression of TJ proteins and their localization in epidermal layer, however, the morphological features of TJ, showing tight junctional contacts and the process of TJ formation have yet to be investigated. In this study, we systematically examined and identified TJ-related proteins and TJ structure in three-dimensional (3D) human skin equivalents reconstructed by layer-by-layer (LbL) cell coating technique (LbL-3D Skin). We demonstrate localization of TJ-related proteins and time course of formation of TJ structure with typical junctional morphology in LbL-3D Skin. These data provide evidence that the LbL-3D Skin is an in vitro model with structure and function extremely similar to living skin., (© 2021 John Wiley & Sons Ltd.)

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.

Acid-soluble, undenatured, type I collagen (BSC) isolated, for the first time, from gilthead bream skin and the novel fabricated 3D porous wound dressing were analyzed for physicochemical and biological properties, in order to offer a safe alternative to commercial bovine collagen (BC) products. SDS-polyacrylamide analysis confirmed the purity of BSC preparation. The hydroxyproline content and temperature of denaturation of BSC were lower than those of BC, in accordance with the structural data recorded by FT-IR spectroscopy. However, certain concentrations of BSC stimulated the cell metabolism of L929 fibroblasts in a higher proportion than BC. The 3D wound dressing presented high porosity and low surface hydrophobicity that could help cell attachment and growth. The rapid biodegradation of BSC wound dressing could explain the improved in vitro cell migration and wound closure rate. In conclusion, the skin of gilthead bream from the Black Sea coast represented a valuable source for the biomedical industry, providing biocompatible, biodegradable collagen and 3D porous wound dressing, as novel material with enhanced wound healing activity., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

UV-B and IR-A radiation are important inducers of biological changes in skin involving ROS generation. The overloading of antioxidant defense mechanisms by ROS production could lead to photoaging and photocarcinogenesis processes. Various traditional usages are reported for Aralia nudicaulis L. extracts, including treatment of dermatological disorders. Antioxidant and anti-inflammatory properties have already been reported for other Aralia species possibly due to the presence of phenolic compounds. However, the phenolic composition and the potential activity of A. nudicaulis rhizomes extract against oxidative stress and UV/IR damages have not been investigated. The main aims of this study were to prepare a fraction enriched in phenolic compounds (FEPC) from A. nudicaulis rhizomes, to identify its major phenolic compounds and to assess its potential for protective effects against oxidative stress induced by UV-B, IR-A or inflammation. A quantitative LC-MS study of FEPC shows that chlorogenic, caffeic and protocatechuic acids are the main phenolic compounds present, with concentrations of 15.6%, 15.3% and 4.8% of the total composition, respectively. With a validated analytical method, those compounds were quantified over different stages of the growing period. As for biological potential, first this extract demonstrates antioxidant and anti-inflammatory activities. Furthermore, ROS generation induced by IR-A and UV-B were strongly inhibited by A. nudicaulis extract, suggesting that Aralia nudicaulis L. rhizome extract could protect dermal cells against oxidative stress induced by UV-B and IR-A.

The population decline of the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from an animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we established and characterized fibroblast cell cultures from the skin sample of a newborn common hippopotamus in this study. By combining the tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 34 h according to the growth curve. Karyotyping based on Giemsa staining showed that the cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. The amplified mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with that of the registered H. amphibius complete mitochondrial DNA, confirming the species of origin of the cells. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4, and vimentin. In conclusion, we generated the fibroblast cell cultures from a common hippopotamus and identified their characteristics using multiple techniques. We believe the cryopreserved cells could be useful genetic materials for future research., (© 2021 International Federation for Cell Biology.)

To date, placental trophoblasts have been of interest in the fields of obstetrics and gynecology, mainly due to their involvement in the formation of a connection between the mother and fetus that aids in placental development and fetal survival. However, the regenerative capacities of trophoblasts for application in regenerative medicine and tissue engineering are poorly understood. Here, we aim to determine the skin regeneration and anti-aging capacities of trophoblast-derived conditioned medium (TB-CM) and exosomes (TB-Exos) using human normal dermal fibroblasts (HNDFs). TB-CM and TB-Exos treatments significantly elevated the migration and proliferation potencies of HNDF cells in a dose- and time-dependent manner. When RNA sequencing (RNA-seq) was used to investigate the mechanism underlying TB-CM-induced cell migration on scratch-wounded HNDFs, the increased expression of genes associated with C-X-C motif ligand (CXCL) chemokines, toll-like receptors, and nuclear factor-kappa B (NF-κB) signaling was observed. Furthermore, treatment of intrinsically/extrinsically senescent HNDFs with TB-CM resulted in an enhanced rejuvenation of HNDFs via both protection and restoration processes. Gene expression of extracellular matrix components in the skin dermis significantly increased in TB-CM- and TB-Exos-treated HNDFs. These components are involved in the TB-CM and Exo-mediated regeneration and anti-aging of HNDFs. Thus, this study demonstrated the regenerative and anti-aging efficacies of trophoblast-derived secretomes, suggesting their potential for use in interventions for skin protection and treatment.

Microfluidic-based tissues-on-chips (TOCs) have thus far been restricted to modelling simple epithelia as a single cell layer, but likely due to technical difficulties, no TOCs have been reported to include both an epithelial and a stromal component despite the biological importance of the stroma for the structure and function of human tissues. We present, for the first time, a novel approach to generate 3D multilayer tissue models in microfluidic platforms. As a proof of concept, we modelled skin, including a dermal and an epidermal compartment. To accomplish this, we developed a parallel flow method enabling the deposition of bilayer tissue in the upper chamber, which was subsequently maintained under dynamic nutrient flow conditions through the lower chamber, mimicking the function of a blood vessel. We also designed and built an inexpensive, easy-to-implement, versatile, and robust vinyl-based device that overcomes some of the drawbacks present in PDMS-based chips. Preliminary tests indicate that this biochip will allow the development and maintenance of multilayer tissues, which opens the possibility of better modelling of the complex cell-cell and cell-matrix interactions that exist in and between the epithelium and mesenchyme, allowing for better-grounded tissue modelling and drug screening.

Collagen type I is the most abundant form of collagen in human tissues, and is composed of two identical α-1 type I chains and an α-2 type I chain organized in a triple helical structure. A previous study has shown that human collagen α-2 type I (hCOL1A2) promotes collagen synthesis, wound healing, and elastin production in normal human dermal fibroblasts (HDFs). However, the biological effects of human collagen α-1 type I (hCOL1A1) on various skin properties have not been investigated. Here, we isolate and identify the hCOL1A1-collagen effective domain (CED) which promotes collagen type I synthesis. Recombinant hCOL1A1-CED effectively induces cell proliferation and collagen biosynthesis in HDFs, as well as increased cell migration and elastin production. Based on these results, hCOL1A1-CED may be explored further for its potential use as a preventative agent against skin aging. [BMB Reports 2021; 54(6): 329-334].

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P < 0.05 was considered significant., Results: A skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p < 0.001 and p < 0.01 respectively), as well as between the control and treated conditions (p < 0.01 and p < 0.001 respectively). The scratch assay induced IL8 upregulation after 3 h (p < 0.05) which continued to increase up to day 1 (p < 0.05). On the other hand, the administration of TNF led to the downregulation of IL8 (p < 0.01), followed by an upregulation on day 2. IL8 gene expression decreased in the scratched condition after day 1 as the natural healing process took place and was lower than in the treated condition on day 8 (p < 0.05). Both untreated and treated conditions showed a downregulation of TGFA 3 h after scratch when compared with the control condition (p < 0.01). Administration of rTNF showed significant downregulation of TGFA after 24 h when compared with the control (p < 0.01) and treated conditions (p < 0.05)., Conclusion: This study suggests that a leukocyte-depleted PRP-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion.

Background: When vascular endothelial cells are subjected to external stimuli, paracrine hormones and cytokines act on adjacent cells. The regulation of the biological behaviour of cells is closely related to the maintenance of organ function and the occurrence and development of disease. However, it is unclear whether vascular endothelial cells affect the biological behaviour of cells involved in wound repair through autocrine and paracrine mechanisms and ultimately play a role in wound healing. We aimed to verify the effect of the autocrine and paracrine functions of vascular endothelial cells on wound healing., Materials and Methods: ELISA was used to detect platelet-derived growth factor, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor in human umbilical vascular endothelial cell-conditioned medium (HUVEC-CM). Different concentrations of HUVEC-CM were used to treat different stem cells. CCK-8 and scratch assays were used to detect the proliferation and migration ability of each cell. A full-thickness dorsal skin defect model was established in mice, and skin wound healing was observed after the local injection of HUVEC-CM, endothelial cell medium (ECM), or normal saline. H&E staining and immunofluorescence were used to observe the gross morphology of the wound tissue, the epithelial cell migration distance, and the expression of CD3 and CD31., Results: HUVEC-CM promotes the proliferation and migration of epidermal stem cells, skin fibroblasts, bone marrow mesenchymal stem cells, and HUVECs themselves. Furthermore, HUVEC-CM can promote angiogenesis in mouse skin wounds and granulation tissue formation and can accelerate wound surface epithelialization and collagen synthesis, thereby promoting wound healing., Conclusion: Our results clearly suggest that it is practicable and effective to promote wound healing with cytokines secreted by vascular endothelial cells in a mouse model., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2021 Yang Lu et al.)

Dermal fibroblasts play a key role in maintaining homoeostasis and functionality of the skin. Their contractility plays a role in changes observed during ageing, especially in processes such as wound healing, inflammation, wrinkling and scar tissue formation as well as structural changes on extracellular matrix. Although alternations in skin physiology and morphology have been previously described, there remains a paucity of information about the influence of chronological ageing on dermal fibroblast contractility. In this study, we applied a novel nano-biomechanical technique on cell-embedded collagen hydrogels in combination with mathematical modelling and numerical simulation to measure contraction forces of normal human dermal fibroblasts (NHDF). We achieved quantitative differentiation of the contractility of cells derived from 'young' (< 30 years old) and 'aged' (> 60 years old) donors. Transforming growth factor β1 (TGF-β1) was used to stimulate the fibroblasts to assess their contractile potential. NHDF from aged donors exhibited a greater basal contractile force, while in contrast, NHDF from young donors have shown a significantly larger contractile force in response to TGF-β1 treatment. These findings validate our nano-biomechanical measurement technique and provide new insights for considering NHDF contractility in regenerative medicine and as a biomarker of dermal ageing processes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Delayed wound healing is one of the most challenging complications of diabetes mellitus (DM) in clinical medicine, and it is related to the excessive generation of reactive oxygen species (ROS). Photobiomodulation (PBM) can promote wound healing in many ways, so it can be used as a method for the treatment of delayed healing of DM wounds. In this study, we investigated the effect of PBM on ROS homeostasis in human embryonic skin fibroblast cells (CCC-ESFs) cultured in high glucose concentrations. The CCC-ESFs were cultured in vitro and divided into two groups, including the control group and the 635 nm laser irradiation group. After 2 days of high glucose treatment, the experimental group was irradiated with different doses of laser for 3 days. First, we measured the cellular proliferation, and the results showed that laser irradiation could promote cellular proliferation. Then, we measured the generation of ROS, the activities of total superoxide dismutase (SOD), and total antioxidant capacity (TAC) of the cells; the results showed that high glucose destroyed cells by inducing high concentration of ROS, the balance of oxidation, and antioxidation cause oxidative stress damage to cells. PBM can increase the antioxidant capacity of cells, reducing the high concentration of ROS induced by high glucose. Finally, we measured the levels of mitochondrial membrane potential (∆ψ m ) and the secretion of nuclear factor kappa-B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β); the results showed that PBM can reduce apoptosis and regulate the inflammatory state. We conclude that PBM can maintain the ROS homeostasis, increase the TAC of cells, and trigger the cellular proliferation, and the response of CCC-ESFs to PBM was dose-dependent.

Background: Aged human skin is primarily attributable to the loss of collagen. Hepatocyte growth factor (HGF) acts as an anti-fibrotic factor by suppression of collagen production. In aged human skin, HGF is elevated in dermal fibroblasts and thus contributes to dermal aging (thin dermis) by suppression of collagen production., Objective: We aimed to investigate the underlying mechanisms of age-related elevation of HGF expression., Methods: Collagen fibrils in the aged skin dermis are fragmented and disorganized, which impairs collagen-fibroblast interaction, resulting in reduced fibroblast spreading and size. To explore the connection between reduced dermal fibroblast size and age-related elevation of HGF expression, we manipulate dermal fibroblast size, and cell-size dependent regulation of HGF was investigated by laser capture microdissection, immunostaining, capillary electrophoresis immunoassay, and quantitative RT-PCR., Results: We found that reduced fibroblast size is responsible for age-related elevation of HGF expression. Further investigation indicated that cell size-dependent upregulation of HGF expression was mediated by impeded YAP/TAZ nuclear translocation and their target gene, CCN2. Conversely, restoration of dermal fibroblast size rapidly reversed cell-size-dependent upregulation of HGF in a YAP/TAZ-dependent manner. Finally, we confirmed that elevated HGF expression is accompanied by the reduced expression of YAP/TAZ and CCN2 in the aged human skin in vivo., Conclusion: Age-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner. These data reveal a novel mechanism by which reduction of fibroblast size upregulates HGF expression, which in turn contributes to loss of collagen, a prominent feature of aged human skin., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Collagen is the major structural protein in the extracellular matrix of skin produced by fibroblasts. UV exposure results in infiltration of neutrophils within the epidermis and dermis, inducing collagen damage and contributing to the process of photo-aging. Collagen-3 is an integral structural component with collagen-1, and is an important regulator of collagen-1 fibrillogenesis. Addition of neutrophils activated with TNFα to normal human dermal fibroblast cultures, but not their supernatant, caused significant collagen-3 damage. To study whether Lumenato can protect from collagen-3 damage, it was added to co-cultures of Normal human dermal fibroblasts and neutrophils activated with TNFα. Lumenato prevented collagen-3 damage induced by activated neutrophils in a dose-dependent manner in the co-cultures. Lumenato also induced a low rate of collagen-3 synthesis in a dose-dependent manner detected by pro-collagen-3 secretion, but did not affect fibroblast cell number. Although Lumenato inhibited MMP-8, MMP-9, and elastase secreted from neutrophils, its main effect was in inhibiting both NADPH oxidase-producing superoxides and MPO activity-producing halides in a dose-dependent manner that correlated with protection from collagen-3 damage. In conclusion, the results suggest that Lumenato induces low levels of collagen-3 that may contribute for skin health and is very effective in defending the co-cultures from collagen-3 damage by inhibiting free radicals secreted from neutrophils, thus, indicating Lumenato's possible potential for skin protection., Competing Interests: The authors have read the journal's policy, and the authors of the study have the following competing interests to declare: Lycored Ltd. partially supported the costs of materials of the research in the form of a grant (number 2016-2020) awarded to RL. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The authors would like to declare the following patents/patent applications associated with this research: A patent Application No. PCT/IL2020/051086 was submitted based on the research results.

Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.

Stem cell transplantation is a promising therapy for wound healing, but the low retention and survival of transplanted stem cells limit their application. Injectable hydrogels exert beneficial effects in skin tissue engineering. In this study, an injectable hydrogel composed of sodium alginate (SA) and collagen type I (Col) was synthesized as a tissue scaffold to improve the efficacy of stem cells in a full-thickness excision wound model. Our results showed that SA/Col hydrogel was injectable, biodegradable, and exhibited low immunogenicity, which could promote the retention and survival of hUC-MSCs in vivo. SA/Col loaded with hUC-MSCs showed reduced wound size (p < 0.05). Histological and immunofluorescence results confirmed that SA/Col loaded with hUC-MSCs significantly promoted the formation of granulation, enhanced collagen deposition and angiogenesis, increased VEGF and TGF-β1 expression (p < 0.05), and mitigated inflammation evidenced by lower production of TNF-α and IL-1β and higher release of IL-4 and IL-10 (p < 0.05). Furthermore, SA/Col loaded with hUC-MSCs significantly lowered the expression of NLRP3 inflammasome-related proteins (p < 0.05). Taken together, our results suggest that SA/Col loaded with hUC-MSCs promotes skin wound healing via partly inhibiting NLRP3 pathway, which has potential to the treatment of skin wounds.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

Cell-based therapies have become a popular approach in the field of regenerative medicine. Human fibroblast cells, one of the cell types widely used in clinical applications, have been used for skin regeneration and wound healing procedures. Furthermore, they are utilized for aesthetic purposes since fibroblasts lose their abilities such as collagen synthesis with age. Here, we describe detailed procedures for isolation, culture, cryopreservation, and preparation of fibroblasts derived from adult human skin as a final product under good manufacturing practice-compliant conditions.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells., (© 2020 Wiley Periodicals LLC.)

The tissue reaction of pig skin to implantation of decellularized and recellularized dermal matrices on a formed wound defect was evaluated by histological methods on days 2, 5, 8, 16, and 20 after surgery. Differences in tissue response to different matrices were identified. In experimental wounds coated with decellularized dermal matrices, we observed the formation of a scar tissue, which required autodermoplasty on day 12 of the experiment. In wounds coated with recellularized dermal matrices, all layers of the skin completely recovered by day 20 after surgery with the formation of full dermal and epidermal layers. Our findings suggest that reparative morphological changes in the wound depend on the presence of fibroblasts in the implanted dermal matrix.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding protein-5 (IGFBP-5) is a conserved member of the IGFBP family of proteins that is overexpressed in SSc and IPF lung tissues. In this study, we investigated the functional role of IGFBP-5 in the development of fibrosis in vivo using a transgenic model. We generated transgenic mice ubiquitously expressing human IGFBP-5 using CRISPR/Cas9 knock-in. Our data show that the heterozygous and homozygous mice are viable and express human IGFBP-5 (h IGFBP-5 ). Transgenic mice had increased expression of extracellular matrix (ECM) genes, especially Col3a1 , Fn, and Lox in lung and skin tissues of mice expressing higher transgene levels. Histologic analysis of the skin tissues showed increased dermal thickness, and the lung histology showed subtle changes in the heterozygous and homozygous mice as compared with the wild-type mice. These changes were more pronounced in animals expressing higher levels of h IGFBP-5 . Bleomycin increased ECM gene expression in wild-type mice and accentuated an increase in ECM gene expression in transgenic mice, suggesting that transgene expression exacerbated bleomycin-induced pulmonary fibrosis. Primary lung fibroblasts cultured from lung tissues of homozygous transgenic mice showed significant increases in ECM gene expression and protein levels, further supporting the observation that IGFBP-5 resulted in a fibrotic phenotype in fibroblasts. In summary, transgenic mice expressing human IGFBP-5 could serve as a useful animal model for examining the function of IGFBP-5 in vivo.

Diabetes mellitus, a complex metabolic disorder, leads to many health complications like kidney failure, diabetic heart disease, stroke, and foot ulcers. Treatment approaches of diabetes and identification of the mechanisms underlying diabetic complications of the skin have gained importance due to continued rapid increase in the diabetes incidence. A thick and pre-vascularized in vitro 3D type 2 diabetic human skin model (DHSM) was developed in this study. The methacrylated gelatin (GelMA) hydrogel was produced by photocrosslinking and its pore size (54.85 ± 8.58 μm), compressive modulus (4.53 ± 0.67 kPa) and swelling ratio (17.5 ± 2.2%) were found to be suitable for skin tissue engineering. 8% GelMA hydrogel effectively supported the viability, spreading and proliferation of human dermal fibroblasts. By isolating dermal fibroblasts, human umbilical vein endothelial cells and keratinocytes from type 2 diabetic patients, an in vitro 3D type 2 DHSM, 12 mm in width and 1.86 mm thick, was constructed. The skin model consisted of a continuous basal epidermal layer and a dermal layer with blood capillary-like structures, ideal for evaluating the effects of anti-diabetic drugs and wound healing materials and factors. The functionality of the DHSM was showed by applying a therapeutic hydrogel into its central wound; especially fibroblast migration to the wound site was evident in 9 d. We have demonstrated that DHSM is a biologically relevant model with sensitivity and predictability in evaluating the diabetic wound healing potential of a therapeutic material.

Keloid, a common dermal fibroproliferative disorder, is benign skin tumors characterized by the aggressive fibroblasts proliferation and excessive accumulation of extracellular matrix. However, common therapeutic approaches of keloid have limited effectiveness, emphasizing the momentousness of developing innovative mechanisms and therapeutic strategies. Epigenetics, representing the potential link of complex interactions between genetics and external risk factors, is currently under intense scrutiny. Accumulating evidence has demonstrated that multiple diverse and reversible epigenetic modifications, represented by DNA methylation, histone modification, and non-coding RNAs (ncRNAs), play a critical role in gene regulation and downstream fibroblastic function in keloid. Importantly, abnormal epigenetic modification manipulates multiple behaviors of keloid-derived fibroblasts, which served as the main cellular components in keloid skin tissue, including proliferation, migration, apoptosis, and differentiation. Here, we have reviewed and summarized the present available clinical and experimental studies to deeply investigate the expression profiles and clarify the mechanisms of epigenetic modification in the progression of keloid, mainly including DNA methylation, histone modification, and ncRNAs (miRNA, lncRNA, and circRNA). Besides, we also provide the challenges and future perspectives associated with epigenetics modification in keloid. Deciphering the complicated epigenetic modification in keloid is hopeful to bring novel insights into the pathogenesis etiology and diagnostic/therapeutic targets in keloid, laying a foundation for optimal keloid ending.

Introduction: Hyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin's HA-HYAL interactions on the molecular and cellular levels., Objective: To analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin., Materials and Methods: Chip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase "Dessau") on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo., Results: Genome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 h ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels., Conclusion: HYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.

Herbal medicines are attractive agents for human care. In this study, we found that the alcohol extract of Ajuga taiwanensis (ATE) screened from a chemical bank exhibited potent capacity for suppressing senescence associated biomarkers, including SA-β-gal and up-regulated p53 in old human dermal fibroblasts (HDFs) without induction of significant cytotoxicity up to 100 µg/ml. Concomitantly, cells re-entered the cell cycle by reducing G1 phase arrest and increasing cell growth rate. The ATE was further partitioned to obtain the sub-fractions of n-butanol (BuOH), ethyl acetate (EA) and water. The BuOH and water sub-fractions exhibited less effects on prohibition of cell growth than the EA sub-fraction. All of these sub-fractions exhibited the ability on suppressing SA-β-gal and p53 of old HDFs as low as 5-10 µg/ml. Under the activity guided fractionation and isolation, a major active constituent named AT-1 was isolated. The AT-1 was further identified as 8-O-acetylharpagide by structural analysis, and it could suppress SA-β-gal and p53 of old HDFs below 10 µM. In addition, the intracellular reactive oxygen species (ROS) levels of old HDFs were suppressed by ATE, the sub-fractions of BuOH and water, and AT-1. However, the EA sub-fraction showed little ability on suppression of ROS. Furthermore, we performed an in vivo study using aging mice to be fed with ATE and the sub-fractions followed by immunohistochemical (IHC) staining. The expression of p53 and SA-β-gal was significantly reduced in several tissue sections, including skin, liver, kidney, and spleen. Taken together, current data demonstrated that A. taiwanensis could suppress cellular senescence in HDFs, and might be used for health care.

Purpose: An electric field (EF) can be used to change the mechanical properties of cells and skin tissues. We demonstrate EF-induced elasticity changes in human dermal fibroblasts (HDFs) and a human skin equivalent and identify the underlying principles related to the changes., Methods: HDFs and human skin equivalent were stimulated with electric fields of 1.0 V/cm. Change in cellular elasticity was determined by using atomic force microscopy. Effects of EF on the biomechanical and chemical properties of a human skin equivalent were analyzed. In cells and tissues, the effects of EF on biomarkers of cellular elasticity were investigated at the gene and protein levels., Results: In HDFs, the cellular elasticity was increased and the expression of biomarkers of cellular elasticity was regulated by the EF. Expression of the collagen protein in the human skin equivalent was changed by EF stimulation; however, changes in density and microstructure of the collagen fibrils were not significant. The viscoelasticity of the human skin equivalent increased in response to EF stimulation, but molecular changes were not observed in collagen., Conclusions: Elasticity of cells and human skin equivalent can be regulated by electrical stimulation. Especially, the change in cellular elasticity was dependent on cell age., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)