Characterization of hair follicle development in engineered skin substitutes.

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells.

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized ß-catenin (hK') were used to prepare ESS epithelium. After 10 days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6 weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

The endogenous protease inhibitor TIMP-1 mediates protection and recovery from cutaneous photodamage.

UVB exposure is well known to induce skin photodamage and photoaging that correlates with qualitative and quantitative deterioration of the dermal extracellular matrix (ECM) because of the upregulation of matrix metalloproteinases (MMPs). Although inhibitory effects of tissue inhibitor of metalloproteinases (TIMPs) on most MMPs have been reported, the protective role of TIMP-1 against photodamage is poorly understood. To address this, TIMP-1 function was augmented or abolished in a human skin xenograft photodamage model after the confirmation of significantly diminished TIMP-1 expression both in photoaged and intrinsically aged skins. During a chronic UVB exposure regimen, pre-treatment with a lentiviral vector overexpressing TIMP-1 or concomitant administration of an anti-TIMP-1-neutralizing antibody (NAB) led to photoprotection or more severe photodamage, respectively. Overexpression of TIMP-1 resulted in significant inhibition of UVB-induced ECM degradation, as well as suppression of decreased skin elasticity and roughness, whereas the NAB-mediated inhibition of TIMP-1 had opposite effects. Furthermore, UVB-induced production of the pro-inflammatory cytokine, tumor necrosis factor α, was inhibited by TIMP-1 treatment of human keratinocytes. Taken together, these data shed light on the important role of TIMP-1 in protection and recovery from cutaneous photodamage because of its suppression of ECM degradation and inflammation.

Effects of IGF-binding protein 5 in dysregulating the shape of human hair.

The hair follicle has a unique dynamic property to cyclically regenerate throughout life. Despite significant progress in hair structure and hair shape determination using animal models, the mechanisms controlling the architecture and the shape of the human hair remain largely unexplored. In this study, comparison of the genetic expression of several human genes, especially those involved in growth, development, and differentiation, between Caucasian curly hair and naturally straight hair was performed. Thereafter, analyses using human recombinant and lentiviral vector technologies were conducted to further dissect and elucidate a molecular mechanism that regulates hair growth and development, particularly in controlling the shape of the hair shaft. Overexpression of IGF-binding protein 5 (IGFBP-5) in the human hair xenografts obtained from straight- and curly-haired individuals was found to result in the decreased expression of several extracellular matrix proteins and disassembly of adhesional junctions, resulting in twisted hair shafts as well as an unusual deposition of hair cuticle that may be derived from the disturbance of normal proliferation and differentiation. This study provides evidence that IGFBP-5 has an effect on human hair shape, and that lentiviral transduction regimen can be used for functional analysis of genes involved in human hair morphogenesis.

Characterization of tight junctions and their disruption by UVB in human epidermis and cultured keratinocytes.

It has not been confirmed whether tight junctions (TJs) function as a paracellular permeability barrier in adult human skin. To clarify this issue, we performed a TJ permeability assay using human skin obtained from abdominal plastic surgery. Occludin, a marker protein of TJs, was expressed in the granular layer, in which a subcutaneously injected paracellular tracer, Sulfo-NHS-LC-Biotin (556.59 Da), was halted. Incubation with ochratoxin A decreased the expression of claudin-4, an integral membrane protein of TJs, and the diffusion of paracellular tracer was no longer prevented at the TJs. These results demonstrate that human epidermis possesses TJs that function as an intercellular permeability barrier at least against small molecules (∼550 Da). UVB irradiation of human skin xenografts and human skin equivalents (HSEs) resulted in functional deterioration of TJs. Immunocytochemical staining of cultured keratinocytes showed that occludin was localized into dot-like shapes and formed a discontinuous network when exposed to UVB irradiation. Furthermore, UVB irradiation downregulated the active forms of Rac1 and atypical protein kinase C, suggesting that their inactivation caused functional deterioration of TJs. In conclusion, TJs function as a paracellular barrier against small molecules (∼550 Da) in human epidermis and are functionally deteriorated by UVB irradiation.

Essential role of microfibrillar-associated protein 4 in human cutaneous homeostasis and in its photoprotection.

UVB-induced cutaneous photodamage/photoaging is characterized by qualitative and quantitative deterioration in dermal extracellular matrix (ECM) components such as collagen and elastic fibers. Disappearance of microfibrillar-associated protein 4 (MFAP-4), a possible limiting factor for cutaneous elasticity, was documented in photoaged dermis, but its function is poorly understood. To characterize its possible contribution to photoprotection, MFAP-4 expression was either augmented or inhibited in a human skin xenograft photodamage murine model and human fibroblasts. Xenografted skin with enhanced MFAP-4 expression was protected from UVB-induced photodamage/photoaging accompanied by the prevention of ECM degradation and aggravated elasticity. Additionally, remarkably increased or decreased fibrillin-1-based microfibril development was observed when fibroblasts were treated with recombinant MFAP-4 or with MFAP-4-specific siRNA, respectively. Immunoprecipitation analysis confirmed direct interaction between MFAP-4 and fibrillin-1. Taken together, our findings reveal the essential role of MFAP-4 in photoprotection and offer new therapeutic opportunities to prevent skin-associated pathologies.

Stem cell factor-KIT signalling plays a pivotal role in regulating pigmentation in mammalian hair.

Hair greying is one of the most distinct but least comprehended features of senescence. The signalling of stem cell factor (SCF) and its receptor KIT has been documented to regulate essential roles in the maintenance of embryonic melanocyte lineages and postnatal cutaneous melanogenesis, although little is known about its detailed mechanisms in postnatal hair pigmentation. To address this, anagen human hair follicles and C57BL/6 murine pelage were analysed in this study. Molecular biological analyses of murine follicular skin indicated a significant increase of membrane-bound SCF expression, reaching its peak 8-16 days after anagen induction in concert with the escalation of cutaneous tyrosinase activity and corresponding pigmentation. Administration of KIT-neutralizing antibody abolished MITF and tyrosinase expressions, resulting in a reversible hair depigmentation in murine regenerated hair and human hair organ culture. Quantitative RT-PCR of human hair follicles indicated that KIT expression as well as the expression of several melanogenic factors, including MITF, was significantly lower in unpigmented than in pigmented follicles. Taken together, these data revealed a pivotal role of SCF-KIT signalling in the maintenance of human hair follicle melanogenesis during the anagen cycle and its involvement in physiological ageing of the hair follicle pigmentary unit.

Mechanistic effects of long-term ultraviolet B irradiation induce epidermal and dermal changes in human skin xenografts.

UVB irradiation has been reported to induce photoaging and suppress systemic immune function that could lead to photocarcinogenesis. However, because of the paucity of an UVB-induced photodamaged skin model, precise and temporal mechanism(s) underlying the deleterious effects of long-term UVB exposure on human skin have yet to be delineated. In this study, we established a model using human skin xenografted onto severe combined immunodeficient mice, which were subsequently challenged by repeated UVB irradiation for 6 weeks. Three-dimensional optical image analysis of skin replicas and noninvasive biophysical measurements illustrated a significant increase in skin surface roughness, similar to premature photoaging, and a significant loss of skin elasticity after long-term UVB exposure. Resembling authentically aged skin, UVB-exposed samples exhibited significant increases in epithelial keratins (K6, K16, K17), elastins, and matrix metalloproteinases (MMP-1, MMP-9, MMP-12) as well as degradation of collagens (I, IV, VII). The UVB-induced deterioration of fibrous keratin intermediate filaments was also observed in the stratum corneum. Additionally, similarities in gene expression patterns between our model and chronologically aged skin substantiated the plausible relationship between photodamage and chronological age. Furthermore, severe skin photodamage was observed when neutralizing antibodies against TIMP-1, an endogenous inhibitor of MMPs, were administered during the UVB exposure regimen. Taken together, these findings suggest that our skin xenograft model recapitulates premature photoaged skin and provides a comprehensive tool with which to assess the deleterious effects of UVB irradiation.

Functional analysis of keratinocytes in skin color using a human skin substitute model composed of cells derived from different skin pigmentation types.

Skin color is one of the most distinct features in the human race. To assess the mechanisms of skin color variation, human skin substitutes (HSS) were constructed by grafting mixtures of cultured keratinocytes and melanocytes from a combination of donor skin types, together with light skin derived fibroblasts, into chambers inserted onto the back skin of severe combined immunodeficient (SCID) mice. The resulting complexion coloration of the HSS was relatively darker and lighter when dark and light skin derived keratinocytes, respectively, were combined with melanocytes derived from either light or dark skin. The melanin content in the epidermis and the maturation stage of melanosomes in basal keratinocytes were significantly increased in the HSS composed of dark compared to light skin derived keratinocytes. In addition, the ratio of individual/clustered melanosomes in recipient keratinocytes was increased in the former as opposed to the latter HSS. The genetic expression of endothelin-1, proopiomelanocortin, microphthalmia-associated transcription factor, tyrosinase, GP100, and MART1 were increased in HSS composed of dark vs. light skin derived keratinocytes. These data suggest that our HSS is a promising melanogenic model that demonstrates the role of the keratinocyte in regulating in part both melanogenesis and distribution of transferred melanosomes.

Comparative efficacy and safety of deoxyarbutin, a new tyrosinase-inhibiting agent.

Several tyrosinase inhibitors have been developed and utilized to ameliorate various cutaneous hyperpigmentary disorders and complexion discolorations. Deoxyarbutin (dA) (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), designed using quantitative structure-activity relationships (QSAR), demonstrates effective inhibition of mushroom tyrosinase and skin-lightening capability (1). However, its comparative safety, effectiveness, and reversibility to other known tyrosinase inhibitors in human melanocytes had not been determined. The effect of dA was assessed in cultured human skin cells, on xenographs, and with a clinical trial. Using cultured human melanocytes, the maximum concentration of dA that allowed 95% viability was fourfold greater than for hydroquinone (HQ), indicating that dA is less cytotoxic/cytostatic than HQ. The viability of cultured human keratinocytes and fibroblasts was also less compromised by increasing concentrations of dA as opposed to HQ. At the maximum concentration allowing normal cellular viability, dA effectively inhibited tyrosinase activity and melanin content in human melanocytes, whereas HQ was marginally inhibitory. Upon removal of dA, tyrosinase activity and melanin content was normalized within five days. Topical application of dA on human xenografts resulted in a gradual and visually apparent skin lightening effect during an eight-week period. In a clinical trial, dA facilitated fading of pre-tanned skin to a statistically significant greater extent than either HQ or no treatment. These results demonstrate that dA is a potentially safe, effective, and reversible tyrosinase inhibitor.

Interaction between stem cell factor and endothelin-1: effects on melanogenesis in human skin xenografts.

The two paracrine melanogenic cytokines, stem cell factor (SCF) and endothelin-1 (ET-1), have been demonstrated to play pivotal roles in skin pigmentation including UVB-induced pigmentation and senile lentigo. However, little is known regarding their interactive effect on skin pigmentation. In order to investigate their roles in vivo, facultative pigmentation of human skin xenografts on severe combined immunodeficient (SCID) mice was assessed. After 1 week of acclimation in a pathogen-free barrier, dermatomed fresh cadaveric skin was surgically grafted onto the back of the mice and allowed to heal for 5-6 weeks prior to cytokine administration. Intradermal injections of SCF at 0.7 or 2.0 microg significantly increased skin pigmentation when compared to vehicle control. Despite the lack of a dose-dependent pigmentation response following ET-1 administration, the combination of 0.2 microg SCF and 0.1 microg ET-1 demonstrated a statistically significant increase in tyrosinase gene expression substantiated by the enhancement of melanin content and skin pigmentation compared to treatment with SCF alone or ET-1 alone. These findings establish an in vivo interaction between SCF and ET-1 with regard to their capacity to effect an increase in skin pigmentation.

An in vivo mouse model of human skin substitute containing spontaneously sorted melanocytes demonstrates physiological changes after UVB irradiation.

Human skin substitutes (HSS) have been developed for repairing burns and other acute or chronic wounds. But although the clinical utility of HSS is well known, scant attention has been paid to their cosmetic properties, especially with regard to color compatibility with the patient's complexion. In this study, we generated an HSS from mixed cell slurries containing keratinocytes and fibroblasts with and without melanocytes on the back of severe combined immunodeficient mice by means of a spontaneous cell-sorting technique. At 16 wk after grafting, Caucasian donor-derived HSS with melanocytes were macroscopically clearly darker than those without melanocytes, and a more darkly pigmented HSS was produced when cells from donors of African descent were seeded. Immunohistochemistry of c-kit, S-100, and HMB45, as well as Fontana-Masson staining and transmission electron microscopy (TEM) demonstrated that melanocytes spontaneously localized to the basal layer. Melanosome transfer to keratinocytes was correctly reorganized, and melanin was evenly dispersed in the basal and suprabasal layers. Colorimetric analysis showed a significantly lower L-value by day 14 following irradiation with 120 mJ per cm2 ultraviolet-B (UVB) (p<0.01), whereas epidermal thickness increased by 50% 1 d after exposure (p<0.01), indicating a normal physiological response to UVB irradiation. These findings suggest that HSS with spontaneously sorted melanocytes offer a means of treating both the structural and cosmetic aspects of skin conditions and trauma, such as pigmentary disorders and skin wounds, by allowing manipulation of the color and population of donor melanocytes.