Human Skin Repair Research Articles

Regenerative Medicine to Improve Outcomes of Ventral Hernia Reconstruction (REPAIR Study) Phase 1: Find the Best Decellularization Protocol for the Human Dermis.

Background: Tissue regeneration is a complex process that allows wounds to heal. Many options are currently available to help human skin repair and to reduce the recurrence of hernias. The aim of this study is to analyze the best decellularization protocol for allogenic human dermal tissues. Methods: Dermal flaps from donors were used and compared with a control group. Each flap was subjected to seven different decellularization protocols and washed with a sequence of five solutions. The samples were then subjected to four control tests (such as Nile Red), and long-term contacts were analyzed to assess whether the decellularized dermis samples could support the growth of human fibroblasts. Results: All the samples had an average residual viability of 60%. Except for one sample, the decellularization treatments were able to reduce cell viability significantly. The Nile Red test showed a significant reduction in phospholipid content (mean 90%, p-value < 0.05) in all treatments. The cell growth increased in a linear manner. As described in the literature, sodium-dodecyl-sulfate (SDS) caused an interference between the test and the detergent. Conclusions: This paper shows the first step to finding the best decellularization protocol for allografting human dermal tissues. Further biocompatibility tests and DNA quantification are necessary.

LGR5 is a conserved marker of hair follicle stem cells in multiple species and is present early and throughout follicle morphogenesis

Hair follicle stem cells are key for driving growth and homeostasis of the hair follicle niche, have remarkable regenerative capacity throughout hair cycling, and display fate plasticity during cutaneous wound healing. Due to the need for a transgenic reporter, essentially all observations related to LGR5-expressing hair follicle stem cells have been generated using transgenic mice, which have significant differences in anatomy and physiology from the human. Using a transgenic pig model, a widely accepted model for human skin and human skin repair, we demonstrate that LGR5 is a marker of hair follicle stem cells across species in homeostasis and development. We also report the strong similarities and important differences in expression patterns, gene expression profiles, and developmental processes between species. This information is important for understanding the fundamental differences and similarities across species, and ultimately improving human hair follicle regeneration, cutaneous wound healing, and skin cancer treatment.

Circular RNA Signatures of Human Healing and Nonhealing Wounds

Venous ulcers (VUs) have complex and obscure pathogenicity, and effective VU therapies are still lacking. Circular RNAs (circRNAs) have emerged as powerful gene regulators with important roles in health and disease. In this study, we used paired total RNA and small RNA sequencing to profile circRNAs, protein-coding mRNAs, and microRNAs expression in a unique collection of clinical samples: healthy skin and acute wounds at inflammatory and proliferative phases and wound-edge VU biopsies. We unravel a dynamically changed expression pattern of circRNAs during human skin repair and their abnormal expression signature in VU, which are presented as a searchable web resource (www.xulandenlab.com/humanwounds-circrna). We analyzed the coexpression relationship between the circRNAs and mRNAs with weighted correlation network analysis and constructed circRNA‒mRNA‒microRNA networks. This allowed us to expose the regulatory networks specific to the inflammatory and proliferative phases of wound repair and VU, the biological processes the circRNAs may regulate, and the circRNAs that could sponge microRNAs in human wounds. Importantly, we found that hsa-CHST15_0003 and hsa-TNFRSF21_0001, two circRNAs upregulated in VU, hampered epidermal keratinocyte migration while promoting proliferation by modulating gene networks underpinning these cellular processes. This study paves the way to decipher the functional significance of circRNAs in tissue repair.

A Novel Xenograft Model Demonstrates Human Fibroblast Behavior During Skin Wound Repair and Fibrosis.

Objective: Xenografts of human skin in immunodeficient mice provide a means of assessing human skin physiology and its response to wounding. Approach: We describe a novel xenograft model using full-thickness human neonatal foreskin to examine human skin wound repair. Full-thickness 8 mm human neonatal foreskin biopsies were sutured into the dorsum of NOD scid gamma (NSG; NOD.Cg-Prkdc scidIl2rgtm1Wjl/SzJ) pups as subcutaneous grafts. At postnatal day 21 the subcutaneous grafts were exposed to cutaneous grafts. Following maturation of 2 months, xenografts were then wounded with 5 mm linear incisions and monitored until postwound day (PWD) 14 to study skin repair and fibrosis. To explore whether our model can be used to test the efficacy of topical therapies, wounded xenografts were injected with antifibrotic fibroblast growth factor 2 (FGF2) for the first four consecutive PWDs. Xenografts were harvested for analysis by histology and fluorescence-activated cell sorting (FACS). Results: Xenografts were successfully engrafted with evidence of mouse-human anastomoses and resembled native neonatal foreskin at the gross and microscopic level. Wounded xenografted skin scarred with human collagen and an expansion of CD26-positive human fibroblasts. Collagen scar was quantitated by neural network analysis, which revealed distinct clustering of collagen fiber networks from unwounded skin and wounded skin at PWD7 and PWD14. Collagen fiber networks within FGF2-treated wounds at PWD14 resembled those in untreated wounded xenografts at PWD7, suggesting that FGF2 treatment at time of wounding can reduce fibrosis. Innovation and Conclusion: This novel xenograft model can be used to investigate acute fibrosis, fibroblast heterogeneity, and the efficacy of antifibrotic agents during wound repair in human skin.

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Fluorescent cell tracer dye permits real-time assessment of re-epithelialization in a serum-free ex vivo human skin wound assay.

Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re-epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re-epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome.

Comprehensive molecular characterization of microneedling therapy in a human three-dimensional skin model.

Background and objectivesMicroneedling therapy is a widely used technique in dermatology. However, little is known about the underlying molecular effects of this therapy on extracellular matrix remodeling, wound healing, and inflammation. The aim of this study was to examine morphological and molecular changes caused by microneedling treatment in a standardized in vitro full-thickness 3D model of human skin.Materials and methodsA microneedling device was used to treat full-thickness 3D skin models. Specimens were harvested at specified time points and qRT-PCR and microarray studies were performed. Frozen sections were examined histologically.ResultsMicroneedling treatment caused morphological changes in the skin model resulting in an almost complete recovery of the epidermis five days after treatment. Microarray analysis identified an upregulation of genes that are associated with tissue remodeling and wound healing (e.g. COL3A1, COL8A1, TIMP3), epithelial proliferation and differentiation (KRT13, IGF1), immune cell recruitment (CCL11), and a member of the heat shock protein family (HSPB6). On the other hand, we detected a downregulation of pro-inflammatory cytokines (e.g. IL1α, IL1β, IL24, IL36γ, IL36RN), and antimicrobial peptides (e.g. S100A7A, DEFB4). These data were confirmed by independent RT-PCR analyses.ConclusionWe present for the first time the direct molecular effects of microneedling therapy on epidermal keratinocytes and dermal fibroblasts using a standardized 3D skin model. Treatment resulted in histological alterations and changed the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling. This data suggests that skin microneedling plays a role in dermal remodeling, increases epidermal differentiation, and might also have a direct effect on collagen synthesis. These findings may increase our understanding of the molecular mechanisms of human skin repair induced by microneedling therapy and will allow comparisons with competing applications, such as ablative laser therapies.

Tailor-made mannosylerythritol lipids: current state and perspectives.

Mannosylerythritol lipids (MELs) are a type of glycolipid biosurfactant produced by basidiomycetous yeasts, most notably those belonging to the genera Pseudozyma and Ustilago. Mannosylerythritol lipids are environmentally friendly and possess many unique functions, such as gene delivery, bio-activation, and human skin repair, and thus have potential applications in cosmetic, pharmaceutical, agriculture, food, and environmental industries. However, MELs will require overcoming same issues related to the commercialization, e.g., expansion of the structure and function variety and cost reduction. In the past decade, various studies have attempted to tailor production of targeted MELs in order to expand the utility of these biosurfactants. Moreover, the rapid development of genomic sequencing techniques will enhance our ability to modify MEL producers. In this review, we focus on current research into the tailored production of MELs, including conventional and advanced approaches.

Possible Contribution of Fibrocytes to Increased Type I Collagen Synthesis during the Early Stage of Dermal Wound Repair in Human Skin

Possible Contribution of Fibrocytes to Increased Type I Collagen Synthesis during the Early Stage of Dermal Wound Repair in Human Skin

Ectodysplasin A Pathway Contributes to Human and Murine Skin Repair

The highly conserved ectodysplasin A (EDA)/EDA receptor signaling pathway is critical during development for the formation of skin appendages. Mutations in genes encoding components of the EDA pathway disrupt normal appendage development, leading to the human disorder hypohidrotic ectodermal dysplasia. Spontaneous mutations in the murine Eda (Tabby) phenocopy human X-linked hypohidrotic ectodermal dysplasia. Little is known about the role of EDA signaling in adult skin homeostasis or repair. Because wound healing largely mimics the morphogenic events that occur during development, we propose a role for EDA signaling in adult wound repair. Here we report a pronounced delay in healing in Tabby mice, demonstrating a functional role for EDA signaling in adult skin. Moreover, pharmacological activation of the EDA pathway in both Tabby and wild-type mice significantly accelerates healing, influencing multiple processes including re-epithelialization and granulation tissue matrix deposition. Finally, we show that the healing promoting effects of EDA receptor activation are conserved in human skin repair. Thus, targeted manipulation of the EDA/EDA receptor pathway has clear therapeutic potential for the future treatment of human pathological wound healing.

Identification of Drugs that Regulate Dermal Stem Cells and Enhance Skin Repair.

SummaryHere, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs), a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

Enhancements of skin cell proliferations and migrations via 6-dehydrogingerdione.

Human skin protects the body from mechanical and chemical damages, and skin wound healing is a costly procedure and worldwide issue. A Zingiber officinale compound, 6-dehydrogingerdione (6-DG), is presented as a novel biofunctional healing agent for human skin wound repair. The effectiveness on cell growth/migration, growth factor, collagen amount, and enzymatic activity was assessed. 6-DG treatment accelerated cellular proliferation and migration dose-dependently. Enzyme-linked immunosorbent assay (ELISA) showed that 6-DG brought about higher growth factor productions on transforming growth factor-β (TGF-β), platelet-derived growth factor-αβ (PDGF-αβ), and vascular endothelial growth factors (VEGF). Under phorbol 12-myristate 13-acetate (PMA) incubation, 6-DG increased fibroblast collagen yield obviously, reduced matrix metalloproteinase-1 (MMP-1) protein expression, and recovered tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. 6-DG also blocked the mitogen-activated protein kinase (MAPK) pathway by suppressing c-Jun protein levels and extracellular signal-regulated kinases (ERK) phosphorylation in fibroblasts. From all of the above, 6-DG has potential to be a novel agent for human skin repair.

Abstract B52: The role of p38 activation in nucleotide excision repair in human skin

Abstract Keratinocyte-based skin malignancies are diagnosed over one million times a year in this country alone despite widespread knowledge about UV avoidance and sunscreen use. The majority bear evidence of UV-induced mutations in key cancer-related genes such as TP53, highlighting the importance of environmental sun and tanning bed exposure in their pathophysiology. Accumulation of UV-induced DNA mutations, and ultimately skin cancer risk, is determined not only by UV exposure but also by the ability of keratinocytes to repair mutagenic damage caused by UV radiation. Xeroderma pigmentosum patients, in whom DNA repair is defective due to homozygous loss of any one of at least eight enzymes in the nucleotide excision repair (NER) pathway, suffer exceptionally high incidences of UV-induced skin cancers, illustrating the importance of NER in resistance to skin cancer. There is little understanding of the factors that determine the efficiency of NER in the skin. We have found that NER can be modified exogenously by inducing a cAMP-based pathway in the skin. Specifically, topically-applied forskolin, an activator of adenylate cyclase reproducibly enhances clearance of UV-induced cyclobutane dimers in human skin explants and in human keratinocytes. Pharmacologic induction of cAMP via forskolin efficiently activates the protein kinase A (PKA) signaling cascade and leads to downstream phosphorylation of the cAMP responsive binding element (CREB) transcription factor. Intriguingly, we and others have found that UV radiation also results in robust CREB phosphorylation in human skin and human keratinocytes, but in a PKA-independent manner. Furthermore, UVB induced CREB phosphorylation displays similar kinetics to UVB induced p38 activation and is diminished upon p38 inhibition. Together, these data suggests the p38 damage response pathway, recruited by UV, directly phosphorylates CREB without involving the cAMP second messenger. Currently, we are determining the effects of p38 inhibition on NER activity. We hypothesize that inhibiting p38 will lead to decreased NER activity in human skin and this loss of NER can be overcome with the addition of forskolin. Our observations presented here suggest that the p38/CREB signaling axis may be a major determinant of NER efficiency in the skin and raise the exciting possibility of a novel skin cancer preventative approach outside of UV-avoidance. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B52.

Skin efficacy of liposomes composed of internal wool lipids rich in ceramides

Ceramides from intercellular lipids of skin stratum corneum are known to play an essential role in maintaining and structuring the lipid barrier of the skin. Internal wool lipids (IWL), which are also rich in ceramides, have a composition similar to that of the stratum corneum lipids. IWL extracted with chloroform/methanol azeotrope at the laboratory scale have been shown to be capable of forming liposomes with a stable bilayer structure. Furthermore, topical application of these IWL liposomes on intact and compromised skin has been demonstrated to improve barrier skin properties.In this study we evaluated the effect on human skin repair of different IWL extract compositions obtained by two extraction methodologies. The formation and characteristics of the liposomes prepared were greatly influenced by the IWL composition, primarily the sterol sulfate content. The IWL liposomes improved skin barrier integrity and increased skin hydration when applied onto intact skin. These improvements were slightly enhanced in the case of IWL liposomes that were richer in polar lipids.

Members of the Family Zingiberaceae Promote Tropoelastin and Procollagen Type 1 Synthesis in Cultured Human Fibroblasts

Recent interest into the mechanisms of dermal healing following injury by photo damage or chronologic aging denotes the need of fast reliable benchmark-assays of skin repair to screen the many novel compounds currently available. Considerable attention has been given to botanical extracts and their antioxidant properties. Herein we report on two members of the ginger family (Zingiberaceae) as potential skin repair agents. Ginger (Zingiber officinale) and turmeric (Curcuma longa) have a long history of use in folk medicine with known medicinal properties (anti-inflammatory, antioxidant, anti-tumor, and anti-bacterial). Human dermal fibroblasts were treated with either a CO2 extract of ginger or curcumin and culture supernatants (72 hr) assayed by enzyme-linked immunosorbent assay (ELISA) for tropoelastin and procollagen type 1 synthesis. Both ginger and curcumin significantly increased tropoelastin (p<0.05 & p<0.05, respectively) and procollagen type 1 (p<0.05 & p<0.05, respectively) synthesis in vitro. These results suggest that members of the family Zingiberaceae may share a common constituent that is responsible for increased tropoelastin and procollagen type 1 synthesis and thus may be potentially valuable reagents for use in human skin repair. Work supported by NIH RO1 HL07097.

An In Vivo Model of Wound Healing in Genetically Modified Skin-Humanized Mice

Cutaneous wound-healing disorders are a major health problem that requires the development of innovative treatments. Whithin this context, the search for reliable human wound-healing models that allow us to address both mechanistic and therapeutic matters is warranted. In this study, we have developed a novel invivo wound-healing model in a genetically modified human context. Our model is based on the regeneration of human skin on the back of nude mice by transplantation of a cultured bioengineered skin equivalent previously designed in our laboratory. In this setting, human keratinocytes in the epidermal compartment were genetically modified with a retroviral vector encoding the enhanced green fluorescent protein (EGFP). After stable engraftment of the EGFP skin was achieved (9-12 wk after grafting), a small circular full thickness wound was performed on this mature human skin. A wide variety of parameters involved in wound healing were monitored, including tissue architecture, cell proliferation, epidermal differentiation, dermal remodelling, and basement membrane regeneration. Wounded gene-targeted skin-humanized mice re-capitulated native skin wound-healing features. In addition, when keratinocyte growth factor (KGF), a growth factor that has been shown to improve wound healing, was added to wounds during 3 d, the re-epithelialization was significantly accelerated. The present wound-healing model system provides a suitable in vivo tool to test gene transfer strategies for human skin repair. It also serves as a complementary platform for studies in genetically modified mice and as a model to evaluate pharmaceutical therapeutic approaches for impaired wound healing.

Clinical application of nerve growth factor on human corneal ulcer.

Nerve growth factor (NGF) is a neurotrophic and immunomodulatory factor contributing of the control of cutaneous morphogenesis, wound healing and inflammatory responses. Following the evidence that topical administration of NGF leads to healing of human corneal ulcers, we investigate the therapeutic action of NGF on immune and/or autoimmune cutaneous ulcers. We found that 1-10 micrograms of highly purified murine NGF dissolved in 50 microliters of physiological solution and topically applied to skin ulcer leads, after 4-12 weeks of daily treatment (depending on the size and depth of the lesion) to complete healing of the ulcer. Thus, NGF was able to promote complete repair in human skin and corneal ulcers which were poorly or non-responsive to conventional topical and systemic treatments. No side effects were observed and a follow up after 4 months showed no signs of relapse. These findings indicate that failure of cutaneous tissues to produce sufficient amounts of NGF might represent a prominent mechanism implicated in the clinical manifestation of ocular ulcers.

XPD exon 10 and 23 polymorphisms and DNA repair in human skin in situ

Forty-four Finnish volunteers who were previously studied with regard to the repair rate of UV-specific cyclobutane pyrimidine dimers in the skin were genotyped for XPD polymorphisms at codons 312 (exon 10 G-->A, Asp-->Asn) and 751 (exon 23 A-->C, Lys-->Gln). The repair rate was measured at 24 h for two different cyclobutane dimers. The data did not show consistent XPD genotype-specific differences in DNA repair rates among all subjects. The combined exon 10 AA and exon 23 CC genotype was associated with an approximately 50% depression of repair rate but this was of borderline statistical significance. However, the exon 23 C allele was associated with depressed repair among subjects aged 50 years or older and the result was consistent with both dimers.

SKH-1 hairless mice repair UV-induced pyrimidine dimers in epidermal DNA

The SKH-1 hairless mouse strain has been used extensively as a model for human photocarcinogenesis, photoimmunology and photoaging, but little is known about DNA repair in living mouse skin. Mice were irradiated with UV-B light at doses which produce mild to severe sunburn, and the frequency of pyrimidine dimers in epidermal DNA was measured immediately and 6 h after irradiation using T4 endonuclease V treatment and alkaline agarose gel electrophoresis. The results demonstrate significant removal of pyrimidine dimers in mouse skin in vivo, with a dimer half-life of 7.4 h. These findings are similar to the repair of dimers in human skin in vivo. The SKH-1 hairless mouse is thus a useful model for pyrimidine dimer repair in human skin.