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Sample records for human dermal papilla

  1. Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles.

    PubMed

    Topouzi, Helena; Logan, Niall J; Williams, Greg; Higgins, Claire A

    2017-06-01

    The dermal papilla is a cluster of mesenchymal cells located at the base of the hair follicle which have a number of important roles in the regulation of hair growth. As a consequence, in vitro models of these cells are widely used to study the molecular mechanisms which underlie hair follicle induction, growth and maintenance. While dermal papilla from rodent hair follicles can be digested prior to cell isolation, the unique extracellular matrix composition found in human dermal papilla renders enzymes such as trypsin and collagenase insufficient for digestion of the dermal papilla into a single cell suspension. As such, to grow human dermal papilla cells in vitro, the papilla has to first be isolated via a micro-dissection approach from the follicle. In this article we describe the micro-dissection and culture methods, which we use within our laboratory, for the study of human dermal papilla cells. © 2017 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd.

  2. Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells.

    PubMed

    Kiratipaiboon, Chayanin; Tengamnuay, Parkpoom; Chanvorachote, Pithi

    2015-12-15

    Although the growth of unwanted hair or hirsutism is a harmless condition, many people find it bothersome and embarrassing. Maintaining stem cell features of dermal papilla cells is a critical biological process that keeps the high rate of hair growth. Glycyrrhizic acid has been reported to impair hair growth in some studies; however, its underlying mechanism has not yet been investigated. This study aimed to explore the effect and underlying mechanism of glycyrrhizic acid on stemness of human dermal papilla cells. The stem cell molecular markers, epithelial to mesenchymal markers and Wnt/β-catenin-associated proteins of human dermal papilla cell line and primary human dermal papilla cells were analysed by western blot analysis and immunocytochemistry. The present study demonstrated that glycyrrhizic acid significantly depressed the stemness of dermal papilla cells in dose- and time-dependent manners. Clonogenicity and stem cell markers in the glycyrrhizic acid-treated cells were found to gradually decrease in the culture in a time-dependent manner. Our results demonstrated that glycyrrhizic acid exerted the stem cell suppressing effects through the interruption of ATP-dependent tyrosine kinase/glycogen synthase kinase3β-dependent mechanism which in turn down-regulated the β-catenin signalling pathway, coupled with decreased its down-stream epithelial-mesenchymal transition and self-renewal transcription factors, namely, Oct-4, Nanog, Sox2, ZEB1 and Snail. The effect of glycyrrhizic acid on the reduction of stem cell features was also observed in the primary dermal papilla cells directly obtained from human hair follicles. These results revealed a novel molecular mechanism of glycyrrhizic acid in regulation of dermal papilla cells and provided the evidence supporting the use of this compound in suppressing the growth of unwanted hair. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice.

    PubMed

    Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

    2017-01-01

    Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice.

  4. Hair Follicle Generation by Injections of Adult Human Follicular Epithelial and Dermal Papilla Cells into Nude Mice

    PubMed Central

    Nilforoushzadeh, Mohammadali; Rahimi Jameh, Elham; Jaffary, Fariba; Abolhasani, Ehsan; Keshtmand, Gelavizh; Zarkob, Hajar; Mohammadi, Parvaneh; Aghdami, Nasser

    2017-01-01

    Objective Dermal papilla and hair epithelial stem cells regulate hair formation and the growth cycle. Damage to or loss of these cells can cause hair loss. Although several studies claim to reconstitute hairs using rodent cells in an animal model, additional research is needed to develop a stable human hair follicle reconstitution protocol. In this study, we have evaluated hair induction by injecting adult cultured human dermal papilla cells and a mixture of hair epithelial and dermal papilla cells in a mouse model. Materials and Methods In this experimental study, discarded human scalp skins were used to obtain dermal papilla and hair epithelial cells. After separation, cells were cultured and assessed for their characteristics. We randomly allocated 15 C57BL/6 nude mice into three groups that received injections in their dorsal skin. The first group received cultured dermal papilla cells, the second group received a mixture of cultured epithelial and dermal papilla cells, and the third group (control) received a placebo [phosphate-buffered saline (PBS-)]. Results Histopathologic examination of the injection sites showed evidence of hair growth in samples that received cells compared with the control group. However, the group that received epithelial and dermal papilla cells had visible evidence of hair growth. PKH tracing confirmed the presence of transplanted cells in the new hair. Conclusion Our data showed that injection of a combination of adult human cultured dermal papilla and epithelial cells could induce hair growth in nude mice. This study emphasized that the combination of human adult cultured dermal papilla and epithelial cells could induce new hair in nude mice. PMID:28670518

  5. Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth

    PubMed Central

    Higgins, Claire A.; Chen, James C.; Cerise, Jane E.; Jahoda, Colin A. B.; Christiano, Angela M.

    2013-01-01

    De novo organ regeneration has been observed in several lower organisms, as well as rodents; however, demonstrating these regenerative properties in human cells and tissues has been challenging. In the hair follicle, rodent hair follicle-derived dermal cells can interact with local epithelia and induce de novo hair follicles in a variety of hairless recipient skin sites. However, multiple attempts to recapitulate this process in humans using human dermal papilla cells in human skin have failed, suggesting that human dermal papilla cells lose key inductive properties upon culture. Here, we performed global gene expression analysis of human dermal papilla cells in culture and discovered very rapid and profound molecular signature changes linking their transition from a 3D to a 2D environment with early loss of their hair-inducing capacity. We demonstrate that the intact dermal papilla transcriptional signature can be partially restored by growth of papilla cells in 3D spheroid cultures. This signature change translates to a partial restoration of inductive capability, and we show that human dermal papilla cells, when grown as spheroids, are capable of inducing de novo hair follicles in human skin. PMID:24145441

  6. Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells.

    PubMed

    Bassino, Eleonora; Gasparri, Franco; Giannini, Valentina; Munaron, Luca

    2015-05-01

    Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell-based co-cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF-1, two well-known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.

  7. Malva verticillata seed extracts upregulate the Wnt pathway in human dermal papilla cells.

    PubMed

    Lee, E Y; Choi, E-J; Kim, J A; Hwang, Y L; Kim, C-D; Lee, M H; Roh, S S; Kim, Y H; Han, I; Kang, S

    2016-04-01

    Mesenchymal-epithelial interactions are important in controlling hair growth and the hair cycle. The β-catenin pathway of dermal papilla cells (DPCs) plays a pivotal role in morphogenesis and normal regeneration of hair follicles. Deletion of β-catenin in the dermal papilla reduces proliferation of the hair follicle progenitor cells that generate the hair shaft and induces an early onset of the catagen phase. In this study, a modulator of the Wnt/β-catenin activity was studied in oriental herb extracts on cultured human DPCs. The effect of Malva verticillata (M. verticillata) seeds on human DPCs was investigated by a Wnt/β-catenin reporter activity assay system (β-catenin-TCF/LEF reporter gene) and cell proliferation analysis. The synthesis of the factors related to hair growth and cycling was measured at both the mRNA and the protein level by semi-quantitative PCR and Western blot analysis, respectively. An extract from M. verticillata seeds increased Wnt reporter activity in a concentration-dependent manner and also led to increased β-catenin levels in cultured human DPCs. Myristoleic acid, identified as an effective compound of M. verticillata seeds, stimulated the proliferation of DPCs in a dose-dependent manner and increased transcription levels of the downstream targets: IGF-1, KGF, VEGF and HGF. Myristoleic acid also enhanced the phosphorylation of MAPKs (Akt and p38). Overall, the data suggest that this extract of M. verticillata seeds could be a good candidate for treating hair loss by modulating the Wnt/β-catenin pathway in DPCs. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  8. TGF-beta is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis.

    PubMed

    Inoue, Keita; Aoi, Noriyuki; Yamauchi, Yuji; Sato, Takahiro; Suga, Hirotaka; Eto, Hitomi; Kato, Harunosuke; Tabata, Yasuhiko; Yoshimura, Kotaro

    2009-01-01

    Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming growth factor (TGF)-beta(2) to be highly expressed in cultured hDPCs. Keratinocyte conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(2) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D(3) analogue was found to promote TGF-beta(2) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(2) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(2) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs.

  9. Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression.

    PubMed

    Wakame, Koji; Okawa, Hiroshi; Komatsu, Ken-Ich; Nakata, Akifumi; Sato, Keisuke; Ingawa, Hiroyuki; Kohchi, Chie; Nishizawa, Takashi; Soma, Gen-Ichiro

    2016-07-01

    The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 μg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  10. Cryptomphalus aspersa Mollusc Egg Extract Promotes Regenerative Effects in Human Dermal Papilla Stem Cells

    PubMed Central

    Alameda, María Teresa; Morel, Esther; Parrado, Concepción; González, Salvador; Juarranz, Ángeles

    2017-01-01

    The aim of this study was to test, by an in vitro approach, whether a natural extract derived from eggs of the mollusc Cryptomphalus aspersa (e-CAF) that seems to present regenerative properties, can enhance the mobilization of human hair dermal papilla cells (HHDPCs) and play a role on tissue repair and regeneration. We have tested HHDPCs proliferation by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) assay; cell migration by using a wound healing assay, as well as the modulation of the expression of cytoskeletal (F-actin and vimentin) and cell adhesion to the extracellular matrix (ECM) (vinculin and P-FAK) proteins. We also explored whether e-CAF could lead HHDPCs to keratinocytes and/or fibroblasts by evaluating the expression of specific markers. We have compared these e-CAF effects with those induced by TGFβ1, implicated in regulation of cell proliferation and migration. e-CAF promotes proliferation and migration of HHDPCs cells in a time- and dose-dependent manner; it also increases the migratory behavior and the expression of adhesion molecules. These results support the fact that e-CAF could play a role on skin regeneration and be used for the prevention or repair of damaged tissue, either due to external causes or as a result of cutaneous aging. PMID:28230777

  11. Cryptomphalus aspersa Mollusc Egg Extract Promotes Regenerative Effects in Human Dermal Papilla Stem Cells.

    PubMed

    Alameda, María Teresa; Morel, Esther; Parrado, Concepción; González, Salvador; Juarranz, Ángeles

    2017-02-21

    The aim of this study was to test, by an in vitro approach, whether a natural extract derived from eggs of the mollusc Cryptomphalus aspersa (e-CAF) that seems to present regenerative properties, can enhance the mobilization of human hair dermal papilla cells (HHDPCs) and play a role on tissue repair and regeneration. We have tested HHDPCs proliferation by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) assay; cell migration by using a wound healing assay, as well as the modulation of the expression of cytoskeletal (F-actin and vimentin) and cell adhesion to the extracellular matrix (ECM) (vinculin and P-FAK) proteins. We also explored whether e-CAF could lead HHDPCs to keratinocytes and/or fibroblasts by evaluating the expression of specific markers. We have compared these e-CAF effects with those induced by TGFβ₁, implicated in regulation of cell proliferation and migration. e-CAF promotes proliferation and migration of HDDPCs cells in a time- and dose-dependent manner; it also increases the migratory behavior and the expression of adhesion molecules. These results support the fact that e-CAF could play a role on skin regeneration and be used for the prevention or repair of damaged tissue, either due to external causes or as a result of cutaneous aging.

  12. Herbal Extracts Induce Dermal Papilla Cell Proliferation of Human Hair Follicles

    PubMed Central

    Rastegar, Hosein; Aghaei, Mahmoud; Barikbin, Behrooz; Ehsani, Amirohushang

    2015-01-01

    Background The number of people suffering from balding or hair thinning is increasing, despite the advances in various medical therapies. Therefore, it is highly important to develop new therapies to inhibit balding and increase hair proliferation. Objective We investigated the effects of herbal extracts commonly used for improving balding in traditional medicine to identify potential agents for hair proliferation. Methods The expression levels of 5α-reductase isoforms (type I and II) were analyzed using quantitative real-time reverse transcription polymerase chain reaction in the human follicular dermal papilla cells (DPCs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide and bromodeoxyuridine tests were used to evaluate the cell proliferation effect of herbal extracts in DPCs. The expression levels of extracellular signal-regulated kinase (ERK), Akt, cyclin D1, cyclin-dependent kinase 4 (Cdk4), B-cell lymphoma (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using western blot analysis. Results The 5α-reductase isoform mRNAs and proteins were detected in the cultured DPCs, and the expression level of 5α-R2 in DPCs in the presence of the herbal extracts was gradually decreased. Herbal extracts were found to significantly increase the proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. These results show that the herbal extracts tested affected the protein expressions of ERK, Akt, cyclin D1, Cdk4, Bcl-2, and Bax in DPCs. Conclusion These results suggest that herbal extracts exert positive effects on hair proliferation via ERK, Akt, cyclin D1, and Cdk4 signaling in DPCs; they also suggest that herbal extracts could be a great alternative therapy for increasing hair proliferation. PMID:26719634

  13. Hair-inducing ability of human dermal papilla cells cultured under Wnt/β-catenin signalling activation.

    PubMed

    Soma, Tsutomu; Fujiwara, Shigeyoshi; Shirakata, Yuji; Hashimoto, Koji; Kishimoto, Jiro

    2012-04-01

    It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/β-catenin activation results in maintenance of the hair-inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/β-catenin signalling stimulation by GSK-3β inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu-positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P-cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/β-catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell-based therapy. © 2012 John Wiley & Sons A/S.

  14. Characterization of Ovine Dermal Papilla Cell Aggregation

    PubMed Central

    Sari, Agnes Rosarina Prita; Rufaut, Nicholas Wolfgang; Jones, Leslie Norman; Sinclair, Rodney Daniel

    2016-01-01

    Context: The dermal papilla (DP) is a condensation of mesenchymal cells at the proximal end of the hair follicle, which determines hair shaft size and regulates matrix cell proliferation and differentiation. DP cells have the ability to regenerate new hair follicles. These cells tend to aggregate both in vitro and in vivo. This tendency is associated with the ability of papilla cells to induce hair growth. However, human papilla cells lose their hair-inducing activity in later passage number. Ovine DP cells are different from human DP cells since they do not lose their aggregative behavior or hair-inducing activity in culture. Nonetheless, our understanding of ovine DP cells is still limited. Aim: The aim of this study was to observe the expression of established DP markers in ovine cells and their association with aggregation. Subjects and Methods: Ovine DP cells from three different sheep were compared. Histochemistry, immunoflourescence, and polymerase chain reaction experiments were done to analyze the DP markers. Results: We found that ovine DP aggregates expressed all the 16 markers evaluated, including alkaline phosphatase and versican. Expression of the versican V0 and V3 isoforms, neural cell adhesion molecule, and corin was increased significantly with aggregation, while hey-1 expression was significantly decreased. Conclusions: Overall, the stable expression of numerous markers suggests that aggregating ovine DP cells have a similar phenotype to papillae in vivo. The stability of their molecular phenotype is consistent with their robust aggregative behavior and retained follicle-inducing activity after prolonged culture. Their phenotypic stability in culture contrasts with DP cells from other species, and suggests that a better understanding of ovine DP cells might provide opportunities to improve the hair-inducing activity and therapeutic potential of human cells. PMID:27625564

  15. Valproic Acid Induces Hair Regeneration in Murine Model and Activates Alkaline Phosphatase Activity in Human Dermal Papilla Cells

    PubMed Central

    Lee, Soung-Hoon; Yoon, Juyong; Shin, Seung Ho; Zahoor, Muhamad; Kim, Hyoung Jun; Park, Phil June; Park, Won-Seok; Min, Do Sik; Kim, Hyun-Yi; Choi, Kang-Yell

    2012-01-01

    Background Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. Methodology/ Principal Findings Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl2 also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. Conclusions/ Significance Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth. PMID:22506014

  16. VEGF induces proliferation of human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERK

    SciTech Connect

    Li, Wei; Man, Xiao-Yong; Li, Chun-Ming; Chen, Jia-Qi; Zhou, Jiong; Cai, Sui-Qing; Lu, Zhong-Fa; Zheng, Min

    2012-08-15

    Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DP cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF{sub 165} stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF{sub 165}-induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF{sub 165}. Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: Black-Right-Pointing-Pointer We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. Black-Right-Pointing-Pointer VEGF{sub 165} stimulated proliferation of human DP cells in a dose-dependent manner. Black-Right-Pointing-Pointer This stimulation was through VEGFR-2-mediated activation of ERK.

  17. Hair growth-promoting effect of Geranium sibiricum extract in human dermal papilla cells and C57BL/6 mice.

    PubMed

    Boisvert, William A; Yu, Miri; Choi, Youngbin; Jeong, Gi Hee; Zhang, Yi-Lin; Cho, Sunghun; Choi, Changsun; Lee, Sanghyun; Lee, Bog-Hieu

    2017-02-13

    Geranium sibiricum L. has been used as a medicinal plant to treat diarrhea, bacterial infection, and cancer in Bulgaria, Peru, and Korea. However, its hair growth-promoting effect was not investigated so far. This study examined the effects of Geranium sibiricum L. extract (GSE) on hair growth, using in vitro and in vivo models. Antioxidant, proliferation and migration assay of GSE was performed with human dermal papilla cells (hDPCs). Hair-growth promoting effect was measured in animal model. Relative expression of interleukin-1, vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor beta 1 was determined by real time RT-PCR. Expression of Ki-67 and stem cell factor were analyzed by immunohistochemistry. GSE treatment proliferated and migrated human dermal papilla cells (hDPCs) more than treatment of 10 μM minoxidil. GSE significantly stimulated the expression of Ki-67 protein and the mRNA levels of hepatocyte growth factor and vascular endothelial growth factor in hDPCs. Topical application of 1,000 ppm GSE for 3 weeks promoted more significant hair growth on shaved C57BL/6 mice than did 5% minoxidil. The histological morphology of hair follicles demonstrated an active anagen phase with the induction of stem cell factor. GSE treatment significantly reduced the number of mast cells and the expression of transforming growth factor beta 1 in mouse skin tissues. These results demonstrated that GSE promotes hair growth in vitro and in vivo by regulating growth factors and the cellular response.

  18. Effect of intense pulsed light treatment on human skin in vitro: analysis of immediate effects on dermal papillae and hair follicle stem cells.

    PubMed

    Larouche, D; Kim, D H; Ratté, G; Beaumont, C; Germain, L

    2013-10-01

    Hair follicles house a permanent pool of epithelial stem cells. Intense pulsed light (IPL) sources have been successfully used for hair removal, but long-term hair reduction may require several treatments. Many questions remain regarding the impact of IPL treatment on the structure of the hair follicle, more specifically on hair follicular stem cells and dermal papilla cells, a group of specialized cells that orchestrate hair growth. To characterize the destruction of human hair follicles and surrounding tissues following IPL treatment, with more attention paid to the bulge and the bulb regions. Human scalp specimens of Fitzpatrick skin phototype II were exposed ex vivo to IPL pulses and were then processed for histological analysis, immunodetection of stem cell-associated keratin 19, and revelation of the endogenous alkaline phosphatase activity expressed in dermal papilla cells. Histological analysis confirmed that pigmented structures, such as the melanin-rich matrix cells of the bulb in anagen follicles and the hair shaft, are principally targeted by IPL treatment, while white hairs and epidermis remained unaffected. Damage caused by heat sometimes extended over the dermal papilla cells, while stem cells were mostly spared. IPL epilation principally targets pigmented structures. Our results suggest that, under the tested conditions, collateral damage does not deplete stem cells. Damage at the dermal papilla was observed only with high-energy treatment modalities. Extrapolated to frequently treated hairs, these observations explain why some hairs grow back after a single IPL treatment. © 2013 British Association of Dermatologists.

  19. Identification of key genes induced by platelet-rich plasma in human dermal papilla cells using bioinformatics methods

    PubMed Central

    Shen, Haiyan; Cheng, Hanxiao; Chen, Haihua; Zhang, Jufang

    2016-01-01

    Dermal papilla cells (DPCs) are located at the base of hair follicles, and are known to induce hair follicle regeneration. Platelet-rich plasma (PRP) functions in hair follicle regeneration. To investigate the influence of PRP on DPCs, the present study analyzed RNA-seq data of human hair dermal papilla cells (HHDPCs) that were treated or untreated by PRP. The data included in the RNA-seq were from two normal and two treated HHDPC samples. Following identification by Cuffdiff software, differentially expressed genes (DEGs) underwent enrichment analyses, and protein-protein interaction networks were constructed using Cytoscape software. Additionally, transcription factor (TF)-DEG and TF-long non-coding RNA (lncRNA) regulatory networks were constructed. A total of 178 differentially expressed lncRNA were screened, 365 were upregulated and 142 were downregulated. Notably, upregulated cyclin dependent kinase 1 (CDK1) (degree=76), polo-like kinase 1 (PLK1) (degree=65), cell division cycle 20 (degree=50), cyclin B1 (degree=49), aurora kinase B (degree=47), cyclin dependent kinase 2 (degree=46) and downregulated v-myc avian myelocytomatosis viral oncogene homolog (MYC) (degree=12) had higher degrees in networks. In addition, CCAAT/enhancer binding protein β, E2F transcription factor 1 (E2F1), early growth response 1 and MYC may be key TFs for their target genes, and were enriched in pathways associated with the cell cycle. They may also be involved in cell proliferation via various interactions with other genes, for example CDK1-PLK1 and E2F1→CDK1. These dysregulated genes induced by PRP may affect proliferation of HHDPCs. PMID:27922680

  20. Different patterns of 5{alpha}-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

    SciTech Connect

    Liu, Shicheng Yamauchi, Hitoshi

    2008-04-18

    Androgens regulate hair growth, and 5{alpha}-reductase (5{alpha}R) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5{alpha}R activity. Real-time RT-PCR revealed that the highest level of 5{alpha}R1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5{alpha}R1 mRNA was observed in fibroblasts. Minimally detectable levels of 5{alpha}R2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5{alpha}R1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5{alpha}R1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5{alpha}R assay using [{sup 14}C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5{alpha}R activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17{beta}-HSD rather than 5{alpha}R among the three cell types. The 5{alpha}R1 inhibitor MK386 exhibited a more potent inhibitory effect on 5{alpha}R activity than finasteride (5{alpha}R2 inhibitor) in bDPCs.

  1. Squarticles as a lipid nanocarrier for delivering diphencyprone and minoxidil to hair follicles and human dermal papilla cells.

    PubMed

    Aljuffali, Ibrahim A; Sung, Calvin T; Shen, Feng-Ming; Huang, Chi-Ting; Fang, Jia-You

    2014-01-01

    Delivery of diphencyprone (DPCP) and minoxidil to hair follicles and related cells is important in the treatment of alopecia. Here we report the development of "squarticles," nanoparticles formed from sebum-derived lipids such as squalene and fatty esters, for use in achieving targeted drug delivery to the follicles. Two different nanosystems, nanostructured lipid carriers (NLC) and nanoemulsions (NE), were prepared. The physicochemical properties of squarticles, including size, zeta potential, drug encapsulation efficiency, and drug release, were examined. Squarticles were compared to a free control solution with respect to skin absorption, follicular accumulation, and dermal papilla cell targeting. The particle size of the NLC type was 177 nm; that of the NE type was 194 nm. Approximately 80% of DPCP and 60% of minoxidil were entrapped into squarticles. An improved drug deposition in the skin was observed in the in vitro absorption test. Compared to the free control, the squarticles reduced minoxidil penetration through the skin. This may indicate a minimized absorption into systemic circulation. Follicular uptake by squarticles was 2- and 7-fold higher for DPCP and minoxidil respectively compared to the free control. Fluorescence and confocal images of the skin confirmed a great accumulation of squarticles in the follicles and the deeper skin strata. Vascular endothelial growth factor expression in dermal papilla cells was significantly upregulated after the loading of minoxidil into the squarticles. In vitro papilla cell viability and in vivo skin irritancy tests in nude mice suggested a good tolerability of squarticles to skin. Squarticles provide a promising nanocarrier for topical delivery of DPCP and minoxidil.

  2. Minoxidil activates β-catenin pathway in human dermal papilla cells: a possible explanation for its anagen prolongation effect.

    PubMed

    Kwack, Mi Hee; Kang, Bo Mi; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

    2011-06-01

    It is believed that the length of the actively growing phase of the anagen hair cycle mainly contributes to hair length. Recent studies showed that maintenance of β-catenin activity in the dermal papilla cells (DPCs) enables hair follicles to keep actively growing. Topical minoxidil treatment promotes hair growth in men with androgenetic alopecia, suggesting that minoxidil may prolong the actively growing phase of the anagen hair cycle. To investigate whether minoxidil prolongs the anagen hair cycle in mice and, if so, to investigate whether minoxidil activates β-catenin pathway in human DPCs. Dorsal skins of C57BL/6 mice were depilated to synchronize the hair cycle. After 10 days, 3% minoxidil were topically applied daily for 10 days. Sections of back skins were stained with hematoxylin and eosin. Hair follicles were graded and hair cycle score (HCS) was calculated. Cultured human DPCs were transiently transfected with the β-catenin responsive TCF reporter plasmid (pTopflash) and corresponding negative control reporter (pFopflash) to assess the activity of β-catenin signaling by minoxidil. Immunofluorescence staining and immunoblot were performed to examine the expression and localization of β-catenin in the presence or absence of minoxidil. Phosphorylation of GSK3β, PKA and PKB were also examined by immunoblot after minoxidil treatment. RT-PCR analysis and immunoblot were employed to investigate the expression of β-catenin pathway targets in DPCs, such as Axin2, Lef-1, and EP2. Modest extension of anagen phase thereby delay of catagen progression was observed by application of minoxidil in mice. Minoxidil stimulated the transcriptional activity of pTopflash but not pFopflash. Nuclear accumulation of β-catenin was also observed after minoxidil treatment. Immunoblot further showed that minoxidil treatment increases the phosphorylation of GSK3β, PKA and PKB. Moreover, minoxidil induced Axin2, Lef-1, and EP2 expression. Our results strongly suggest that

  3. Ability of transplanted cultured epithelium to respond to dermal papillae.

    PubMed

    Xing, L; Kobayashi, K

    2001-10-01

    Cultured epithelium has been used successfully in the treatment of extensive burns. Regenerated epidermis, however, lacks such as hair follicles and sweat glands that are common in mammalian skin. We attempted to determine whether cultured epithelium could be induced to form hair follicles by dermal papillae, which are most important for the morphogenesis and growth of hair follicles. We cultivated adult rat sole keratinocytes, obtained the cultured epithelium, and prepared recombinants consisting of cultured epithelium and fresh dermal papillae with or without the sole dermis. These recombinants were then transplanted underneath the dermis of the dorsal skin of syngeneic rats or athymic mice. Histologic examination revealed that the transplanted cultured epithelium formed the follicular structures with sebaceous gland-like structure following induction of the dermal papillae, especially when supported by the dermis. We concluded that transplanted cultured epithelium of adult rat sole keratinocytes can respond to growth signals from adult dermal papillae.

  4. Do androgens influence hair growth by altering the paracrine factors secreted by dermal papilla cells?

    PubMed

    Randall, V A; Hibberts, N A; Thornton, M J; Merrick, A E; Hamada, K; Kato, S; Jenner, T J; de Oliveira, I; Messenger, A G

    2001-01-01

    Androgens regulate many aspects of human hair growth in both sexes. After puberty they transform tiny vellus follicles in many areas, e.g. the face, to terminal ones producing long, thick, pigmented hairs. In genetically predisposed individuals, androgens also cause the reverse transformation of terminal scalp follicles into vellus ones, causing balding. In the current hypothesis for androgen action, androgens control most follicular cells indirectly acting via the mesenchyme-derived dermal papilla which regulates many aspects of follicular activity. In this model androgens binding to androgen receptors in dermal papilla cells alter their production of regulatory molecules which influence other follicular components; these molecules may be soluble paracrine factors and/or extracellular matrix proteins. This hypothesis is supported by immunohistochemical localisation of androgen receptors in dermal papilla cell nuclei and the demonstrations that androgen receptor content and testosterone metabolism patterns of cultured dermal papilla cells from various body sites reflect hair growth in androgen-insensitivity syndromes. The next question is whether androgens alter the paracrine factors secreted by dermal papilla cells. Cultured dermal papilla cells do release soluble, proteinaceous factors into their media which stimulate the growth of keratinocytes and other dermal papilla cells. This mitogenic potential can cross species from humans to rodents. Importantly, testosterone in vitro stimulates the mitogenic potential of beard cells, but in contrast inhibits production by balding scalp cells reflecting their in vivo androgenic responses. Since androgens in vitro do alter the secretion of paracrine factors the current focus lies in identifying specific factors produced, e.g. IGF-I and stem cell factor (SCF), using ELISA and RT-PCR, and comparing their expression in cells from follicles with varying responses to androgens in vivo or under androgen stimulation in vitro

  5. Identification and localization of insulin-like growth factor-binding protein (IGFBP) messenger RNAs in human hair follicle dermal papilla.

    PubMed

    Batch, J A; Mercuri, F A; Werther, G A

    1996-03-01

    The role of the insulin-like growth factors (IGFs) in hair follicle biology has recently been recognized, although their actions, sites of production, and modulation by the insulin-like growth factor-binding proteins (IGFBPs) have not to date been defined. IGF-I is essential for normal hair growth and development, and may be important in regulation of the hair growth cycle. In many culture systems, IGF-I actions are modulated by the IGFBPs. Thus, if IGFBPs are produced in the human hair follicle, they may play a role in targeting IGF-I to its receptor or may modulate IGF-I action by interaction with matrix proteins. We have used in situ hybridization to localize messenger RNA for the six IGFBPs in anagen hair follicles. Anti-sense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-micrometer sections of adult facial skin were probed. Messenger RNA for IGFBP-3, -4, and -5 were identified, with predominantly IGFBP-3 and -5 mRNA found in the dermal papilla, and to a lesser extent IGFBP-4 mRNA. IGFBP-4 mRNA was also found at the dermal papilla/epithelial matrix border. Messenger RNAs for both IGFBP-4 and -5 were also demonstrated in the dermal sheath surrounding the hair follicle. Messenger RNAs for IGFBP-1, -2, and -6 were not identified. These studies demonstrate specific localization of IGFBP mRNAs in hair follicles, suggesting that they each play specific roles in the local modulation of IGF action during the hair growth cycle.

  6. Surface Tension Guided Hanging-Drop: Producing Controllable 3D Spheroid of High-Passaged Human Dermal Papilla Cells and Forming Inductive Microtissues for Hair-Follicle Regeneration.

    PubMed

    Lin, Bojie; Miao, Yong; Wang, Jin; Fan, Zhexiang; Du, Lijuan; Su, Yongsheng; Liu, Bingcheng; Hu, Zhiqi; Xing, Malcolm

    2016-03-09

    Human dermal papilla (DP) cells have been studied extensively when grown in the conventional monolayer. However, because of great deviation from the real in vivo three-dimensional (3D) environment, these two-dimensional (2D) grown cells tend to lose the hair-inducible capability during passaging. Hence, these 2D caused concerns have motivated the development of novel 3D culture techniques to produce cellular microtissues with suitable mimics. The hanging-drop approach is based on surface tension-based technique and the interaction between surface tension and gravity field that makes a convergence of liquid drops. This study used this technique in a converged drop to form cellular spheroids of dermal papilla cells. It leads to a controllable 3Dspheroid model for scalable fabrication of inductive DP microtissues. The optimal conditions for culturing high-passaged (P8) DP spheroids were determined first. Then, the morphological, histological and functional studies were performed. In addition, expressions of hair-inductive markers including alkaline phosphatase, α-smooth muscle actin and neural cell adhesion molecule were also analyzed by quantitative RT-PCR, immunostaining and immunoblotting. Finally, P8-DP microtissues were coimplanted with newborn mouse epidermal cells (EPCs) into nude mice. Our results indicated that the formation of 3D microtissues not only endowed P8-DP microtissues many similarities to primary DP, but also confer these microtissues an enhanced ability to induce hair-follicle (HF) neogenesis in vivo. This model provides a potential to elucidate the native biology of human DP, and also shows the promising for the controllable and scalable production of inductive DP cells applied in future follicle regeneration.

  7. Human placental extract exerts hair growth-promoting effects through the GSK-3β signaling pathway in human dermal papilla cells.

    PubMed

    Kwon, Tae-Rin; Oh, Chang Taek; Choi, Eun Ja; Park, Hye Min; Han, Hae Jung; Ji, Hyi Jeong; Kim, Beom Joon

    2015-10-01

    Human placental extract (HPE) is widely used in Korea to relieve fatigue. However, its effects on human dermal papilla cells (hDPCs) remain unknown. In the present study, in an effort to develop novel therapies to promote hair growth, we screened HPE. We demonstrate that HPE has hair growth‑promoting activities and induces β‑catenin expression through the inhibition of glycogen synthase kinase‑3β (GSK‑3β) by phosphorylation in hDPCs. Treatment with HPE significantly increased the viability of the hDPCs in a concentration‑dependent manner, as shown by bromodeoxyuridine (BrdU) assay. HPE also significantly increased the alkaline phosphatase (ALP) expression levels. The increased β‑catenin levels and the inhibition of GSK‑3β (Ser9) by phosphorylation suggested that HPE promoted the hair-inductive capacity of hDPCs. We compared the effects of treatment with HPE alone and treatment with HPE in conjunction with minoxidil (MXD). We found that HPE plus MXD effectively inhibited GSK‑3β by phosphorylation (Ser9) in the hDPCs. Moreover, we demonstrated that HPE was effective in inducing root hair elongation in rat vibrissa hair follicles, and that treatment with HPE led to a delay in catagen progression. Overall, our findings suggest that HPE promotes hair growth and may thus provide the basis of a novel therapeutic strategy for the clinical treatment of hair loss.

  8. Implication of microRNA regulation in para-phenylenediamine-induced cell death and senescence in normal human hair dermal papilla cells

    PubMed Central

    LEE, OK-KYU; CHA, HWA JUN; LEE, MYUNG JOO; LIM, KYUNG MI; JUNG, JAE WOOK; AHN, KYU JOONG; AN, IN-SOOK; AN, SUNGKWAN; BAE, SEUNGHEE

    2015-01-01

    Para-phenylenediamine (PPD) is a major component of hair coloring and black henna products. Although it has been largely demonstrated that PPD induces allergic reactions and increases the risk of tumors in the kidney, liver, thyroid gland and urinary bladder, the effect on dermal papilla cells remains to be elucidated. Therefore, the current study evaluated the effects of PPD on growth, cell death and senescence using cell-based assays and microRNA (miRNA) microarray in normal human hair dermal papilla cells (nHHDPCs). Cell viability and cell cycle analyses demonstrated that PPD exhibited a significant cytotoxic effect on nHHDPCs through inducing cell death and G2 phase cell cycle arrest in a dose-dependent manner. It was additionally observed that treatment of nHHDPCs with PPD induced cellular senescence by promoting cellular oxidative stress. In addition, the results of the current study indicated that these PPD-mediated effects were involved in the alteration of miRNA expression profiles. Treatment of nHHDPCs with PPD altered the expression levels of 74 miRNAs by ≥2-fold (16 upregulated and 58 downregulated miRNAs). Further bioinformatics analysis determined that these identified miRNA target genes were likely to be involved in cell growth, cell cycle arrest, cell death, senescence and the induction of oxidative stress. In conclusion, the observations of the current study suggested that PPD was able to induce several cytotoxic effects through alteration of miRNA expression levels in nHHDPCs. PMID:25776079

  9. Induction of hair follicle dermal papilla cell properties in human induced pluripotent stem cell-derived multipotent LNGFR(+)THY-1(+) mesenchymal cells

    PubMed Central

    Veraitch, Ophelia; Mabuchi, Yo; Matsuzaki, Yumi; Sasaki, Takashi; Okuno, Hironobu; Tsukashima, Aki; Amagai, Masayuki; Okano, Hideyuki; Ohyama, Manabu

    2017-01-01

    The dermal papilla (DP) is a specialised mesenchymal component of the hair follicle (HF) that plays key roles in HF morphogenesis and regeneration. Current technical difficulties in preparing trichogenic human DP cells could be overcome by the use of highly proliferative and plastic human induced pluripotent stem cells (hiPSCs). In this study, hiPSCs were differentiated into induced mesenchymal cells (iMCs) with a bone marrow stromal cell phenotype. A highly proliferative and plastic LNGFR(+)THY-1(+) subset of iMCs was subsequently programmed using retinoic acid and DP cell activating culture medium to acquire DP properties. The resultant cells (induced DP-substituting cells [iDPSCs]) exhibited up-regulated DP markers, interacted with human keratinocytes to up-regulate HF related genes, and when co-grafted with human keratinocytes in vivo gave rise to fibre structures with a hair cuticle-like coat resembling the hair shaft, as confirmed by scanning electron microscope analysis. Furthermore, iDPSCs responded to the clinically used hair growth reagent, minoxidil sulfate, to up-regulate DP genes, further supporting that they were capable of, at least in part, reproducing DP properties. Thus, LNGFR(+)THY-1(+) iMCs may provide material for HF bioengineering and drug screening for hair diseases. PMID:28220862

  10. Epigallocatechin Gallate-Mediated Alteration of the MicroRNA Expression Profile in 5α-Dihydrotestosterone-Treated Human Dermal Papilla Cells

    PubMed Central

    Shin, Shanghun; Kim, Karam; Lee, Myung Joo; Lee, Jeongju; Choi, Sungjin; Kim, Kyung-Suk; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Youn, Hae Jeong; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan

    2016-01-01

    Background Dihydrotestosterone (DHT) induces androgenic alopecia by shortening the hair follicle growth phase, resulting in hair loss. We previously demonstrated how changes in the microRNA (miRNA) expression profile influenced DHT-mediated cell death, cell cycle arrest, cell viability, the generation of reactive oxygen species (ROS), and senescence. Protective effects against DHT have not, however, been elucidated at the genome level. Objective We showed that epigallocatechin gallate (EGCG), a major component of green tea, protects DHT-induced cell death by regulating the cellular miRNA expression profile. Methods We used a miRNA microarray to identify miRNA expression levels in human dermal papilla cells (DPCs). We investigated whether the miRNA expression influenced the protective effects of EGCG against DHT-induced cell death, growth arrest, intracellular ROS levels, and senescence. Results EGCG protected against the effects of DHT by altering the miRNA expression profile in human DPCs. In addition, EGCG attenuated DHT-mediated cell death and growth arrest and decreased intracellular ROS levels and senescence. A bioinformatics analysis elucidated the relationship between the altered miRNA expression and EGCG-mediated protective effects against DHT. Conclusion Overall, our results suggest that EGCG ameliorates the negative effects of DHT by altering the miRNA expression profile in human DPCs. PMID:27274631

  11. Ginsenoside Rg3 up-regulates the expression of vascular endothelial growth factor in human dermal papilla cells and mouse hair follicles.

    PubMed

    Shin, Dae Hyun; Cha, Youn Jeong; Yang, Kyeong Eun; Jang, Ik-Soon; Son, Chang-Gue; Kim, Bo Hyeon; Kim, Jung Min

    2014-07-01

    Crude Panax ginseng has been documented to possess hair growth activity and is widely used to treat alopecia, but the effects of ginsenoside Rg3 on hair growth have not to our knowledge been determined. The aim of the current study was to identify the molecules through which Rg3 stimulates hair growth. The thymidine incorporation for measuring cell proliferation was determined. We used DNA microarray analysis to measure gene expression levels in dermal papilla (DP) cells upon treatment with Rg3. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in human DP cells were measured by real-time polymerase chain reaction and immunohistochemistry, respectively. We also used immunohistochemistry assays to detect in vivo changes in VEGF and 3-stemness marker expressions in mouse hair follicles. Reverse transcription polymerase chain reaction showed dose-dependent increases in VEGF mRNA levels on treatment with Rg3. Immunohistochemical analysis showed that expression of VEGF was significantly up-regulated by Rg3 in a dose-dependent manner in human DP cells and in mouse hair follicles. In addition, the CD8 and CD34 were also up-regulated by Rg3 in the mouse hair follicles. It may be concluded that Rg3 might increase hair growth through stimulation of hair follicle stem cells and it has the potential to be used in hair growth products.

  12. Hair follicle signaling networks: a dermal papilla-centric approach.

    PubMed

    Ramos, Raul; Guerrero-Juarez, Christian F; Plikus, Maksim V

    2013-10-01

    Functional testing of dermal papilla (DP) signaling inputs into hair follicle (HF) morphogenesis and regeneration is becoming possible with the advent of new Cre lines. Targeted deletion of the signature genes in early DP precursors has revealed significant signaling redundancy during HF morphogenesis. Furthermore, the DP lineage commitment program can be exploited for generating highly inductive DP cells to be used in HF bioengineering assays.

  13. Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism.

    PubMed

    Luanpitpong, Sudjit; Nimmannit, Ubonthip; Chanvorachote, Pithi; Leonard, Stephen S; Pongrakhananon, Varisa; Wang, Liying; Rojanasakul, Yon

    2011-08-01

    Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.

  14. iTRAQ-Based Quantitative Proteomic Comparison of Early- and Late-Passage Human Dermal Papilla Cell Secretome in Relation to Inducing Hair Follicle Regeneration

    PubMed Central

    Huang, Keng; Cai, Bo-Zhi; Zeng, Yang; Xu, Yan-Ming; Liu, Yang; Yuan, Yan-Ping; Lin, Chang-Min

    2016-01-01

    Alopecia is an exceedingly prevalent problem that lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential, we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs, and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM did not. In order to identify the key factors responsible for hair induction, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1. Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results indicate DPC-secreted proteins play important roles in HF regeneration, with SDF1, MMP3, biglycan, and LTBP1 being potential key inductive factors secreted by dermal papilla cells in the regeneration of hair follicles. PMID:27907131

  15. 3-Deoxysappanchalcone Promotes Proliferation of Human Hair Follicle Dermal Papilla Cells and Hair Growth in C57BL/6 Mice by Modulating WNT/β-Catenin and STAT Signaling

    PubMed Central

    Kim, Young Eun; Choi, Hyung Chul; Lee, In-Chul; Yuk, Dong Yeon; Lee, Hyosung; Choi, Bu Young

    2016-01-01

    3-Deoxysappanchalcone (3-DSC) has been reported to possess anti-allergic, antiviral, anti-inflammatory and antioxidant activities. In the present study, we investigated the effects of 3-DSC on the proliferation of human hair follicle dermal papilla cells (HDPCs) and mouse hair growth in vivo. A real-time cell analyzer system, luciferase assay, Western blot and real-time polymerase chain reaction (PCR) were employed to measure the biochemical changes occurring in HDPCs in response to 3-DSC treatment. The effect of 3-DSC on hair growth in C57BL/6 mice was also examined. 3-DSC promoted the proliferation of HDPCs, similar to Tofacitinib, an inhibitor of janus-activated kinase (JAK). 3-DSC promoted phosphorylation of β-catenin and transcriptional activation of the T-cell factor. In addition, 3-DSC potentiated interleukin-6 (IL-6)-induced phosphorylation and subsequent transactivation of signal transducer and activator of transcription-3 (STAT3), thereby increasing the expression of cyclin-dependent kinase-4 (Cdk4), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). On the contrary, 3-DSC attenuated STAT6 mRNA expression and IL4-induced STAT6 phosphorylation in HDPCs. Finally, we observed that topical application of 3-DSC promoted the anagen phase of hair growth in C57BL/6 mice. 3-DSC stimulates hair growth possibly by inducing proliferation of follicular dermal papilla cells via modulation of WNT/β-catenin and STAT signaling. PMID:27795451

  16. Standardized Scalp Massage Results in Increased Hair Thickness by Inducing Stretching Forces to Dermal Papilla Cells in the Subcutaneous Tissue

    PubMed Central

    Kobayashi, Kazuhiro; Hama, Takanori; Murakami, Kasumi; Ogawa, Rei

    2016-01-01

    Objective: In this study, we evaluated the effect of scalp massage on hair in Japanese males and the effect of stretching forces on human dermal papilla cells in vitro. Methods: Nine healthy men received 4 minutes of standardized scalp massage per day for 24 weeks using a scalp massage device. Total hair number, hair thickness, and hair growth rate were evaluated. The mechanical effect of scalp massage on subcutaneous tissue was analyzed using a finite element method. To evaluate the effect of mechanical forces, human dermal papilla cells were cultured using a 72-hour stretching cycle. Gene expression change was analyzed using DNA microarray analyses. In addition, expression of hair cycle-related genes including IL6, NOGGIN, BMP4, and SMAD4 were evaluated using real-time reverse transcription-polymerase chain reaction. Results: Standardized scalp massage resulted in increased hair thickness 24 weeks after initiation of massage (0.085 ± 0.003 mm vs 0.092 ± 0.001 mm). Finite element method showed that scalp massage caused z-direction displacement and von Mises stress on subcutaneous tissue. In vitro, DNA microarray showed gene expression change significantly compared with nonstretching human dermal papilla cells. A total of 2655 genes were upregulated and 2823 genes were downregulated. Real-time reverse transcription-polymerase chain reaction demonstrated increased expression of hair cycle–related genes such as NOGGIN, BMP4, SMAD4, and IL6ST and decrease in hair loss–related genes such as IL6. Conclusions: Stretching forces result in changes in gene expression in human dermal papilla cells. Standardized scalp massage is a way to transmit mechanical stress to human dermal papilla cells in subcutaneous tissue. Hair thickness was shown to increase with standardized scalp massage. PMID:26904154

  17. Clinically applicable transplantation procedure of dermal papilla cells for hair follicle regeneration.

    PubMed

    Aoi, Noriyuki; Inoue, Keita; Kato, Harunosuke; Suga, Hirotaka; Higashino, Takuya; Eto, Hitomi; Doi, Kentaro; Araki, Jun; Iida, Takuya; Katsuta, Tomoya; Yoshimura, Kotaro

    2012-02-01

    Dermal papilla cells (DPCs) interact with epithelial stem cells and induce hair folliculogenesis. Cell-based therapies using expanded DPCs for hair regeneration have been unsuccessful in humans. Two major challenges remain: first, expanded DPCs obtained from adult hair follicles have functional limitations; second, a clinically applicable method is needed for transplanting DPCs. This study aimed to identify an efficient, minimally invasive and economical DPC transplantation procedure for use in clinical settings. Five clinically applicable transplantation procedures were tested, termed the Pinhole, Laser, Slit, Non-vascularized sandwich (NVS) and Hemi-vascularized sandwich (HVS) methods. Labelled rat dermal papilla tissue was transplanted into rat sole skin, and hair follicle regeneration was evaluated histologically. Regenerated follicles and labelled DPCs were detected for all methods, although some follicles showed abnormal growth, i.e. a cystic or inverted appearance. The HVS method, pioneered here, resulted in significantly larger number of regenerated follicles that were more mature and regular than those observed using the other methods. Moreover, hair growth was detected after expanded adult-derived DPC transplantation using the HVS method. These results suggest that direct contact of epithelial and dermal components and better vascularization/oxygenation of the recipient site are critical for hair regeneration in cell-based therapies. Copyright © 2011 John Wiley & Sons, Ltd.

  18. Dkk2/Frzb in the dermal papillae regulates feather regeneration

    PubMed Central

    Chu, Qiqi; Cai, Linyan; Fu, Yu; Chen, Xi; Yan, Zhipeng; Lin, Xiang; Zhou, Guixuan; Han, Hao; Widelitz, Randall B.; Chuong, Cheng-ming; Wu, Wei; Yue, Zhicao

    2015-01-01

    Avian feathers have robust growth and regeneration capability. To evaluate the contribution of signaling molecules and pathways in these processes, we profiled gene expression in the feather follicle using an absolute quantification approach. We identified hundreds of genes that mark specific components of the feather follicle: the dermal papillae (DP) which controls feather regeneration and axis formation, the pulp mesenchyme (Pp) which is derived from DP cells and nourishes the feather follicle, and the ramogenic zone epithelium (Erz) where a feather starts to branch. The feather DP is enriched in BMP/TGF-β signaling molecules and inhibitors for Wnt signaling including Dkk2/Frzb. Wnt ligands are mainly expressed in the feather epithelium and pulp. We find that while Wnt signaling is required for the maintenance of DP marker gene expression and feather regeneration, excessive Wnt signaling delays regeneration and reduces pulp formation. Manipulating Dkk2/Frzb expression by lentiviral-mediated overexpression, shRNA-knockdown, or by antibody neutralization resulted in dual feather axes formation. Our results suggest that the Wnt signaling in the proximal feather follicle is fine-tuned to accommodate feather regeneration and axis formation. PMID:24463139

  19. Dkk2/Frzb in the dermal papillae regulates feather regeneration.

    PubMed

    Chu, Qiqi; Cai, Linyan; Fu, Yu; Chen, Xi; Yan, Zhipeng; Lin, Xiang; Zhou, Guixuan; Han, Hao; Widelitz, Randall B; Chuong, Cheng-ming; Wu, Wei; Yue, Zhicao

    2014-03-15

    Avian feathers have robust growth and regeneration capability. To evaluate the contribution of signaling molecules and pathways in these processes, we profiled gene expression in the feather follicle using an absolute quantification approach. We identified hundreds of genes that mark specific components of the feather follicle: the dermal papillae (DP) which controls feather regeneration and axis formation, the pulp mesenchyme (Pp) which is derived from DP cells and nourishes the feather follicle, and the ramogenic zone epithelium (Erz) where a feather starts to branch. The feather DP is enriched in BMP/TGF-β signaling molecules and inhibitors for Wnt signaling including Dkk2/Frzb. Wnt ligands are mainly expressed in the feather epithelium and pulp. We find that while Wnt signaling is required for the maintenance of DP marker gene expression and feather regeneration, excessive Wnt signaling delays regeneration and reduces pulp formation. Manipulating Dkk2/Frzb expression by lentiviral-mediated overexpression, shRNA-knockdown, or by antibody neutralization resulted in dual feather axes formation. Our results suggest that the Wnt signaling in the proximal feather follicle is fine-tuned to accommodate feather regeneration and axis formation.

  20. A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration

    PubMed Central

    Zhang, Peipei; Kling, Russell E; Ravuri, Sudheer K; Kokai, Lauren E; Rubin, J Peter; Chai, Jia-ke

    2014-01-01

    Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration. PMID:25383178

  1. Comparison of Calcium and Barium Microcapsules as Scaffolds in the Development of Artificial Dermal Papillae

    PubMed Central

    Liu, Yang; Lin, Changmin; Zeng, Yang; Li, Haihong; Cai, Bozhi; Huang, Keng; Yuan, Yanping; Li, Yu

    2016-01-01

    This study aimed to develop and evaluate barium and calcium microcapsules as candidates for scaffolding in artificial dermal papilla. Dermal papilla cells (DPCs) were isolated and cultured by one-step collagenase treatment. The DPC-Ba and DPC-Ca microcapsules were prepared by using a specially designed, high-voltage, electric-field droplet generator. Selected microcapsules were assessed for long-term inductive properties with xenotransplantation into Sprague-Dawley rat ears. Both barium and calcium microcapsules maintained xenogenic dermal papilla cells in an immunoisolated environment and induced the formation of hair follicle structures. Calcium microcapsules showed better biocompatibility, permeability, and cell viability in comparison with barium microcapsules. Before 18 weeks, calcium microcapsules gathered together, with no substantial immune response. After 32 weeks, some microcapsules were near inflammatory cells and wrapped with fiber. A few large hair follicles were found. Control samples showed no marked changes at the implantation site. Barium microcapsules were superior to calcium microcapsules in structural and mechanical stability. The cells encapsulated in hydrogel barium microcapsules exhibited higher short-term viability. This study established a model to culture DPCs in 3D culture conditions. Barium microcapsules may be useful in short-term transplantation study. Calcium microcapsules may provide an effective scaffold for the development of artificial dermal papilla. PMID:27123456

  2. Comparison of Calcium and Barium Microcapsules as Scaffolds in the Development of Artificial Dermal Papillae.

    PubMed

    Liu, Yang; Lin, Changmin; Zeng, Yang; Li, Haihong; Cai, Bozhi; Huang, Keng; Yuan, Yanping; Li, Yu

    2016-01-01

    This study aimed to develop and evaluate barium and calcium microcapsules as candidates for scaffolding in artificial dermal papilla. Dermal papilla cells (DPCs) were isolated and cultured by one-step collagenase treatment. The DPC-Ba and DPC-Ca microcapsules were prepared by using a specially designed, high-voltage, electric-field droplet generator. Selected microcapsules were assessed for long-term inductive properties with xenotransplantation into Sprague-Dawley rat ears. Both barium and calcium microcapsules maintained xenogenic dermal papilla cells in an immunoisolated environment and induced the formation of hair follicle structures. Calcium microcapsules showed better biocompatibility, permeability, and cell viability in comparison with barium microcapsules. Before 18 weeks, calcium microcapsules gathered together, with no substantial immune response. After 32 weeks, some microcapsules were near inflammatory cells and wrapped with fiber. A few large hair follicles were found. Control samples showed no marked changes at the implantation site. Barium microcapsules were superior to calcium microcapsules in structural and mechanical stability. The cells encapsulated in hydrogel barium microcapsules exhibited higher short-term viability. This study established a model to culture DPCs in 3D culture conditions. Barium microcapsules may be useful in short-term transplantation study. Calcium microcapsules may provide an effective scaffold for the development of artificial dermal papilla.

  3. Shh maintains dermal papilla identity and hair morphogenesis via a Noggin–Shh regulatory loop

    PubMed Central

    Woo, Wei-Meng; Zhen, Hanson H.; Oro, Anthony E.

    2012-01-01

    During hair follicle morphogenesis, dermal papillae (DPs) function as mesenchymal signaling centers that cross-talk with overlying epithelium to regulate morphogenesis. While the DP regulates hair follicle formation, relatively little is known about the molecular basis of DP formation. The morphogen Sonic hedgehog (Shh) is known for regulating hair follicle epithelial growth, with excessive signaling resulting in basal cell carcinomas. Here, we investigate how dermal-specific Shh signaling contributes to DP formation and hair growth. Using a Cre-lox genetic model and RNAi in hair follicle reconstitution assays, we demonstrate that dermal Smoothened (Smo) loss of function results in the loss of the DP precursor, the dermal condensate, and a stage 2 hair follicle arrest phenotype reminiscent of Shh−/− skin. Surprisingly, dermal Smo does not regulate cell survival or epithelial proliferation. Rather, molecular screening and immunostaining studies reveal that dermal Shh signaling controls the expression of a subset of DP-specific signature genes. Using a hairpin/cDNA lentiviral system, we show that overexpression of the Shh-dependent gene Noggin, but not Sox2 or Sox18, can partially rescue the dermal Smo knockdown hair follicle phenotype by increasing the expression of epithelial Shh. Our findings suggest that dermal Shh signaling regulates specific DP signatures to maintain DP maturation while maintaining a reciprocal Shh–Noggin signaling loop to drive hair follicle morphogenesis. PMID:22661232

  4. PDGF-AA-induced filamentous mitochondria benefit dermal papilla cells in cellular migration.

    PubMed

    Mifude, C; Kaseda, K

    2015-06-01

    Human dermal papilla cells (HDPCs) play essential roles in hair follicular morphogenesis and postnatal hair growth cycles. Previous reports demonstrated that platelet-derived growth factor-AA (PDGF-AA) enhanced the formation of dermal condensates in hair follicular development. Additionally, PDGF-AA induces/maintains the anagen phase of the hair cycle. It is likely that mitochondrial morphology and functions are tightly coupled with maintenance of these energy-demanding activities. However, little is known about the mitochondrial regulation in HDPCs. Thus, we investigated the PDGF-involved mitochondrial regulation in HDPCs. The mitochondrial morphologies of HDPCs were examined in the presence or absence of PDGF-AA under a fluorescent microscope. ATP production and cellular motility were investigated. The relationship between mitochondrial morphology and the cellular functions was discussed. We observed that primary HDPCs contained mitochondria with filamentous and/or rounded morphologies. Both types of mitochondria showed similar membrane potentials. Interestingly, in the presence of PDGF-AA, but not PDGF-BB, the balance between the two morphologies shifted towards the filamentous form. Concomitantly, both mitochondrial enzymatic activity and total cellular ATP level were augmented by PDGF-AA. These two parameters were closely correlated, suggesting the mitochondrial involvement in the PDGF-augmented ATP production. Moreover, PDGF-AA accelerated the migration of HDPCs in a gap-filling assay, but did not change the rate of cellular proliferation. Notably, filamentous mitochondria dominated migrating HDPCs. PDGF-AA benefits HDPCs in the process of migration, by increasing the number of filamentous mitochondria. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  5. Combination of herbal extracts and platelet-rich plasma induced dermal papilla cell proliferation: involvement of ERK and Akt pathways.

    PubMed

    Rastegar, Hosein; Ahmadi Ashtiani, Hamidreza; Aghaei, Mahmoud; Ehsani, Amirohushang; Barikbin, Behrooz

    2013-06-01

    Recently, platelet-rich plasma (PRP) has attracted attention in various medical fields, including plastic surgery, treatment for problematic wounds, and dermatology. Specifically, PRP has been tested during hair transplantation to reduce swelling and pain and to increase hair density. We examined the effects of PRP and herbal extracts combination in order to identify potential stimulants of hair growth. PRP was prepared using the double-spin method and applied to dermal papilla cells (DPCs). MTT viability test and BrdU cell proliferation assay were used to study the effect of herbal extracts and PRP on proliferation of DPCs. To understand the mechanisms of herbal extracts and PRP involved in the regulation of hair growth, we evaluated signaling pathways and measured the expressions of ERK and Akt, by Western blot. Combination of herbal extracts and PRP was found to induce significant proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. The present study shows that herbal extracts and PRP affect the expressions of extracellular signal-regulated kinase (ERK) and Akt in DPCs. In this study, we have shown that combination of herbal extracts and PRP plays an active role in promoting the proliferation of human dermal papilla (DP) cells via the regulation of ERK and Akt proteins, and this may be applicable to the future development of herbal extracts and PRP combination therapeutics to enhance hair growth. © 2013 Wiley Periodicals, Inc.

  6. Human taste papilla stimulation: stability of quality judgments over time.

    PubMed

    McCutcheon, N B; Saunders, J

    1972-01-14

    Human taste papillae are sensitive to two or more chemical stimuli. Individual papillae produce stable response patterns (quality and intensity) over a month's time. Hence, the response pattern does not appear to be affected by turnover of receptor cells.

  7. Quantitative analysis of intrinsic skin aging in dermal papillae by in vivo harmonic generation microscopy

    PubMed Central

    Liao, Yi-Hua; Kuo, Wei-Cheng; Chou, Sin-Yo; Tsai, Cheng-Shiun; Lin, Guan-Liang; Tsai, Ming-Rung; Shih, Yuan-Ta; Lee, Gwo-Giun; Sun, Chi-Kuang

    2014-01-01

    Chronological skin aging is associated with flattening of the dermal-epidermal junction (DEJ), but to date no quantitative analysis focusing on the aging changes in the dermal papillae (DP) has been performed. The aim of the study is to determine the architectural changes and the collagen density related to chronological aging in the dermal papilla zone (DPZ) by in vivo harmonic generation microscopy (HGM) with a sub-femtoliter spatial resolution. We recruited 48 Asian subjects and obtained in vivo images on the sun-protected volar forearm. Six parameters were defined to quantify 3D morphological changes of the DPZ, which we analyzed both manually and computationally to study their correlation with age. The depth of DPZ, the average height of isolated DP, and the 3D interdigitation index decreased with age, while DP number density, DP volume, and the collagen density in DP remained constant over time. In vivo high-resolution HGM technology has uncovered chronological aging-related variations in DP, and sheds light on real-time quantitative skin fragility assessment and disease diagnostics based on collagen density and morphology. PMID:25401037

  8. Sprouty / FGF signaling regulates the proximal-distal feather morphology and the size of dermal papillae

    PubMed Central

    Yue, Zhicao; Jiang, Ting Xin; Wu, Ping; Widelitz, Randall B; Chuong, Cheng Ming

    2013-01-01

    In a feather, there are distinct morphologies along the proximal-distal axis. The proximal part is a cylindrical stalk (calamus), whereas the distal part has barb and barbule branches. Here we focus on what molecular signaling activity can modulate feather stem cells to generate these distinct morphologies. We demonstrate the drastic tissue remodeling during feather cycling which includes initiation, growth and resting phases. In the growth phase, epithelial components undergo progressive changes from the collar growth zone to the ramogenic zone, to maturing barb branches along the proximal- distal axis. Mesenchymal components also undergo progressive changes from the dermal papilla, to the collar mesenchyme, to the pulp along the proximal- distal axis. Over-expression of Spry4, a negative regulator of receptor tyrosine kinases, promotes barb branch formation at the expense of the epidermal collar. It even induces barb branches from the follicle sheath (equivalent to the outer root sheath in hair follicles). The results are feathers with expanded feather vane regions and small or missing proximal feather shafts (the calamus). Spry4 also expands the pulp region while reducing the size of dermal papillae, leading to a failure to regenerate. In contrast, over-expressing Fgf10 increases the size of the dermal papillae, expands collar epithelia and mesenchyme, but also prevents feather branch formation and feather keratin differentiation. These results suggest that coordinated Sprouty/FGF pathway activity at different stages is important to modulate feather epidermal stem cells to form distinct feather morphologies along the proximal-distal feather axis. PMID:23000358

  9. Dominant-negative Sox18 function inhibits dermal papilla maturation and differentiation in all murine hair types.

    PubMed

    Villani, Rehan; Hodgson, Samantha; Legrand, Julien; Greaney, Jessica; Wong, Ho Yi; Pichol-Thievend, Cathy; Adolphe, Christelle; Wainwight, Brandon; Francois, Mathias; Khosrotehrani, Kiarash

    2017-05-15

    SOX family proteins SOX2 and SOX18 have been reported as being essential in determining hair follicle type; however, the role they play during development remains unclear. Here, we demonstrate that Sox18 regulates the normal differentiation of the dermal papilla of all hair types. In guard (primary) hair dermal condensate (DC) cells, we identified transient Sox18 in addition to SOX2 expression at E14.5, which allowed fate tracing of primary DC cells until birth. Similarly, expression of Sox18 was detected in the DC cells of secondary hairs at E16.5 and in tertiary hair at E18.5. Dominant-negative Sox18 mutation (opposum) did not prevent DC formation in any hair type. However, it affected dermal papilla differentiation, restricting hair formation especially in secondary and tertiary hairs. This Sox18 mutation also prevented neonatal dermal cells or dermal papilla spheres from inducing hair in regeneration assays. Microarray expression studies identified WNT5A and TNC as potential downstream effectors of SOX18 that are important for epidermal WNT signalling. In conclusion, SOX18 acts as a mesenchymal molecular switch necessary for the formation and function of the dermal papilla in all hair types. © 2017. Published by The Company of Biologists Ltd.

  10. Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline

    PubMed Central

    Chi, Woo; Wu, Eleanor; Morgan, Bruce A.

    2013-01-01

    Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss. PMID:23487317

  11. Oct4 and klf4 reprogram dermal papilla cells into induced pluripotent stem cells.

    PubMed

    Tsai, Su-Yi; Clavel, Carlos; Kim, Soo; Ang, Yen-Sin; Grisanti, Laura; Lee, Dung-Fang; Kelley, Kevin; Rendl, Michael

    2010-02-01

    Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells by only four transcription factors (Oct4, Sox2, Klf4, and c-Myc) has great potential for tissue-specific regenerative therapies, eliminating the ethical issues surrounding the use of embryonic stem cells and the rejection problems of using non-autologous cells. The reprogramming efficiency generally is very low, however, and the problems surrounding the introduction of viral genetic material are only partially investigated. Recent efforts to reduce the number of virally expressed transcription factors succeeded at reprogramming neural stem cells into iPS cells by overexpressing Oct4 alone. However, the relative inaccessibility and difficulty of obtaining neural cells in humans remains to be resolved. Here we report that dermal papilla (DP) cells, which are specialized skin fibroblasts thought to instruct hair follicle stem cells, endogenously express high levels of Sox2 and c-Myc, and that these cells can be reprogrammed into iPS cells with only Oct4 and Klf4. Moreover, we show that DP cells are reprogrammed more efficiently than skin and embryonic fibroblasts. iPS cells derived from DP cells expressed pluripotency genes and differentiated into cells from all germ layers in vitro and widely contributed to chimeric mice in vivo, including the germline. Our work establishes DP cells as an easily accessible source to generate iPS cells with efficiency and with less genetic material. This opens up the possibility of streamlined generation of skin-derived, patient-specific pluripotent stem cells and of ultimately replacing the remaining two factors with small molecules for safe generation of transplantable cells.

  12. Ascorbigen induces dermal papilla cell proliferation in vitro, but fails to modulate chemotherapy-induced alopecia in vivo.

    PubMed

    Wang, Chih-Hsiu; Huang, Hsu-Shan; Dai, Niann-Tzyy; Sheu, Ming-Jen; Chang, Deh-Ming

    2013-12-01

    Ascorbigen (ABG) is the predominant indole-derived compound from Brassica vegetables. In this study, we attempted to evaluate the effects of ABG on hair growth. To this end, we examined the proliferation of isolated human dermal papilla (DP) cells and keratinocytes after incubation in various concentrations (0-1.25 mM) of ABG. Furthermore, hair shaft regrowth was monitored in a mouse model of chemotherapy-induced alopecia (CIA), and hematoxylin and eosin staining was performed for histological analyses. We found that 1.25 mM ABG induced a 1.2-fold increase in the growth of DP cells, but not keratinocytes. However, ABG did not exert significant protective effects against CIA in the mouse model. These findings suggest that ABG may not be able to counteract CIA and that further investigation of the therapeutic potential of ABG in disease models is required.

  13. Preventable effect of L-threonate, an ascorbate metabolite, on androgen-driven balding via repression of dihydrotestosterone-induced dickkopf-1 expression in human hair dermal papilla cells.

    PubMed

    Kwack, Mi Hee; Ahn, Ji Sup; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

    2010-10-01

    In a previous study, we recently claimed that dihydrotestosterone (DHT)-inducible dickkopf-1 (DKK-1) expression is one of the key factors involved in androgen-potentiated balding. We also demonstrated that L-ascorbic acid 2-phosphate (Asc 2-P) represses DHT-induced DKK-1 expression in cultured dermal papilla cells (DPCs). Here, we investigated whether or not L-threonate could attenuate DHT-induced DKK-1 expression. We observed via RT-PCR analysis and enzyme-linked immunosorbent assay that DHT-induced DKK-1 expression was attenuated in the presence of L-threonate. We also found that DHT-induced activation of DKK-1 promoter activity was significantly repressed by L-threonate. Moreover, a co-culture system featuring outer root sheath (ORS) keratinocytes and DPCs showed that DHT inhibited the growth of ORS cells, which was then significantly reversed by L-threonate. Collectively, these results indicate that L-threonate inhibited DKK-1 expression in DPCs and therefore is a good treatment for the prevention of androgen-driven balding.

  14. Long noncoding RNA expression in dermal papilla cells contributes to hairy gene regulation.

    PubMed

    Lin, Chang-Min; Liu, Yang; Huang, Keng; Chen, Xian-Cai; Cai, Bo-Zhi; Li, Hai-Hong; Yuan, Yan-Ping; Zhang, Huan; Li, Yu

    2014-10-24

    Dermal papilla (DP) cells may be the source of dermal-derived signaling molecules involved in hair-follicle development and postnatal hair cycling. Early-passage DP cells can induce hair growth in vivo, but, on further culture, this ability is lost. The cellular mechanisms underlying the hair-follicle induction property of early-passage DP cells are unclear. Long noncoding RNAs (lncRNAs) are an important class of genes involved in various biological functions. They are aberrantly expressed and play roles in the regulation of the Wnt signaling pathway, a critical point in maintaining hair-induction activity. LncRNA microarray revealed 1683 upregulated and 1773 downregulated lncRNAs in passage-4 DP cells compare with passage-10 DP cells. To investigate the relation between lncRNAs and coding genes in WNT signaling, we constructed a coding-noncoding gene co-expression network using lncRNAs and coding genes that were differentially expressed between the passage-4 and -10 DP cells. RP11-766N7.3, H19 and HOTAIR are specific lncRNAs that were aberrantly expressed in DP cells and played an important role in regulating Wnt signaling. This study may provide potential targets for discovering the hair-follicle induction mechanism of early-passage DP cells.

  15. Contact between dermal papilla cells and dermal sheath cells enhances the ability of DPCs to induce hair growth.

    PubMed

    Yamao, Mikaru; Inamatsu, Mutsumi; Ogawa, Yuko; Toki, Hiroshi; Okada, Taro; Toyoshima, Koh-ei; Yoshizato, Katsutoshi

    2010-12-01

    We previously showed that cultured rat dermal papilla cells (DPCs) retain their hair-inducing capacity on afollicular epidermal cell (EPCs). Here, we examined the hair growth-inducing capacity of differently subcultured DPCs by transplanting them, along with rat EPCs, onto the backs of nude mice (graft chamber assay). DPCs at passage (p) 6 (DPCs(p6) or, more generally, low-passage DPCs) induced hair formation. However, DPCs(p>30) (high-passage DPCs) had no such activity and induced only subepidermal hair follicles (HFs) that were not encapsulated by the dermal sheath (DS). Thus, we examined the effect of DS cells (DSCs(p=1)) on the ability of DPCs(p=60) to induce hair growth by testing a mixture of these two cell types (cotransplant) in the graft chamber assay, in which DSCs(p=1) and DPCs(p=60) were labeled with enhanced green fluorescent protein (EGFP) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), respectively. These cotransplants generated hairs as actively as did DPCs(p=6) transplants. Their HFs were encapsulated with EGFP(+)-DS and had DPs consisting largely of EGFP(+)-DPCs (47%) and DiI(+)-DPCs (43%), indicating a major contribution of DSC(p=1)-derived DPCs to HF induction. In addition, the results of in vitro coculture of DPCs(p=60) and DSCs(p=1) suggest that high-passage DPCs stimulate the expression of certain trichogenic genes in DSCs.

  16. Dermal Contributions to Human Interfollicular Epidermal Architecture and Self-Renewal

    PubMed Central

    Lawlor, Kynan T.; Kaur, Pritinder

    2015-01-01

    The human interfollicular epidermis is renewed throughout life by populations of proliferating basal keratinocytes. Though interfollicular keratinocyte stem cells have been identified, it is not known how self-renewal in this compartment is spatially organized. At the epidermal-dermal junction, keratinocytes sit atop a heterogeneous mix of dermal cells that may regulate keratinocyte self-renewal by influencing local tissue architecture and signalling microenvironments. Focusing on the rete ridges and complementary dermal papillae in human skin, we review the identity and organisation of abundant dermal cells types and present evidence for interactions between the dermal microenvironment and the interfollicular keratinocytes. PMID:26602926

  17. Follicular dermal papilla structures by organization of epithelial and mesenchymal cells in interfacial polyelectrolyte complex fibers.

    PubMed

    Lim, Tze Chiun; Leong, Meng Fatt; Lu, Hongfang; Du, Chan; Gao, Shujun; Wan, Andrew C A; Ying, Jackie Y

    2013-09-01

    The hair follicle is a regenerating organ that produces a new hair shaft during each growth cycle. Development and cycling of the hair follicle is governed by interactions between the epithelial and mesenchymal components. Therefore, development of an engineered 3D hair follicle would be useful for studying these interactions to identify strategies for treatment of hair loss. We have developed a technique suitable for assembly of different cell types in close proximity in fibrous hydrogel scaffolds with resolutions of ∼50 μm. By assembly of dermal papilla (DP) and keratinocytes, structures similar to the native hair bulb arrangement are formed. Gene expression of these constructs showed up-regulation of molecules involved in epithelial-mesenchymal interactions of the hair follicle. Implantation of the follicular structures in SCID mice led to the formation of hair follicle-like structures, thus demonstrating their hair inductive ability. The transparency of the fiber matrix and the small dimensions of the follicular structures allowed the direct quantitation of DP cell proliferation by confocal microscopy, clearly illustrating the promoting or inhibitory effects of hair growth regulating agents. Collectively, our results suggested a promising application of these 3D engineered follicular structures for in vitro screening and testing of drugs for hair growth therapy.

  18. Canonical Wnts, specifically Wnt-10b, show ability to maintain dermal papilla cells

    SciTech Connect

    Ouji, Yukiteru Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide

    2013-08-30

    Highlights: •First report on effects of various Wnts on DP cells. •Wnt-10b promoted trichogenesis, while Wnt-3a showed to a limited extent. •Canonical Wnts, specifically Wnt-10b, is important for DP cells maintenance. -- Abstract: Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.

  19. Oxidative stress-associated senescence in dermal papilla cells of men with androgenetic alopecia.

    PubMed

    Upton, James H; Hannen, Rosalind F; Bahta, Adiam W; Farjo, Nilofer; Farjo, Bessam; Philpott, Michael P

    2015-05-01

    Dermal papilla cells (DPCs) taken from male androgenetic alopecia (AGA) patients undergo premature senescence in vitro in association with the expression of p16(INK4a), suggesting that DPCs from balding scalp are more sensitive to environmental stress than nonbalding cells. As one of the major triggers of senescence in vitro stems from the cell "culture shock" owing to oxidative stress, we have further investigated the effects of oxidative stress on balding and occipital scalp DPCs. Patient-matched DPCs from balding and occipital scalp were cultured at atmospheric (21%) or physiologically normal (2%) O2. At 21% O2, DPCs showed flattened morphology and a significant reduction in mobility, population doubling, increased levels of reactive oxygen species and senescence-associated β-Gal activity, and increased expression of p16(INK4a) and pRB. Balding DPCs secreted higher levels of the negative hair growth regulators transforming growth factor beta 1 and 2 in response to H2O2 but not cell culture-associated oxidative stress. Balding DPCs had higher levels of catalase and total glutathione but appear to be less able to handle oxidative stress compared with occipital DPCs. These in vitro findings suggest that there may be a role for oxidative stress in the pathogenesis of AGA both in relation to cell senescence and migration but also secretion of known hair follicle inhibitory factors.

  20. Assembling Composite Dermal Papilla Spheres with Adipose-derived Stem Cells to Enhance Hair Follicle Induction

    PubMed Central

    Huang, Chin-Fu; Chang, Ya-Ju; Hsueh, Yuan-Yu; Huang, Chia-Wei; Wang, Duo-Hsiang; Huang, Tzu-Chieh; Wu, Yi-Ting; Su, Fong-Chin; Hughes, Michael; Chuong, Cheng-Ming; Wu, Chia-Ching

    2016-01-01

    Intradermal adipose tissue plays an essential role for hair follicles (HFs) regeneration by regulating hair cycles. However, the effect of reconstruction of HFs and the involvement of adipose-related cells are poorly understood. We investigated assembly strategies for the interactions of dermal papilla (DP) cells with adipose-derived stem cells (ASCs) in promoting hair formation. DP cells lose DP traits during adherent culture, but preserved DP markers with a unified sphere diameter by seeding on chitosan-coated microenvironments. Next, ASCs isolated from rats were co-cultured with DP spheres by different assembling approaches to determine their interactions; a mixed sphere of ASCs with DP cells (MA-DPS), or a core-shell structure, outer ASCs shell and an inner DP core (CSA-DPS). CSA-DPS exhibited superior DP characteristics compared to MA-DPS. Conditional medium from ASCs, but not differentiated adipocytes, promoted DP markers and functional alkaline phosphatase activity from the DP cells. In vivo patch assay showed the core-shell assembling of CSA-DPS can reconstruct cellular arrangements and microenvironmental niches as dominated by PPARα signal in ASCs to induce the greater hair induction than MA-DPS or DP spheres alone. Therefore, the assembling of a core-shell sphere for DP with ASCs could reconstruct the HF cellular arrangement for hair formation. This paper set the groundwork for further evaluation of the input of other cell types. PMID:27210831

  1. Dermal Papilla Cells Improve the Wound Healing Process and Generate Hair Bud-Like Structures in Grafted Skin Substitutes Using Hair Follicle Stem Cells

    PubMed Central

    Leirós, Gustavo José; Kusinsky, Ana Gabriela; Drago, Hugo; Bossi, Silvia; Sturla, Flavio; Castellanos, María Lía; Stella, Inés Yolanda

    2014-01-01

    Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. The use of dermal papilla cells (DPCs) as a dermal component in a permanent composite skin with human hair follicle stem cells (HFSCs) was evaluated by studying the tissue-engineered skin architecture, stem cell persistence, hair regeneration, and graft-take in nude mice. A porcine acellular dermal matrix was seeded with HFSCs alone and with HFSCs plus human DPCs or dermal fibroblasts (DFs). In vitro, the presence of DPCs induced a more regular and multilayered stratified epidermis with more basal p63-positive cells and invaginations. The DPC-containing constructs more accurately mimicked the skin architecture by properly stratifying the differentiating HFSCs and developing a well-ordered epithelia that contributed to more closely recapitulate an artificial human skin. This acellular dermal matrix previously repopulated in vitro with HFSCs and DFs or DPCs as the dermal component was grafted in nude mice. The presence of DPCs in the composite substitute not only favored early neovascularization, good assimilation and remodeling after grafting but also contributed to the neovascular network maturation, which might reduce the inflammation process, resulting in a better healing process, with less scarring and wound contraction. Interestingly, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human origin, presence of precursor epithelial cells, and expression of a hair differentiation marker. Although preliminary, these findings have demonstrated the importance of the presence of DPCs for proper skin repair. PMID:25161315

  2. Dermal papilla cells improve the wound healing process and generate hair bud-like structures in grafted skin substitutes using hair follicle stem cells.

    PubMed

    Leirós, Gustavo José; Kusinsky, Ana Gabriela; Drago, Hugo; Bossi, Silvia; Sturla, Flavio; Castellanos, María Lía; Stella, Inés Yolanda; Balañá, María Eugenia

    2014-10-01

    Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. The use of dermal papilla cells (DPCs) as a dermal component in a permanent composite skin with human hair follicle stem cells (HFSCs) was evaluated by studying the tissue-engineered skin architecture, stem cell persistence, hair regeneration, and graft-take in nude mice. A porcine acellular dermal matrix was seeded with HFSCs alone and with HFSCs plus human DPCs or dermal fibroblasts (DFs). In vitro, the presence of DPCs induced a more regular and multilayered stratified epidermis with more basal p63-positive cells and invaginations. The DPC-containing constructs more accurately mimicked the skin architecture by properly stratifying the differentiating HFSCs and developing a well-ordered epithelia that contributed to more closely recapitulate an artificial human skin. This acellular dermal matrix previously repopulated in vitro with HFSCs and DFs or DPCs as the dermal component was grafted in nude mice. The presence of DPCs in the composite substitute not only favored early neovascularization, good assimilation and remodeling after grafting but also contributed to the neovascular network maturation, which might reduce the inflammation process, resulting in a better healing process, with less scarring and wound contraction. Interestingly, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human origin, presence of precursor epithelial cells, and expression of a hair differentiation marker. Although preliminary, these findings have demonstrated the importance of the presence of DPCs for proper skin repair. ©AlphaMed Press.

  3. CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth.

    PubMed

    Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A; Andl, Thomas; Zhang, Yuhang

    2016-10-01

    Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  4. Keratinocyte Growth Inhibition through the Modification of Wnt Signaling by Androgen in Balding Dermal Papilla Cells

    PubMed Central

    Kitagawa, Tomoko; Matsuda, Ken-Ichi; Inui, Shigeki; Takenaka, Hideya; Katoh, Norito; Itami, Satoshi; Kishimoto, Saburo; Kawata, Mitsuhiro

    2009-01-01

    Context/Objective: Androgen induces androgenetic alopecia (AGA), which has a regressive effect on hair growth from the frontal region of the scalp. Conversely, Wnt proteins are known to positively affect mammalian hair growth. We hypothesized that androgen reduces hair growth via an interaction with the Wnt signaling system. The objective of this study was to investigate the effect of androgen on Wnt signaling in dermal papilla (DP) cells. Design: The effect of androgen and Wnt3a on keratinocyte proliferation was measured by use of a coculture system consisting of DP cells and keratinocytes. The molecular mechanisms of androgen and Wnt pathway interactions in DP cells were examined by analyzing the expression, intracellular localization, and activity of the androgen receptor (AR) and also downstream Wnt signaling molecules. Results: Wnt3a-dependent keratinocyte growth was suppressed by the addition of dihydrotestosterone in coculture with DP cells that were derived from AGA patients, but growth was not suppressed in coculture with DP cells from non-AGA males. Whereas DP cells from both scalp regions expressed AR protein, the expression levels of AR and cotranslocation with β-catenin, a downstream Wnt signaling molecule, were higher in DP cells of AGA patients than in DP cells from non-AGA males. In addition, significant suppression of Wnt signal-mediated transcription in response to dihydrotestosterone treatment was observed only in DP cells from AGA patients. Conclusion: These results suggest that Wnt signaling in DP cells is regulated by androgen and this regulation plays a pivotal role in androgen’s action on hair growth. PMID:19141591

  5. CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth

    PubMed Central

    Zhou, Linli; Yang, Kun; Carpenter, April; Lang, Richard A.; Andl, Thomas; Zhang, Yuhang

    2016-01-01

    Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment. PMID:27462123

  6. Sprouty/FGF signaling regulates the proximal-distal feather morphology and the size of dermal papillae.

    PubMed

    Yue, Zhicao; Jiang, Ting Xin; Wu, Ping; Widelitz, Randall B; Chuong, Cheng Ming

    2012-12-01

    In a feather, there are distinct morphologies along the proximal-distal axis. The proximal part is a cylindrical stalk (calamus), whereas the distal part has barb and barbule branches. Here we focus on what molecular signaling activity can modulate feather stem cells to generate these distinct morphologies. We demonstrate the drastic tissue remodeling during feather cycling which includes initiation, growth and resting phases. In the growth phase, epithelial components undergo progressive changes from the collar growth zone to the ramogenic zone, to maturing barb branches along the proximal-distal axis. Mesenchymal components also undergo progressive changes from the dermal papilla, to the collar mesenchyme, to the pulp along the proximal-distal axis. Over-expression of Spry4, a negative regulator of receptor tyrosine kinases, promotes barb branch formation at the expense of the epidermal collar. It even induces barb branches from the follicle sheath (equivalent to the outer root sheath in hair follicles). The results are feathers with expanded feather vane regions and small or missing proximal feather shafts (the calamus). Spry4 also expands the pulp region while reducing the size of dermal papillae, leading to a failure to regenerate. In contrast, over-expressing Fgf10 increases the size of the dermal papillae, expands collar epithelia and mesenchyme, but also prevents feather branch formation and feather keratin differentiation. These results suggest that coordinated Sprouty/FGF pathway activity at different stages is important to modulate feather epidermal stem cells to form distinct feather morphologies along the proximal-distal feather axis.

  7. Characterization of Human Fungiform Papillae Cells in Culture

    PubMed Central

    Brand, Joseph G.; Spielman, Andrew I.; Lischka, Fritz W.; Teeter, John H.; Breslin, Paul A.S.; Rawson, Nancy E.

    2011-01-01

    The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells. PMID:21471186

  8. Inhibition of hair growth by testosterone in the presence of dermal papilla cells from the frontal bald scalp of the postpubertal stumptailed macaque.

    PubMed

    Obana, N; Chang, C; Uno, H

    1997-01-01

    Hair-follicle regression in the bald scalps of stumptailed macaques develops after puberty, which corresponds to an elevation of serum testosterone and dihydrotestosterone. Using the cultured cells from the pre- and postpubertal macaques, we examined the role of dermal papilla cells in testosterone-induced inhibition of outer root sheath cell proliferation. Testosterone showed no effects on proliferation of either dermal papilla cells or outer root sheath cells cultured alone. Testosterone-induced inhibition of outer root sheath cell proliferation occurred only in coculture with dermal papilla cells derived from the bald scalps of adult macaques but not with dermal papilla cells from the hairy occipital scalps of adult macaques or the prebald frontal scalps of juvenile macaques. Furthermore, RU 58841, an androgen receptor blocker, antagonized this testosterone-elicited inhibition. Together our data indicate that the inhibitory effect of testosterone on proliferation of epithelial cells is age dependent, and androgen may play an essential role in hair growth either by inducing repressor(s) from dermal papilla cells, which may then inhibit the growth of epithelial cells of the hair follicle, or by inducing growth factor(s) from dermal papilla cells, which, in turn, may trigger the induction of some repressors in epithelial cells, thereby inhibiting the epithelial cell growth. Our animal studies also showed that RU 58841 has a dramatic effect on hair regrowth in the bald frontal scalp of the stumptailed macaque, which may further support our in vitro culture studies showing that antiandrogens can antagonize testosterone-elicited hair growth. In summary, our studies may provide a model for further isolation of androgen-regulated repressor(s)/growth factors, which may help control hair growth and baldness.

  9. Osteogenic differentiation of human dental papilla mesenchymal cells

    SciTech Connect

    Ikeda, Etsuko; Hirose, Motohiro . E-mail: motohiro-hirose@aist.go.jp; Kotobuki, Noriko; Shimaoka, Hideki; Tadokoro, Mika; Maeda, Masahiko; Hayashi, Yoshiko; Kirita, Tadaaki; Ohgushi, Hajime

    2006-04-21

    We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of {beta}-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.

  10. A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin.

    PubMed

    Hunt, David P J; Morris, Paul N; Sterling, Jane; Anderson, Jane A; Joannides, Alexis; Jahoda, Colin; Compston, Alastair; Chandran, Siddharthan

    2008-01-01

    Skin-derived precursor cells (SKPs) are multipotent neural crest-related stem cells that grow as self-renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture-initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate-mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest-derived sphere-forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000-fold enriched for sphere-forming neural crest-derived cells compared with whole facial skin. These "papillaspheres" share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere-forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.

  11. Hair growth promoting effect of dermal papilla like tissues from canine adipose-derived mesenchymal stem cells through vascular endothelial growth factor

    PubMed Central

    LEE, Aeri; BAE, Sohee; LEE, Seung Hoon; KWEON, Oh-Kyeong; KIM, Wan Hee

    2016-01-01

    The purpose of this study was to investigate the protein expression pattern and the in vivo trichogenicity of dermal papilla-like tissues (DPLTs) made from canine adipose-derived mesenchymal stem cells (ASCs) in athymic nude mice. Canine ASCs were isolated and cultured from adipose tissue, and differentiation was induced by culturing ASCs in dermal papilla forming media. DPLTs were embedded in collagen gel, and their structural characteristics and protein expression were evaluated by hematoxylin and eosin stain and immunohistochemistry. Athymic nude mice were divided into two groups (control and DPLTs groups), and DPLTs were injected in skin wounds of mice in the DPLTs group. The trichogenicity of DPLTs was assessed by gross and histological evaluations for 30 days. The fate and the growth factor-secretion effect of DPLTs were evaluated by immunohistochemistry and Western blotting. DPLTs have a compact aggregated structure, form extracellular matrix and highly express the protein specific for dermal papillae, including ALP and versican. New hair follicle formation was remarkable in nude mice of the DPLTs group in gross findings and H&E stain. Vascularization was increased in the DPLTs group, which was the effect of vascular endothelial growth factor secreted by DPLTs in vitro and in vivo. These data suggest that engineered canine DPLTs have characteristics of dermal papillae and have a positive effect on hair regeneration by secreting growth factors. PMID:27647656

  12. Technique to collect fungiform (taste) papillae from human tongue.

    PubMed

    Spielman, Andrew I; Pepino, M Yanina; Feldman, Roy; Brand, Joseph G

    2010-09-18

    The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 2008). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

  13. Differential expression analysis of balding and nonbalding dermal papilla microRNAs in male pattern baldness with a microRNA amplification profiling method.

    PubMed

    Goodarzi, H R; Abbasi, A; Saffari, M; Fazelzadeh Haghighi, M; Tabei, M B; Noori Daloii, M R

    2012-05-01

      Male pattern baldness or androgenetic alopecia is a common disorder affecting almost 50% of men throughout their lifetime, with androgens and genetics having significant contributing aetiologies. In contrast to the positive regulatory effect of androgens on body hair growth, they are thought to alter scalp hair follicle behaviour pathophysiologically, leading to male pattern baldness. However, the exact mechanisms of this paradoxical action have not yet been elucidated. The role of microRNAs, a novel group of noncoding RNAs impacting almost every aspect of biology, health and human diseases, has been documented in hair follicle formation. In addition, their deregulation in cancer of the prostate, a target organ of androgens, has also been well established. To investigate the possible contribution of microRNAs in the pathophysiology of male pattern baldness. We initially screened microRNA expression profiles of balding and nonbalding hair follicle papillae with a sensitive microRNA cloning method, microRNA amplification profiling, and statistically analysed significant differentially expressed microRNAs in balding relative to nonbalding dermal papillae, with real-time polymerase chain reaction as a confirmatory method to quantify expression in eight individuals affected with the disorder.   We detected the significant upregulation of miR-221, miR-125b, miR-106a and miR-410 in balding papilla cells.   We found four microRNAs that could participate in the pathogenesis of male pattern baldness. Regarding the strong therapeutic potential of microRNAs and the easy accessibility of hair follicles for gene therapy, microRNAs are possible candidates for a new generation of revolutionary treatments. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

  14. The Dermal Papilla: An Instructive Niche for Epithelial Stem and Progenitor Cells in Development and Regeneration of the Hair Follicle

    PubMed Central

    Morgan, Bruce A.

    2014-01-01

    The dermal papilla (DP) of the hair follicle is both a chemical and physical niche for epithelial progenitor cells that regenerate the cycling portion of the hair follicle and generate the hair shaft. Here, we review experiments that revealed the importance of the DP in regulating the characteristics of the hair shaft and frequency of hair follicle regeneration. More recent work showed that the size of this niche is dynamic and actively regulated and reduction in DP cell number per follicle is sufficient to cause hair thinning and loss. The formation of the DP during follicle neogenesis provides a context to contemplate the mechanisms that maintain DP size and the potential to exploit these processes for hair preservation or restoration. PMID:24985131

  15. 1α,25-dihydroxyvitamin D3 modulates the hair-inductive capacity of dermal papilla cells: therapeutic potential for hair regeneration.

    PubMed

    Aoi, Noriyuki; Inoue, Keita; Chikanishi, Toshihiro; Fujiki, Ryoji; Yamamoto, Hanako; Kato, Harunosuke; Eto, Hitomi; Doi, Kentaro; Itami, Satoshi; Kato, Shigeaki; Yoshimura, Kotaro

    2012-08-01

    Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.

  16. Effect of extremely low frequency electromagnetic field on MAP2 and Nestin gene expression of hair follicle dermal papilla cells.

    PubMed

    Moraveji, Marzie; Haghighipour, Nooshin; Keshvari, Hamid; Nourizadeh Abbariki, Tannaz; Shokrgozar, Mohammad Ali; Amanzadeh, Amir

    2016-08-19

    In recent years, the extremely low frequency electromagnetic field (ELF-EMF) has attracted a great deal of scientific interest. The ELF-EMF signal is able to control ion transport across ion channels and therefore induce cell differentiation. The purpose of this study was to investigate the effect of ELF-EMF (50 Hz, 1 mT) on MAP2 and Nestin gene expression of dermal papilla mesenchymal cells (DPCs). In order to examine the effect of chemical and electromagnetic factors on gene expression, 4 experimental groups, namely chemical (cell exposure to chemical signals), EMF (exposing cells to ELF-EMF), chemical-EMF (subjecting cells to chemical signals and ELF-EMF) and control (with no treatment) groups, were prepared, treated for 5 days, and studied. To assess the effect of extended test time on the expression of neural differentiation markers (Nestin and MAP2), an EMF group was prepared and treated for a period of 14 consecutive days. The beneficial role of EMF in inducing neural differentiation was shown by real-time PCR analysis. The higher expression of MAP2 after 14 days compared to that after 5 days and decrease of cell proliferation on days 5 to 20 were indicative of the positive effect of extending treatment time on neural differentiation by evaluation of gene expression in EMF group.

  17. Activation of β-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth

    PubMed Central

    Zhou, Linli; Xu, Mingang; Yang, Yongguang; Yang, Kun; Wickett, Randall R.; Andl, Thomas; Millar, Sarah E.

    2016-01-01

    The hair follicle dermal papilla (DP) contains a unique prominin-1/CD133-positive (CD133+) cell subpopulation, which has been shown to possess hair follicle-inducing capability. By assaying for endogenous CD133 expression and performing lineage tracing using CD133-CreERT2; ZsGreen1 reporter mice, we find that CD133 is expressed in a subpopulation of DP cells during the growth phase of the murine hair cycle (anagen), but is absent at anagen onset. However, how CD133+ DP cells interact with keratinocytes to induce hair regenerative growth remains unclear. Wnt/β-catenin has long been recognized as a major signaling pathway required for hair follicle morphogenesis, development, and regeneration. Nuclear Wnt/β-catenin activity is observed in the DP during the hair growth phase. Here we show that induced expression of a stabilized form of β-catenin in CD133+ DP cells significantly accelerates spontaneous and depilation-induced hair growth. However, hair follicle regression is not affected in these mutants. Further analysis indicates that CD133+ DP-expressed β-catenin increases proliferation and differentiation of epithelial matrix keratinocytes. Upregulated Wnt/β-catenin activity in CD133+ DP cells also increases the number of proliferating DP cells in each anagen follicle. Our data demonstrate that β-catenin signaling potentiates the capability of CD133+ DP cells to promote postnatal hair growth. PMID:27472062

  18. Experimental study on repairing of nude mice skin defects with composite skin consisting of xenogeneic dermis and epidermal stem cells and hair follicle dermal papilla cells.

    PubMed

    Qi, Shao-Hai; Liu, Po; Xie, Ju-Lin; Shu, Bin; Xu, Ying-Bin; Ke, Chang-Neng; Liu, Xu-Sheng; Li, Tian-Zeng

    2008-05-01

    To investigate the influence of hair follicle dermal papilla cells (DPCs) on biological features of composite skin. In the test group, xenogeneic acellular dermal matrix was employed as the frame, DPCs were seeded on the subcutaneous side, and epithelial stem cells onto the dermal papilla side of the dermal frame so as to construct a composite skin. In the control group, there was no DPC in the frame. The two kinds of composite skin were employed to cover skin defects on the back of the nude mice. Wound healing was observed 4 weeks after grafting and area was analyzed and contraction rate was calculated. The tissue samples in the grafted area were harvested for HE staining and the state of the composite skin was observed. The stress-strain curve of the sampled skin was measured, so as to calculate the maximal breaking power of the sample. The data were collected and statistically analyzed. HE staining indicated that the epithelial depth was increased (more than 10 layers of cells) in test group, with only 6-7 layers in control group. The skin contraction rate in test group on the 4th week after skin grafting (3.94+/-0.013)% was much lower than that in control group (29.07+/-0.018)% (P<0.05). It was indicated by biomechanical test that the stress-strain curve of the composite skin in the test group was closer to that of normal nude mice skin in comparison to that in control group. The maximal breaking force of the composite skin in test group was (1.835+/-0.035)N (Newton), while that in control group was (1.075+/-0.065)N (P<0.01). Reconstruction of epidermis in composite skin was promoted by dermal DPCs seeded in the dermal matrix frame. As a result, there was less skin contraction in the composite skin with DPCs, so that the biological characteristics of the skin were improved.

  19. Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model.

    PubMed

    Yamao, Mikaru; Inamatsu, Mutsumi; Okada, Taro; Ogawa, Yuko; Ishida, Yuji; Tateno, Chise; Yoshizato, Katsutoshi

    2015-03-01

    No model is available for examining whether in vivo-damaged human hair follicles (hu-HFs) are rescued by transplanting cultured hu-HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo-damaged hu-HFs such as miniaturized hu-HFs in androgenic alopecia are improvable by auto-transplanting hu-HF dermal cells. In this study, we first developed mice with humanized skin composed of hu-keratinocytes and hu-dermal fibroblasts. Then, a 'humanized scalp model mouse' was generated by transplanting hu-scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu-HFs in the model were amputated in situ, and cultured hu-HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation.

  20. Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

    PubMed

    Aljuffali, Ibrahim A; Pan, Tai-Long; Sung, Calvin T; Chang, Shu-Hao; Fang, Jia-You

    2015-08-01

    This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and -46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. Topical use of minoxidil is only one of the very few treatment options for alopecia. Nonetheless, the current delivery method is far from ideal. In this article, the authors developed lipid nanocarriers with anti-platelet-derived growth factor receptor ? antibody to target dermal papilla cells, and showed enhanced uptake of minoxidil. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Androgens modify Wnt agonists/antagonists expression balance in dermal papilla cells preventing hair follicle stem cell differentiation in androgenetic alopecia.

    PubMed

    Leirós, Gustavo José; Ceruti, Julieta María; Castellanos, María Lía; Kusinsky, Ana Gabriela; Balañá, María Eugenia

    2017-01-05

    In androgenetic alopecia, androgens impair dermal papilla-induced hair follicle stem cell (HFSC) differentiation inhibiting Wnt signaling. Wnt agonists/antagonists balance was analyzed after dihydrotestosterone (DHT) stimulation in androgen-sensitive dermal papilla cells (DPC) cultured as spheroids or monolayer. In both culture conditions, DHT stimulation downregulated Wnt5a and Wnt10b mRNA while the Wnt antagonist Dkk-1 was upregulated. Notably, tissue architecture of DPC-spheroids lowers Dkk-1 and enhances Wnt agonists' basal expression; probably contributing to DPC inductivity. The role of Wnt agonists/antagonists as mediators of androgen inhibition of DPC-induced HFSC differentiation was evaluated. Inductive DPC-conditioned medium supplemented with DKK-1 impaired HFSC differentiation mimicking androgens' action. This effect was associated with inactivation of Wnt/β-catenin pathway in differentiating HFSC by both DPC-conditioned media. Moreover, addition of WNT10b to DPC-medium conditioned with DHT, overcame androgen inhibition of HFSC differentiation. Our results identify DKK1 and WNT10b as paracrine factors which modulate the HFSC differentiation inhibition involved in androgen-driven balding.

  2. Clonal Growth of Dermal Papilla Cells in Hydrogels Reveals Intrinsic Differences between Sox2-Positive and -Negative Cells In Vitro and In Vivo

    PubMed Central

    Driskell, Ryan R; Juneja, Vikram R; Connelly, John T; Kretzschmar, Kai; Tan, David W -M; Watt, Fiona M

    2012-01-01

    In neonatal mouse skin, two types of dermal papilla (DP) are distinguished by Sox2 expression: CD133+Sox2+ DP are associated with guard/awl/auchene hairs, whereas CD133+Sox2− DP are associated with zigzag (ZZ) hairs. We describe a three-dimensional hydrogel culture system that supports clonal growth of CD133+Sox2+, CD133+Sox2−, and CD133−Sox2− (non-DP) neonatal dermal cells. All three cell populations formed spheres that expressed the DP markers alkaline phosphatase, α8 integrin, and CD133. Nevertheless, spheres formed by CD133− cells did not efficiently support hair follicle formation in skin reconstitution assays. In the presence of freshly isolated P2 dermal cells, CD133+Sox2+ and CD133+Sox2− spheres contributed to the DP of both AA and ZZ hairs. Hair type did not correlate with sphere size. Sox2 expression was maintained in culture, but not induced significantly in Sox2− cells in vitro or in vivo, suggesting that Sox2+ cells are a distinct cellular lineage. Although Sox2+ cells were least efficient at forming spheres, they had the greatest ability to contribute to DP and non-DP dermis in reconstituted skin. As the culture system supports clonal growth of DP cells and maintenance of distinct DP cell types, it will be useful for further analysis of intrinsic and extrinsic signals controlling DP function. PMID:22189784

  3. Adult Human CD133/1+ Kidney Cells Isolated from Papilla Integrate into Developing Kidney Tubules

    PubMed Central

    Ward, Heather H.; Romero, Elsa; Welford, Angela; Pickett, Gavin; Bacallao, Robert; Gattone, Vincent H.; Ness, Scott A.; Wandinger-Ness, Angela; Roitbak, Tamara

    2011-01-01

    Approximately 60,000 patients in the US are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin+ and CD133/1+ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1+ cells. Isolated CD133/1+ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1+ progenitors from the papilla and cortex, became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. PMID:21255643

  4. Self-assembly of dermal papilla cells into inductive spheroidal microtissues on poly(ethylene-co-vinyl alcohol) membranes for hair follicle regeneration.

    PubMed

    Young, Tai-Horng; Lee, Chiao-Yun; Chiu, Hsien-Ching; Hsu, Chih-Jung; Lin, Sung-Jan

    2008-09-01

    Self-aggregation is key to hair follicle (HF) induction ability of dermal papilla (DP) cells and neogenesis of HF can be achieved by transplanting DP microtissues. However, there is currently lack of a suitable system that allows efficient production of DP microtissues and analysis of DP self-aggregation in vitro. We demonstrate that, at a higher seeding cell density, poly(ethylene-co-vinyl alcohol) (EVAL) membranes facilitate DP self-assembly into many compact spheroidal microtissues that are able to induce new HFs. This self-assembling process is associated with an enhanced cell movement and a declined cell-substrate adhesivity on EVAL. A compromised cell growth is also revealed on EVAL. On the contrary, a more adherent surface allows faster cell expansion but maintains DP cells in a flat morphology. Dynamically, cell migration, intercellular collision and intercellular adhesion contribute to DP microtissue formation on EVAL. Our results suggest that, for large-scale production of DP microtissues for HF regeneration, an adhesive surface is needed for quick cell expansion and a biomaterial with a lower adhesivity is required for self-aggregation. In addition, this system can be a model for investigation of DP self-aggregation in vitro.

  5. Sox2 in the dermal papilla niche controls hair growth by fine-tuning Bmp signaling in differentiating hair shaft progenitors

    PubMed Central

    Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Rezza, Amelie; Barros, Rita; Sennett, Rachel; Mazloom, Amin; Chung, Chi-Yeh; Cai, Xiaoqiang; Cai, Chen-Leng; Pevny, Larysa; Nicolis, Silvia; Ma’ayan, Avi; Rendl, Michael

    2012-01-01

    SUMMARY How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18Cre to target embryonic DP precursors, we ablate the transcription factor Sox2 early and efficiently, resulting in diminished hair shaft outgrowth. We find that DP niche expression of Sox2 controls the migration rate of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased Bmp inhibitor Sostdc1, a direct Sox2 transcriptional target. Subsequently, we identify upregulated Bmp signaling in knockout hair shaft progenitors and demonstrate that Bmps inhibit cell migration, an effect that can be attenuated by Sostdc1. A shorter and Sox2-negative hair type lacks Sostdc1 in the DP and shows reduced migration and increased Bmp activity of hair shaft progenitors. Collectively, our data identify Sox2 as a key regulator of hair growth that controls progenitor migration by fine-tuning Bmp-mediated mesenchymal-epithelial crosstalk. PMID:23153495

  6. Sox2 in the dermal papilla niche controls hair growth by fine-tuning BMP signaling in differentiating hair shaft progenitors.

    PubMed

    Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Rezza, Amelie; Barros, Rita; Sennett, Rachel; Mazloom, Amin Reza; Chung, Chi-Yeh; Cai, Xiaoqiang; Cai, Chen-Leng; Pevny, Larysa; Nicolis, Silvia; Ma'ayan, Avi; Rendl, Michael

    2012-11-13

    How dermal papilla (DP) niche cells regulate hair follicle progenitors to control hair growth remains unclear. Using Tbx18(Cre) to target embryonic DP precursors, we ablate the transcription factor Sox2 early and efficiently, resulting in diminished hair shaft outgrowth. We find that DP niche expression of Sox2 controls the migration speed of differentiating hair shaft progenitors. Transcriptional profiling of Sox2 null DPs reveals increased Bmp6 and decreased BMP inhibitor Sostdc1, a direct Sox2 transcriptional target. Subsequently, we identify upregulated BMP signaling in knockout hair shaft progenitors and demonstrate that Bmp6 inhibits cell migration, an effect that can be attenuated by Sostdc1. A shorter and Sox2-negative hair type lacks Sostdc1 in the DP and shows reduced migration and increased BMP activity of hair shaft progenitors. Collectively, our data identify Sox2 as a key regulator of hair growth that controls progenitor migration by fine-tuning BMP-mediated mesenchymal-epithelial crosstalk.

  7. Site-specific rectocele repair with dermal graft augmentation: comparison of porcine dermal xenograft (Pelvicol) and human dermal allograft.

    PubMed

    Biehl, Roger C; Moore, Robert D; Miklos, John R; Kohli, Neeraj; Anand, Indu S; Mattox, T Fleming

    2008-01-01

    This study is a retrospective chart review comparing 195 women who underwent rectocele repair with either a porcine dermal xenograft or human allogenic cadaveric dermal graft augmentation over a two year period. A site-specific defect repair was completed prior to augmentation with the graft. Examinations were performed preoperatively and postoperatively using the pelvic organ prolapse quantification system. Questionnaires were used to assess constipation and dyspareunia. De novo dyspareunia and cure rates for constipation and dyspareunia were not statistically different between the two groups. Site-specific fascial rectocele repairs with xenograft or allograft augmentation were found to have similar complication rates as well as objective and subjective cure rates.

  8. Human acellular dermal matrix grafts for rhinoplasty.

    PubMed

    Sherris, David A; Oriel, Brad S

    2011-09-01

    Rhinoplasty often relies on graft material for structural support in the form of cartilage, bone grafts, or fascia. In addition, pliable grafts are often helpful for contouring and can function as a barrier. Unfortunately, grafts carry the disadvantage of requiring an additional donor site, with associated complications. Human acellular dermal matrix (ADM) biological implants offer an exciting alternative for structural support and nonstructural implantation in rhinoplasty procedures. To examine the efficacy of ADM placement in rhinoplasty and septoplasty, the authors report the results from a series of 51 patients. In this series, there were no cases of infection, skin discoloration, seroma formation, septal perforation, significant resorption, extrusion, or other complications related to ADM placement. Therefore, the authors believe that ADM offers a safe and effective alternative to traditional grafting methods for functional and aesthetic rhinoplasty.

  9. Human dermal fibroblasts in psychiatry research.

    PubMed

    Kálmán, S; Garbett, K A; Janka, Z; Mirnics, K

    2016-04-21

    In order to decipher the disease etiology, progression and treatment of multifactorial human brain diseases we utilize a host of different experimental models. Recently, patient-derived human dermal fibroblast (HDF) cultures have re-emerged as promising in vitro functional system for examining various cellular, molecular, metabolic and (patho)physiological states and traits of psychiatric disorders. HDF studies serve as a powerful complement to postmortem and animal studies, and often appear to be informative about the altered homeostasis in neural tissue. Studies of HDFs from patients with schizophrenia (SZ), depression, bipolar disorder (BD), autism, attention deficit and hyperactivity disorder and other psychiatric disorders have significantly advanced our understanding of these devastating diseases. These reports unequivocally prove that signal transduction, redox homeostasis, circadian rhythms and gene*environment (G*E) interactions are all amenable for assessment by the HDF model. Furthermore, the reported findings suggest that this underutilized patient biomaterial, combined with modern molecular biology techniques, may have both diagnostic and prognostic value, including prediction of response to therapeutic agents.

  10. l-Ascorbic acid 2-phosphate promotes elongation of hair shafts via the secretion of insulin-like growth factor-1 from dermal papilla cells through phosphatidylinositol 3-kinase.

    PubMed

    Kwack, M H; Shin, S H; Kim, S R; Im, S U; Han, I S; Kim, M K; Kim, J C; Sung, Y K

    2009-06-01

    l-Ascorbic acid 2-phosphate (Asc 2-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice. To investigate whether the promotion of hair growth by Asc 2-P is mediated by insulin-like growth factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc 2-P-induced IGF-1 expression. Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc 2-P treatment in the absence or presence of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc 2-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc 2-P treatment in the absence or presence of LY294002 was examined by Ki-67 immunostaining. IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc 2-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc 2-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc 2-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc 2-P-induced growth of cocultured keratinocytes. LY294002 significantly attenuated Asc 2-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles. These data show that Asc 2-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle growth in vitro via PI3K.

  11. Proteomic analysis of balding and non-balding mesenchyme-derived dermal papilla cells from androgenetic alopecia patients using on-line two-dimensional reversed phase-reversed phase LC-MS/MS.

    PubMed

    Moon, Pyong-Gon; Kwack, Mi Hee; Lee, Jeong-Eun; Cho, Young-Eun; Park, Ji-Hwan; Hwang, Daehee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan; Baek, Moon-Chang

    2013-06-24

    Androgenetic alopecia (AGA) is the most common and progressive disorder of hair loss with psychological effects. However, the exact mechanisms of baldness have not yet been elucidated. Until now, there has been no report using current proteomic approaches to examine balding and non-balding mesenchyme-derived DPCs simultaneously. To achieve the goal of identifying differentially expressed proteins in balding DPCs compared to non-balding DPCs, we present a strategy combining gel-assisted digestion and automatic on-line two-dimensional reversed phase-reversed phase (2D RP-RP) LC-MS/MS with informatics-assisted label-free protein quantitation. This strategy efficiently quantified the proteins of balding and non-balding DPCs from patients, and 128 up-regulated and 12 down-regulated proteins among 690 distinct proteins were identified in balding DPCs compared to non-balding DPCs. Up-regulated proteins belonging to these pathways in the balding DPCs, including argininosuccinate synthase 1 (ASS1), phosphoribosylaminoimidazole carboxylase (PAICS; ADE2), cytoskeleton-associated protein 4 (CKAP4), gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), and S-phase kinase-associated protein 1 (SKP1), were confirmed by Western blot analysis. Gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), plectin-1 (PLEC1), and nonerythroid α-spectrin (SPTAN1) were confirmed by immunofluorescence analysis. This proteomic approach to DPCs should help in understanding the pathogenesis of AGA. The results demonstrated the first deep proteomic approach to mesenchyme-derived dermal papilla cells (DPCs), which are a useful model system to investigate the mechanisms of baldness, from human hair and also identified differentially expressed proteins in balding DPCs compared to non-balding DPCs. This study should help in understanding the pathogenesis of androgenetic alopecia (AGA) and furthermore some proteins differentially expressed could be studied as therapeutic targets for AGA

  12. Dermal absorption of mucopolysaccharide polysulfate (heparinoid) in human and minipig.

    PubMed

    Kumokawa, Tadao; Hirata, Kazumasa; Sato, Keiichi; Kano, Satoshi

    2011-01-01

    Dermal absorption of mucopolysaccharide polysulfate (MPS, the active ingredient of Hirudoid") in human and minipig was investigated by using 14C-labeled MPS. Three types of human and minipig skin samples were used: intact, dried and tape-stripped. At 24 h after application of 14C-MPS to intact human skin on a Franz cell in vitro, the radioactivity was detected in 0.98, 1.34, and 0.08% of the applied dose in stratum corneum, epidermal-dermal skin, and receptor fluid, respectively. In dried human skin, the amount of radioactivity detected was similar to that in intact human skin. By contrast, in tape-stripped human skin, higher radioactivity was detected in epidermal-dermal skin and receptor fluid (2.85 and 0.33% of the applied dose, respectively) than in intact or dried skin. Minipig skin showed 1.5 to 4.5 times greater dermal absorption of 14C-MPS, as compared with human skin. In an in vivo study with minipig, radioactivity was detected at the dosing skin site after dermal administration of 14C-MPS. The stability of 14C-MPS in human skin after dermal application was evaluated by agarose gel electrophoresis and ion-exchange chromatography. It was suggested that 14C-MPS absorbed into human skin would be stable because the chromatogram behaviors of the radioactivity on the two types of method were not shifted. Microautoradiography of human and minipig skins after 14C-MPS dosing showed that radioactivity was widely distributed in the epidermis in the area near hair follicles. The present results clearly demonstrate that MPS is stable and that a small fraction of it is percutaneously absorbed by human and minipig skin.

  13. WNT5A inhibits human dental papilla cell proliferation and migration

    SciTech Connect

    Peng, L.; Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P.; Zhou, X.D.

    2009-12-18

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  14. [Stereology of the dermo-epithelial interface of the matrix-papilla unit of the hair bulb. Computerized processing of tridimensional images].

    PubMed

    Van Neste, D; Thievenaz, H; Dumortier, J L; Dumortier, M

    1986-01-01

    Images of the spatial relations of the matrix-papilla unit of the human hair bulb are shown. Hair bulbs were reconstructed in 3 dimensions by a computer graphics system from measurements made on horizontal sections parallel to the scalp surface. The outlines of hair matrix and dermal papilla of every fifth serial sections were fed into a computer as a segment 30 mu high (5 sections, each 6 mu thick). Processing of these segments into 3 dimensional models, using techniques adapted from those currently used for architectural drawing enabled us to avoid major distortion of the printed pictures. As an end result, the hair bulbs appear as wire frame structures in which the boundaries between consecutive segments have been connected to improve rendering of the dermal papilla interacting with the overlying epithelial hair matrix. This computerized method has been used to evaluate dermal-epithelial stereology of the interacting dermal papilla and epithelial hair matrix in hair bulbs. In a patient with trichorhinophalangeal syndrome the hair bulbs, and especially their dermal papillae, were found to the hypotrophic as compared with a sex- and age-matched control. Even though the pictures are somewhat artifactual in their present form, there are several means of improving the rendering of relief and of the interrelation between the hair follicle and other surrounding structures. This method could be useful to demonstrate disturbed dermal-epithelial interactions in other types of alopecia and to evaluate potential biological effects of drugs used in the treatment of alopecia.

  15. In vivo observation of papillae of the human tongue using confocal laser scanning microscopy.

    PubMed

    Just, Tino; Stave, Joachim; Pau, Hans Wilhelm; Guthoff, Rudolf

    2005-01-01

    The aim of this investigation was to visualize the epithelial structures of the tongue using confocal laser scanning microscopy (LSM). The human tongue epithelium of 28 healthy subjects, aged 21-67 years, mean age 38 years, 14 women and 14 men, was examined in vivo by LSM. Using LSM, a combination of the Heidelberg Retina Tomograph HRT II and the Rostock Cornea Module, up to 800-fold magnifications were obtained. On the tongue surface both filiform and fungiform papillae and their taste pores were easily identified. The epithelium of the tongue with its subcellular structures could be observed up to a depth of 50 microm, cellular structures up to 150 microm and subepithelial vessels up to 300 microm. Additionally the papillary crests and blood flow were visible. Confocal LSM seems suitable for noninvasive in vivo examination of the tongue. The hydraulic z scan, the manual start setting and the measurement of the depth allow a clear classification of the observed structures.

  16. DISPOSITION OF BROMODICHLOROMETHANE IN HUMANS FOLLOWING ORAL AND DERMAL EXPOSURE

    EPA Science Inventory

    DISPOSITION OF BROMODICHLOROMETHANE IN HUMANS FOLLOWING ORAL AND DERMAL EXPOSURE. TL Leavens1, MW Case1, RA Pegram1, BC Blount2, DM DeMarini1, MC Madden1, and JL Valentine3. 1NHEERL, USEPA, RTP, NC, USA; 2CDC, Atlanta, GA, USA; 3RTI, RTP, NC, USA.
    The disinfection byproduct ...

  17. DISPOSITION OF BROMODICHLOROMETHANE IN HUMANS FOLLOWING ORAL AND DERMAL EXPOSURE

    EPA Science Inventory

    DISPOSITION OF BROMODICHLOROMETHANE IN HUMANS FOLLOWING ORAL AND DERMAL EXPOSURE. TL Leavens1, MW Case1, RA Pegram1, BC Blount2, DM DeMarini1, MC Madden1, and JL Valentine3. 1NHEERL, USEPA, RTP, NC, USA; 2CDC, Atlanta, GA, USA; 3RTI, RTP, NC, USA.
    The disinfection byproduct ...

  18. Lateral Ankle Stabilization Using Acellular Human Dermal Allograft Augmentation.

    PubMed

    Parks, Robert M; Parks, Stephanie M

    2015-05-01

    We describe a retrospective study that uses the Broström-type surgical procedure with modifications that augment deficient and torn ligaments with acellular human dermal grafts. At the onset of this study, the most prevalent dermal graft available to us was GraftJacket (Wright Medical Technology, Arlington, Tennessee). Greater than 50% of the study participants were grafted with this product, but more recently other equally effective human dermal grafts have been used with no apparent difference. Thirty-five lateral ankle stabilization procedures were performed in the past 6 years on 33 patients. Eight patients were considered athletes (mean age, 23 years). The balance of the study group consisted of sedentary patients (mean age, 41 years). The mean patient body mass index (calculated as the weight in kilograms divided by the square of the height in meters) was 31. All of the patients were satisfied with their results, with no recurrent instability. Two patients in this group went on to have contralateral ankle stabilization in a similar manner owing to their satisfaction. Complications included two soft-tissue infections. Lateral ankle stabilization using acellular human dermal graft augmentation is a useful tool in the surgical treatment of ankle instability. This procedure offers distinct advantages over traditional methods of ankle repair and can be performed with relatively limited surgical exposure. Ease of operation, consistent results, and limited patient morbidity should allow surgeons to use this procedure independently or adjunctively to improve surgical outcomes.

  19. Comparative morphology of changeable skin papillae in octopus and cuttlefish.

    PubMed

    Allen, Justine J; Bell, George R R; Kuzirian, Alan M; Velankar, Sachin S; Hanlon, Roger T

    2014-04-01

    A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three-dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide-rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue.

  20. Engineering human neo-tendon tissue in vitro with human dermal fibroblasts under static mechanical strain.

    PubMed

    Deng, Dan; Liu, Wei; Xu, Feng; Yang, Yang; Zhou, Guangdong; Zhang, Wen Jie; Cui, Lei; Cao, Yilin

    2009-12-01

    Proper cell source is one of the key issues for tendon engineering. Our previous study showed that dermal fibroblasts could be used to successfully engineer tendon in vivo and tenocytes could engineer neo-tendon in vitro with static strain. This study further investigated the possibility of engineering human neo-tendon tissue in vitro using dermal fibroblasts. Human dermal fibroblasts were seeded on polyglycolic acid (PGA) fibers pre-fixed on a U-shape as a mechanical loading group, or simply cultured in a dish as a tension-free group. In addition, human tenocytes were also seeded on PGA fibers with tension as a comparison to human dermal fibroblasts. The results showed that human neo-tendon tissue could be generated using dermal fibroblasts during in vitro culture under static strain and the tissue structure became more mature with the increase of culture time. Longitudinally aligned collagen fibers and spindle shape cells were observed histologically and collagen fibril diameter and tensile strength increased with time and reached a peak at 14 weeks. In contrast, the dermal fibroblast-PGA constructs failed to form neo-tendon, but formed disorganized fibrous tissue in tension-free condition with significantly weaker strength and poor collagen fiber formation. Interestingly, neo-tendon tissues generated with human dermal fibroblasts were indistinguishable from the counterpart engineered with human tenocytes, which supports the viewpoint that human dermal fibroblasts is likely to replace tenocytes for future tendon graft development in vitro with dynamic mechanical loading in a bioreactor system.

  1. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    PubMed

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte

    2009-11-01

    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

  2. [Human stem cells from apical papilla can regenerate dentin-pulp complex].

    PubMed

    Xiong, Huacui; Chen, Ke; Huang, Yibin; Liu, Caiqi

    2013-10-01

    To regenerate dentin-pulp complex by tissue engineering with human stem cells from apical papilla cells (SCAP) as the seed cells. SCAP was separated from from normal human impacted third molars with immature roots by outgrowth culture. The cells were then cultured in the differentiation medium for 3 weeks or in normal medium for 60 days, and analyzed for mineralization potential by Alizarin red staining. The osteo/odontogenic markers including alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC) and dentin sialoprotein (DSP) were investigated by immunofluorescence staining and reverse transcription-polymerase chain reaction. The co-cultured mixture of SCAP and HA/TCP, or HA/TCP alone was implanted subcutaneously on the back of nude mice for 8 weeks, and the implants were collected and examined by HE and immunohistochemical staining. Round alizarin red-positive nodules formed in the isolated cells after cell culture in the differentiation medium for 3 weeks or in normal medium for 60 days with positive staining for osteo/odontogenic markers. SCAP with HA/TCP could regenerate pulp-dentin complex-like tissue in nude mice. The cells near the dentin-like tissue were positive for DSP. No mineral tissue was found in mice receiving HA/TCP implantation. SCAP may serve as a promising seed cell for dentin-pulp complex tissue engineering.

  3. Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue.

    PubMed

    Madi, Moinecha; Rols, Marie-Pierre; Gibot, Laure

    2015-10-01

    DNA electrotransfer is a successful technic for gene delivery. However, its use in clinical applications is limited since little is known about the mechanisms governing DNA electrotransfer in the complex environment occurring in a tissue. The objectives of this work were to investigate the role of the extracellular matrix (ECM) in that process. Tumor ECM composition was shown to modulate in vivo gene electrotransfer efficiency. In order to assess the effects of ECM composition and organization, as well as intercellular junctions and communication, in normal tissue response to electric pulses, we developed an innovative three-dimensional (3D) reconstructed human connective tissue model. 3D human dermal tissue was reconstructed in vitro by a tissue engineering approach and was representative of in vivo cell organization since cell-cell contacts were present as well as complex ECM. This human cell model presented multiple layers of primary dermal fibroblasts embedded in a native, collagen-rich ECM. This dermal tissue could become a useful tool to study skin DNA electrotransfer mechanisms. As proof of the concept, we show here that the cells within this standardized 3D tissue can be efficiently electropermeabilized by milliseconds electric pulses. We believe that a better comprehension of gene electrotransfer in such a model tissue would help improve electrogene therapy approaches such as the systemic delivery of therapeutic proteins and DNA vaccination.

  4. Human acellular dermal wound matrix: evidence and experience.

    PubMed

    Kirsner, Robert S; Bohn, Greg; Driver, Vickie R; Mills, Joseph L; Nanney, Lillian B; Williams, Marie L; Wu, Stephanie C

    2015-12-01

    A chronic wound fails to complete an orderly and timely reparative process and places patients at increased risk for wound complications that negatively impact quality of life and require greater health care expenditure. The role of extracellular matrix (ECM) is critical in normal and chronic wound repair. Not only is ECM the largest component of the dermal skin layer, but also ECM proteins provide structure and cell signalling that are necessary for successful tissue repair. Chronic wounds are characterised by their inflammatory and proteolytic environment, which degrades the ECM. Human acellular dermal matrices, which provide an ECM scaffold, therefore, are being used to treat chronic wounds. The ideal human acellular dermal wound matrix (HADWM) would support regenerative healing, providing a structure that could be repopulated by the body's cells. Experienced wound care investigators and clinicians discussed the function of ECM, the evidence related to a specific HADWM (Graftjacket(®) regenerative tissue matrix, Wright Medical Technology, Inc., licensed by KCI USA, Inc., San Antonio, TX), and their clinical experience with this scaffold. This article distills these discussions into an evidence-based and practical overview for treating chronic lower extremity wounds with this HADWM.

  5. Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

    PubMed

    Yoon, Hyung Ho; Min, Joongkee; Shin, Nari; Kim, Yong Hwan; Kim, Jin-Mo; Hwang, Yu-Shik; Suh, Jun-Kyo Francis; Hwang, Onyou; Jeon, Sang Ryong

    2013-05-05

    Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson's disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [(18)F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

  6. Angiogenic Potential and Secretome of Human Apical Papilla Mesenchymal Stem Cells in Various Stress Microenvironments.

    PubMed

    Bakopoulou, Athina; Kritis, Aristeidis; Andreadis, Dimitrios; Papachristou, Eleni; Leyhausen, Gabriele; Koidis, Petros; Geurtsen, Werner; Tsiftsoglou, Asterios

    2015-11-01

    Stem cells from the apical papilla (SCAP) of human adult teeth are considered an accessible source of cells with angiogenic properties. The aims of this study were to investigate the endothelial transdifferentiation of SCAP, the secretion of pro- and antiangiogenic factors from SCAP, and the paracrine effects of SCAP when exposed to environmental stress to stimulate tissue damage. SCAP were exposed to serum deprivation (SD), glucose deprivation (GD), and oxygen deprivation/hypoxia (OD) conditions, individually or in combination. Endothelial transdifferentiation was evaluated by in vitro capillary-like formation assays, real-time polymerase chain reaction, western blot, and flow cytometric analyses of angiogenesis-related markers; secretome by antibody arrays and enzyme-linked immunosorbent assays (ELISA); and paracrine impact on human umbilical vein endothelial cells (HUVECs) by in vitro transwell migration and capillary-like formation assays. The short-term exposure of SCAP to glucose/oxygen deprivation (GOD) in the presence, but mainly in deprivation, of serum (SGOD) elicited a proangiogenesis effect indicated by expression of angiogenesis-related genes involved in vascular endothelial growth factor (VEGF)/VEGFR and angiopoietins/Tie pathways. This effect was unachievable under SD in normoxia, suggesting that the critical microenvironmental condition inducing rapid endothelial shift of SCAP is the combination of SGOD. Interestingly, SCAP showed high adaptability to these adverse conditions, retaining cell viability and acquiring a capillary-forming phenotype. SCAP secreted higher numbers and amounts of pro- (angiogenin, IGFBP-3, VEGF) and lower amounts of antiangiogenic factors (serpin-E1, TIMP-1, TSP-1) under SGOD compared with SOD or SD alone. Finally, secretome obtained under SGOD was most effective in inducing migration and capillary-like formation by HUVECs. These data provide new evidence on the microenvironmental factors favoring endothelial

  7. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    PubMed

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  8. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin

    PubMed Central

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-01-01

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts. PMID:27428951

  9. Cytotoxicity of Various Endodontic Materials on Stem Cells of Human Apical Papilla

    PubMed Central

    Saberi, Eshagh Ali; Karkehabadi, Hamed; Mollashahi, Narges Farhad

    2016-01-01

    Introduction: This in vitro study assessed and compared the cytotoxicity of mineral trioxide aggregate (MTA), calcium-enriched mixture (CEM) cement, Biodentine (BD) and octacalcium phosphate (OCP) on stem cells of the human apical papilla (SCAP). Methods and Materials: SCAPs were isolated from two semi-impacted third molars. The cells were cultured in wells of an insert 24-well plate and were then incubated. The plates were then removed from the incubator and randomly divided into four experimental groups that were exposed to 1-mm discs of set MTA, CEM, BD or OCP, and one untreated control group. After 24, 48 and 168 h, the plates were removed from the incubator and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) solution was added to each well. Data were analyzed at different time points using the repeated measures ANOVA followed by Bonferroni test and the level of significance was set at 0.05. Results: Cytotoxicity of the four materials was not significantly different from that of the control group at 24, 48 and 168 h (P>0.05). Two-by-two comparison revealed that cytotoxicity of MTA and CEM cement was significantly different from each other at 168 h (P<0.05) although the cytotoxicity of CEM was less than MTA. Cytotoxicity of OCP and MTA was also significantly different from each other at 48 h and OCP had more favorable biocompatibility than MTA (P<0.05). Conclusion: CEM, OCP, BD and MTA showed acceptable biocompatibility when exposed to SCAP. Over time, CEM showed the least cytotoxicity among the materials under study. PMID:26843872

  10. Effect of microemulsions on cell viability of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  11. Malathion dermal permeability in relation to dermal load: Assessment by physiologically based pharmacokinetic modeling of in vivo human data.

    PubMed

    Bogen, Kenneth T; Singhal, Ankur

    2017-02-01

    Estimates of dermal permeability (Kp), obtained by fitting an updated human PBPK model for malathion to previously reported data on excreted urinary metabolites after 29 volunteers were dermally exposed to measured values of [(14)C]malathion dermal load (L), were used to examine the empirical relationship between Kp and L. The PBPK model was adapted from previously reported human biokinetic and PBPK models for malathion, fit to previously reported urinary excretion data after oral [(14)C]malathion intake by volunteers, and then augmented to incorporate a standard Kp approach to modeling dermal-uptake kinetics. Good to excellent PBPK-model fits were obtained to all of 29 sets of cumulative urinary metabolite-excretion data (ave. [±1 SD] R(2) = 0.953 [±0.064]). Contrary to the assumption that Kp and L are independent typically applied for dermally administered liquids or solutions, the 29 PBPK-based estimates of Kp obtained for malathion exhibit a strong positive association with the 2/3rds power of L (log-log Pearson correlation = 0.925, p = ∼0). Possible explanations of this observation involving physico-chemical characteristics and/or in vivo cutaneous effects of malathion are discussed. The PBPK model presented, and our observation that Kp estimates obtained by fitting this model to human experimental urinary-excretion data correlate well with L(2/3), allow more realistic assessments of absorbed and metabolized dose during or after a variety of scenarios involving actual or potential dermal or multi-route malathion exposures, including for pesticide workers or farmers who apply malathion to crops.

  12. Stress responses of human dermal fibroblasts exposed to zinc pyrithione.

    PubMed

    Rudolf, Emil; Cervinka, Miroslav

    2011-07-28

    Zinc pyrithione is used as a topical agent in a range of medicinal and cosmetic applications. Despite its extensive use and reported beneficial effects in treatment of various dermal problems, its potential toxicity towards skin cells remains relatively underexplored. In this work we investigated effects of nM zinc pyrithione on cell stress response pathways of primary human skin fibroblasts during 24h of exposure. We demonstrate that zinc pyrithione-induced cytotoxity in dermal fibroblasts is dose-dependent and it associates with increased intracellular zinc concentrations and activated stress response pathways including p53 and stress kinase p38. Higher zinc pyrithione concentrations (500nM and above) stimulate oxidative stress and moderate DNA damage which occur in the presence of activated p38 kinase. Cells further upregulate the expression of p53 which increases its transcriptional activity while mitogenic signaling exemplified by mTOR (mammalian target of rapamycin) expression is suppressed and these steps lead to mitochondrial, caspase-dependent apoptosis. Conversely, lower zinc concentrations (125nM) fail to induce oxidative stress and significant DNA damage; however, treated cells still activate p38 and upregulate the expression and transcriptional activity of p53 and its target gene p21 as well as the expression of p16 in the presence of active mTOR pathway and a changed DNA methylation pattern. The end result is premature senescence phenotype. Specific pharmacological inhibitors as well as gene knockdown technology prove that an interaction between p38, p53 and mTOR might be responsible for these observed endpoints. Taken together, exposure of dermal fibroblasts to varying concentrations of zinc pyrithione may result in either cell death-apoptosis or cellular premature senescence which attests to the ability of this compound to affect this type of cells in an in vitro model system.

  13. In vitro dermal absorption of pyrethroid pesticides in human and rat skin

    EPA Science Inventory

    Dermal exposure to pyrethroid pesticides can occur during manufacture and application. This study examined the in vitro dermal absorption of pyrethroids using rat and human skin. Dermatomed skin from adult male Long Evans rats or human cadavers was mounted in flowthrough diffusi...

  14. In vitro dermal absorption of pyrethroid pesticides in human and rat skin

    EPA Science Inventory

    Dermal exposure to pyrethroid pesticides can occur during manufacture and application. This study examined the in vitro dermal absorption of pyrethroids using rat and human skin. Dermatomed skin from adult male Long Evans rats or human cadavers was mounted in flowthrough diffusi...

  15. Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma.

    PubMed

    Chan, E S L; Fernandez, P; Merchant, A A; Montesinos, M C; Trzaska, S; Desai, A; Tung, C F; Khoa, D N; Pillinger, M H; Reiss, A B; Tomic-Canic, M; Chen, J F; Schwarzschild, M A; Cronstein, B N

    2006-08-01

    Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor-deficient and A2A receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.

  16. Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

    PubMed

    Bikfalvi, A; Cramer, E M; Tenza, D; Tobelem, G

    1991-01-01

    Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.

  17. IN VITRO DERMAL ABSORPTION OF PYRETHROID PESTICIDES IN RAT AND HUMAN SKIN

    EPA Science Inventory

    Pyrethriods are a class of neurotoxic pesticides and their use may lead to dermal exposure. This study examined the in vitro dermal absorption of pyrethroids in rat and human skin. Dorsal skin removed from adult male LD rats (hair clipped 24 h previously) was dermatomed and mou...

  18. IN VITRO DERMAL ABSORPTION OF PYRETHROID PESTICIDES IN RAT AND HUMAN SKIN

    EPA Science Inventory

    Pyrethriods are a class of neurotoxic pesticides and their use may lead to dermal exposure. This study examined the in vitro dermal absorption of pyrethroids in rat and human skin. Dorsal skin removed from adult male LD rats (hair clipped 24 h previously) was dermatomed and mou...

  19. A new model for preclinical testing of dermal substitutes for human skin reconstruction.

    PubMed

    Hartmann-Fritsch, Fabienne; Biedermann, Thomas; Braziulis, Erik; Meuli, Martin; Reichmann, Ernst

    2013-05-01

    Currently, acellular dermal substitutes used for skin reconstruction are usually covered with split-thickness skin grafts. The goal of this study was to develop an animal model in which such dermal substitutes can be tested under standardized conditions using a bioengineered dermo-epidermal skin graft for coverage. Bioengineered grafts consisting of collagen type I hydrogels with incorporated human fibroblasts and human keratinocytes seeded on these gels were produced. Two different dermal substitutes, namely Matriderm(®), and an acellular collagen type I hydrogel, were applied onto full-thickness skin wounds created on the back of immuno-incompetent rats. As control, no dermal substitute was used. As coverage for the dermal substitutes either the bioengineered grafts were used, or, as controls, human split-thickness skin or neonatal rat epidermis were used. Grafts were excised 21 days post-transplantation. Histology and immunofluorescence was performed to investigate survival, epidermis formation, and vascularization of the grafts. The bioengineered grafts survived on all tested dermal substitutes. Epidermis formation and vascularization were comparable to the controls. We could successfully use human bioengineered grafts to test different dermal substitutes. This novel model can be used to investigate newly designed dermal substitutes in detail and in a standardized way.

  20. Mycoplasma detection and elimination are necessary for the application of stem cell from human dental apical papilla to tissue engineering and regenerative medicine.

    PubMed

    Kim, Byung-Chul; Kim, So Yeon; Kwon, Yong-Dae; Choe, Sung Chul; Han, Dong-Wook; Hwang, Yu-Shik

    2015-01-01

    Recently, postnatal stem cells from dental papilla with neural crest origin have been considered as one of potent stem cell sources in regenerative medicine regarding their multi-differentiation capacity and relatively easy access. However, almost human oral tissues have been reported to be infected by mycoplasma which gives rise to oral cavity in teeth, and mycoplasma contamination of ex-vivo cultured stem cells from such dental tissues and its effect on stem cell culture has received little attention. In this study, mycoplama contamination was evaluated with stem cells from apical papilla which were isolated from human third molar and premolars from various aged patients undergoing orthodontic therapy. The ex-vivo expanded stem cells from apical papilla were found to express stem cell markers such as Stro-1, CD44, nestin and CD133, but mycoplama contamination was detected in almost all cell cultures of the tested 20 samples, which was confirmed by mycoplasma-specific gene expression and fluorescence staining. Such contaminated mycoplasma could be successfully eliminated using elimination kit, and proliferation test showed decreased proliferation activity in mycoplasma-contaminated cells. After elimination of contaminated mycoplasma, stem cells from apical papilla showed osteogenic and neural lineage differentiation under certain culture conditions. Our study proposes that the evaluation of mycoplasma contamination and elimination process might be required in the use of stem cells from apical papilla for their potent applications to tissue engineering and regenerative medicine.

  1. Comparing Manual Counting to Automated Image Analysis for the Assessment of Fungiform Papillae Density on Human Tongue.

    PubMed

    Piochi, Maria; Monteleone, Erminio; Torri, Luisa; Masi, Camilla; Almli, Valérie Lengard; Wold, Jens Petter; Dinnella, Caterina

    2017-09-01

    The density of fungiform papillae (FPD) on the human tongue is currently taken as index for responsiveness to oral chemosensory stimuli. Visual analysis of digital tongue picture and manual counting by trained operators represents the most popular technique for FPD assessment. Methodological issues mainly due to operator bias are considered among factors accounting for the uncertainty about the relationships between FPD and responsiveness to chemosensory stimuli. The present study describes a novel automated method to count fungiform papillae (FP) from image analysis of tongue pictures. The method was applied to tongue pictures from 133 subjects. Taking the manual count as reference method, a partial least squares regression model was developed to predict FPD from tongue automated analysis output. FPD from manual and automated count showed the same normal distribution and comparable descriptive statistic values. Consistent subject classifications as low and high FPD were obtained according to the median values from manual and automated count. The same results on the effect of FPD variation on taste perception were obtained both using predicted and counted values. The proposed method overcomes count uncertainties due to researcher bias in manual counting and is suited for large population studies. Additional information is provided such as FP size class distribution which would help for a better understanding of the relationships between FPD variation and taste functions. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Hormone-responsive cells derived from human dental papilla: characterization in vitro and in vivo in diffusion chambers.

    PubMed

    Shteyer, A; Gazit, D; Binderman, I; Bab, I A

    1987-01-01

    Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E2 there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.

  3. Human dermal exposure to galaxolide from personal care products.

    PubMed

    Correia, P; Cruz, A; Santos, L; Alves, A

    2013-06-01

    Musks are synthetic fragrances applied on personal care and household products as fixatives, by retarding the release of other fragrances with higher volatility. Galaxolide is the most used polycyclic musk since the 90th decade, and it has been detected in several environmental and biological matrices, particularly in human tissues and fluids. For exposure assessment purposes, large-monitoring data need to be obtained and rapid but reliable analytical techniques are requested. The main objective of this study is to develop and validate a new and fast analytical methodology to quantify galaxolide in personal care products and to apply this method to real matrices like skin care products (creams and lotions), shower products (soap bar), hair care products (shampoo and hair conditioner) and oral care products (toothpaste), to evaluate the human dermal exposure risk. A dispersive solid-phase extraction is proposed, using QuEChERS methodology, followed by HPLC with fluorescence detection. Some extraction parameters were studied, like the ratio of sample/solvent amounts, the homogenization time, the salt addition effect and the used sorbents. The validation parameters of the developed method were the following: a linearity range of 0.005-1.002 mg kg⁻¹ sample, a limit of detection of 0.001 mg kg⁻¹ sample, repeatability between 0.7% and 11.3% (variation coefficient of six standard injections), an intermediate precision of 2.5% (variation coefficient of six independent analysis of the same sample), mean recoveries ranging from 65% (soap bar) to 95% (body cream) and 3% of global uncertainty in most of the working range. The time of analysis, including the extraction steps, is 60 min, allowing a throughput of 4 samples h⁻¹ . Galaxolide was detected in all of the seven analysed products in concentrations ranging from 0.04 ± 0.01 mg kg⁻¹ sample (toothpaste) to 280.78 ± 8.19 mg kg⁻¹ sample (perfumed body cream), which may correspond to a significant estimated

  4. Dermal penetration potential of perfluorooctanoic acid (PFOA) in human and mouse skin.

    PubMed

    Franko, Jennifer; Meade, B J; Frasch, H Frederick; Barbero, Ana M; Anderson, Stacey E

    2012-01-01

    Recent data, using a murine model, have indicated that dermal exposure to perfluorooctanoic acid (PFOA) induces immune modulation, suggesting that this may be an important route of PFOA exposure. To investigate the dermal penetration potential of PFOA, serum concentrations were analyzed in mice following topical application. Statistically significant and dose-responsive increases in serum PFOA concentrations were identified. In vitro dermal penetration studies also demonstrated that PFOA permeates both mouse and human skin. Investigation into the mechanisms mediating PFOA penetration demonstrated that dermal absorption was strongly dependent upon the ionization status of PFOA. In addition, PFOA solid, but not 1% PFOA/acetone solution, was identified as corrosive using a cultured epidermis in vitro model. Despite its corrosive potential, expression of inflammatory cytokines in the skin of topically exposed mice was not altered. These data suggest that PFOA is dermally absorbed and that under certain conditions the skin may be a significant route of exposure.

  5. Human dermal absorption of chlorinated organophosphate flame retardants; implications for human exposure.

    PubMed

    Abou-Elwafa Abdallah, Mohamed; Pawar, Gopal; Harrad, Stuart

    2016-01-15

    Tris-2-chloroethyl phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCIPP) and tris-1,3-dichloropropyl phosphate (TDCIPP) are organophosphate flame retardants (PFRs) widely applied in a plethora of consumer products despite their carcinogenic potential. Human dermal absorption of these PFRs is investigated for the first time using human ex vivo skin and EPISKIN™ models. Results of human ex vivo skin experiments revealed 28%, 25% and 13% absorption of the applied dose (500 ng/cm(2), finite dose) of TCEP, TCIPP and TDCIPP, respectively after 24h exposure. The EPISKIN™ model showed enhanced permeability values (i.e. weaker barrier), that were respectively 16%, 11% and 9% for TCEP, TCIPP and TDCIPP compared to human ex vivo skin. However, this difference was not significant (P>0.05). Estimated permeability constants (Kp, cm/h) showed a significant negative correlation with log Kow for the studied contaminants. The effect of hand-washing on dermal absorption of PFRs was investigated. Washing reduced overall dermal absorption, albeit to varying degrees depending on the physicochemical properties of the target PFRs. Moreover, slight variations of the absorbed dose were observed upon changing the dosing solution from acetone to 20% Tween 80 in water, indicating the potential influence of the dose vehicle on the dermal absorption of PFRs. Finally, estimated dermal uptake of the studied PFRs via contact with indoor dust was higher in UK toddlers (median ΣPFRs=36 ng/kg bw day) than adults (median ΣPFRs=4 ng/kg bw day). More research is required to fully elucidate the toxicological implications of such exposure.

  6. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    SciTech Connect

    Fan, Rong-hui; Zhu, Xiu-mei; Sun, Yao-wen; Peng, Hui-zi; Wu, Hang-li; Gao, Wen-jie

    2016-07-08

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  7. Endogenous gustatory responses and gene expression profile of stably proliferating human taste cells isolated from fungiform papillae.

    PubMed

    Hochheimer, Andreas; Krohn, Michael; Rudert, Kerstin; Riedel, Katja; Becker, Sven; Thirion, Christian; Zinke, Holger

    2014-05-01

    Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities.

  8. Increased human dermal microvascular endothelial cell survival induced by cysteamine.

    PubMed

    Besouw, M; van den Heuvel, L; van Eijsden, R; Bongaers, I; Kluijtmans, L; Dewerchin, M; Levtchenko, E

    2013-11-01

    Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC). After cysteamine exposure (range 0-3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0-10.0 mM). HDMVEC viability and proliferation increased after cysteamine exposure 0.03-3.0 mM (p < 0.01) and 0.03-1.0 mM (p = 0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0-1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p = 0.02) and 1.0 mM (p < 0.01). HDMVEC viability increased after exposure to GSH 1.0-5.0 mM (p < 0.01). Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.

  9. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  10. LPS-Stimulated Human Skin-Derived Stem Cells Enhance Neo-Vascularization during Dermal Regeneration

    PubMed Central

    Rapoport, Daniel H.; Kruse, Charli; Schumann, Sandra; Stang, Felix H.; Siemers, Frank; Matthießen, Anna E.

    2015-01-01

    High numbers of adult stem cells are still required to improve the formation of new vessels in scaffolds to accelerate dermal regeneration. Recent data indicate a benefit for vascularization capacity by stimulating stem cells with lipopolysaccharide (LPS). In this study, stem cells derived from human skin (SDSC) were activated with LPS and seeded in a commercially available dermal substitute to examine vascularization in vivo. Besides, in vitro assays were performed to evaluate angiogenic factor release and tube formation ability. Results showed that LPS-activated SDSC significantly enhanced vascularization of the scaffolds, compared to unstimulated stem cells in vivo. Further, in vitro assays confirmed higher secretion rates of proangiogenic as well as proinflammatoric factors in the presence of LPS-activated SDSC. Our results suggest that combining activated stem cells and a dermal substitute is a promising option to enhance vascularization in scaffold-mediated dermal regeneration. PMID:26565617

  11. The fate of dermally applied [14C]d-limonene in rats and humans.

    PubMed

    Api, Anne Marie; Ritacco, Gretchen; Hawkins, David R

    2013-01-01

    The fate of dermally applied [(14)C]d-limonene was evaluated in humans and Long-Evans rats. In rats, 5 mg/kg body weight of [(14)C]d-limonene applied dermally to the shaved back under occlusion, resulted in the absorption of approximately 12% of the dose. The absorbed d-limonene was completely metabolized and excreted rapidly, primarily from the urine (80%) with a small fraction (20%) excreted in the feces. There was no long-term retention of the test material in body tissues. In humans, following dermal application of 12 mg of [(14)C]d-limonene in ethanol (1 mL) to the back under nonocclusive conditions (for 1 h after application to allow the material to dry, thereafter under occlusion), only 0.16% of the dose was absorbed and the radioactivity was recovered from the urine. Radioactivity in human feces was below the limit of detection. These results indicate that under conditions of simulated use of fragrances and cosmetics, d-limonene has a low potential for dermal absorption and tissue accumulation, and the d-limonene that is absorbed is rapidly excreted in the urine. Based upon these findings and the knowledge that d-limonene possesses a low-systemic toxicity profile, it is reasonable to conclude that dermal exposure to d-limonene from fragrance and cosmetic applications is highly unlikely to result in any clinically significant human toxicity.

  12. NOTCH3 is expressed in human apical papilla and in subpopulations of stem cells isolated from the tissue

    PubMed Central

    Jamal, Mohamed; Chogle, Sami M.; Karam, Sherif M.; Huang, George T.-J.

    2015-01-01

    NOTCH plays a role in regulating stem cell function and fate decision. It is involved in tooth development and injury repair. Information regarding NOTCH expression in human dental root apical papilla (AP) and its residing stem cells (SCAP) is limited. Here we investigated the expression of NOTCH3, its ligand JAG1, and mesenchymal stem cell markers CD146 and STRO-1 in the AP or in the primary cultures of SCAP isolated from AP. Our in situ immunostaining showed that in the AP NOTCH3 and CD146 were co-expressed and associated with blood vessels having NOTCH3 located more peripherally. In cultured SCAP, NOTCH3 and JAG1 were co-expressed. Flow cytometry analysis showed that 7%, 16% and 98% of the isolated SCAP were positive for NOTCH3, STRO-1 and CD146, respectively with a rare 1.5% subpopulation of SCAP co-expressing all three markers. The expression level of NOTCH3 reduced when SCAP underwent osteogenic differentiation. Our findings are the first step towards defining the regulatory role of NOTCH3 in SCAP fate decision. PMID:26989760

  13. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide.

    PubMed

    Feng, Bing; Ma, Lai-ji; Yao, Jin-jing; Fang, Yun; Mei, Yan-ai; Wei, Shao-min

    2013-02-01

    Oat contains different components that possess antioxidant properties; no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level. Therefore, the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide (H(2)O(2)). Kjeldahl determination, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%, of which 97.43% are peptides with a molecular range from 438.56 to 1301.01 Da. Assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay for apoptosis showed that administration of H(2)O(2) in human dermal fibroblasts caused cell damage and apoptosis. Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H(2)O(2), but application oat peptides with H(2)O(2) at same time did not. Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H(2)O(2)-induced decrease of superoxide dismutase (SOD) and the inhibition of malondialdehyde (MDA). The results demonstrate that oat peptides possess antioxidant activity and are effective against H(2)O(2)-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level. Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury.

  14. Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability.

    PubMed

    Allen, Justine J; Bell, George R R; Kuzirian, Alan M; Hanlon, Roger T

    2013-06-01

    Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three-dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer

  15. Triiodothyronine (T3) inhibits hyaluronate synthesis in a human dermal equivalent by downregulation of HAS2.

    PubMed

    Pouyani, Tara; Sadaka, Basma H; Papp, Suzanne; Schaffer, Lana

    2013-03-01

    Triiodothyronine (T3) is a thyroid hormone that can have varying effects on skin. In order to assess the effects of T3 on the human dermis, we prepared dermal equivalents using neonatal dermal cells via the process of self-assembly in the presence of differing concentrations of T3. These dermal equivalents were prepared in the absence of serum and a three dimensional matrix allowing for the direct assessment of different concentrations of T3 on dermal extracellular matrix formation. Three different concentrations of T3 were chosen, 20 pM, which is part of the base medium, 0.2 nM T3 and 2 nM T3. We find that self-assembled dermal equivalents formed under these conditions show a progressive "thinning" with increasing T3 concentrations. While we observed no change in total collagen content, inhibition of hyaluronate (HA) synthesis was observed in the 0.2- and 2-nM T3 constructs as compared to the 20-pM construct. Other glycosaminoglycan synthesis was not affected by increasing T3 concentrations. In order to identify the gene(s) responsible for inhibition of HA synthesis in the 2-nM T3 dermal equivalent, we conducted a differential gene array analysis. The results of these experiments demonstrate the differential expression of 40 genes, of these, 34 were upregulated and 6 genes were downregulated. The results from these experiments suggest that downregulation of HAS2 may be responsible for inhibition of hyaluronate synthesis in the self-assembled 2-nM T3 human dermal matrix.

  16. Coverage of Megaprosthesis with Human Acellular Dermal Matrix after Ewing's Sarcoma Resection: A Case Report.

    PubMed

    Whitfield, Robert M; Rinard, Jeremy; King, David

    2011-01-01

    A 23-year-old female with Ewing's Sarcoma underwent tibial resection and skeletal reconstruction using proximal tibial allograft prosthetic reconstruction with distal femur endoprosthetic reconstruction and rotating hinge. Human acellular dermal matrix, (Alloderm, LifeCell, Branchburg, NJ, USA), was used to wrap the skeletal reconstruction. Soft tissue reconstruction was completed with a rotational gastrocnemius muscle flap and skin graft. Despite prolonged immobilization, the patient quickly regained full range of motion of her skeletal reconstruction. Synthetic mesh, tapes and tubes are used to perform capsule reconstruction of megaprosthesis. This paper describes the role of human acellular dermal matrix in capsule reconstruction around a megaprosthesis.

  17. Nanocrystalline titanium dioxide and magnesium oxide in vitro dermal absorption in human skin.

    PubMed

    van der Merwe, Deon; Tawde, Snehal; Pickrell, John A; Erickson, Larry E

    2009-01-01

    The dermal absorption potential of a nanocrystalline magnesium oxide (MgO) and titanium dioxide (TiO(2)) mixture in dermatomed human skin was assessed in vitro using Bronaugh-type flow-through diffusion cells. Nanocrystalline material was applied to the skin surface at a dose rate of 50 mg/cm(2) as a dry powder, as a water suspension, and as a water/surfactant (sodium lauryl sulfate) suspension, for 8 hours. Dermal absorption of nanocrystalline MgO and TiO(2) through human skin with intact, functional stratum corneum was not detectable under the conditions of this experiment.

  18. [Elaboration of biodegradable polymer substrate for cultivation of human dermal fibroblasts].

    PubMed

    Shved, Iu A; Kukhareva, L V; Zorin, I M; Solov'ev, A Iu; Blinova, M I; Bilibin, A Iu; Pinaev, G P

    2006-01-01

    The influence of polylactic acid (PLA) surface films on the pattern of cell behavior was studied. The human dermal fibroblasts were cultivated on PLA covered glasses. The hydrophobic nature of PLA films depends on the availability of polymer solvent in the film preparation. PLA films obtained from a more polar solvent--aceton--appeared to be more hydrophilic than those obtained from methylene chloride. More hydrophilic polymer films also appeared to be more preferable for cell cultivation, and human dermal fibroblasts demonstrated a better adhesion and proliferation on hydrophilic rather than on hydrophobic PLA films.

  19. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging

    PubMed Central

    Quan, Taihao; Fisher, Gary J

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment (AADM), which contributes to decline of human skin function. PMID:25660807

  20. Effect of topical application of raspberry ketone on dermal production of insulin-like growth factor-I in mice and on hair growth and skin elasticity in humans.

    PubMed

    Harada, Naoaki; Okajima, Kenji; Narimatsu, Noriko; Kurihara, Hiroki; Nakagata, Naomi

    2008-08-01

    Sensory neurons release calcitonin gene-related peptide (CGRP) on activation. We recently reported that topical application of capsaicin increases facial skin elasticity and promotes hair growth by increasing dermal insulin-like growth factor-I (IGF-I) production through activation of sensory neurons in mice and humans. Raspberry ketone (RK), a major aromatic compound contained in red raspberries (Rubus idaeus), has a structure similar to that of capsaicin. Thus, it is possible that RK activates sensory neurons, thereby increasing skin elasticity and promoting hair growth by increasing dermal IGF-I production. In the present study, we examined this possibility in mice and humans. RK, at concentrations higher than 1 microM, significantly increased CGRP release from dorsal root ganglion neurons (DRG) isolated from wild-type (WT) mice and this increase was completely reversed by capsazepine, an inhibitor of vanilloid receptor-1 activation. Topical application of 0.01% RK increased dermal IGF-I levels at 30 min after application in WT mice, but not in CGRP-knockout mice. Topical application of 0.01% RK increased immunohistochemical expression of IGF-I at dermal papillae in hair follicles and promoted hair re-growth in WT mice at 4 weeks after the application. When applied topically to the scalp and facial skin, 0.01% RK promoted hair growth in 50.0% of humans with alopecia (n=10) at 5 months after application and increased cheek skin elasticity at 2 weeks after application in 5 females (p<0.04). These observations strongly suggest that RK might increase dermal IGF-I production through sensory neuron activation, thereby promoting hair growth and increasing skin elasticity.

  1. Oral and dermal pharmacokinetics of triclopyr in human volunteers.

    PubMed

    Carmichael, N G; Nolan, R J; Perkins, J M; Davies, R; Warrington, S J

    1989-11-01

    Blood levels and urinary excretion of triclopyr, the active ingredient in Garlon herbicides, were followed in six volunteers given single oral doses of 0.1 and 0.5 mg/kg body weight. Five of these volunteers later received dermal applications of Garlon 4 herbicide formulation equivalent to 3.7 mg triclopyr/kg body weight applied to the forearm. Following oral administration blood levels peaked at 2-3 h and declined to undetectable levels within 48 h; more than 80% of the dose was found as unchanged triclopyr in the urine. A two-compartment pharmacokinetic model was used to describe the time-course of triclopyr clearance; half-lives for the rapid initial and slower terminal phases were 1.3 h and 5.1 h respectively, and were independent of dose. Due to the slow half-life for dermal absorption (t1/2 = 16.8 h) the rapid initial elimination phase was obscured and the pharmacokinetics could be simplified by a one-compartment model. An average of 1.37% of the applied dose was recovered in the urine; when corrected for recovery after oral administration this was equivalent to an absorption of 1.65%. Triclopyr is slowly absorbed through skin and is rapidly eliminated. It has very low potential to accumulate in man or to be absorbed through the skin in acutely toxic amounts.

  2. The exclusion of human serum albumin by human dermal collagenous fibres and within human dermis.

    PubMed Central

    Bert, J L; Mathieson, J M; Pearce, R H

    1982-01-01

    Preparations of dermal collagenous fibres and slices of human dermis have been equilibrated with 125I-labelled monomeric human serum albumin. The space inaccessible to the albumin in the fibres and in the dermis was determined by subtraction of the accessible space, calculated from the radioactivity of the specimen, from its total fluid. For a fibre preparation examined in detail, the fluid exclusion was independent of the concentration of either albumin or collagen. Binding of albumin to the fibres was not demonstrable. Three fibre preparations excluded albumin from 3.75 +/- 0.96, 3.55 +/- 0.67, and 2.05 +/- 0.39 g of fluid/g of collagen (+/-S.D.). Slices from three specimens of dermis excluded albumin from 1.45 +/- 0.08 g of fluid/g of insoluble solids or 1.57 +/- 0.11 g of fluid/g of collagen (+/-S.D.). Thus the exclusion of albumin by dermis was much less than expected from its content of collagenous fibres. On the basis of these data and the published composition of dermis, the concentration of albumin in the accessible interstitial space was estimated to be close to that in the plasma. PMID:7082298

  3. Evaluation of Cytotoxic Effects of Various Endodontic Irrigation Solutions on the Survival of Stem Cell of Human Apical Papilla

    PubMed Central

    Farhad Mollashahi, Narges; Saberi, Eshaghali; Karkehabadi, Hamed

    2016-01-01

    Introduction: Root canal disinfection is an important step in regenerative endodontic treatments. An ideal irrigating solution must have high antimicrobial activity and minimum cytotoxicity. This study sought to assess the effect of some irrigating solutions on stem cells from the human apical papilla (SCAP) after different periods of exposure. Methods and Materials: Stem cells were isolated from immature, impacted mandibular third molars, transferred to 24-well plates, randomly divided into 6 experimental groups and exposed to BioPure MTAD Cleanser, QMix, 17% EDTA, 2% chlorhexidine (CHX), 5.25% sodium hypochlorite (NaOCl), sterile saline and untreated control group. Cytotoxicity of these solutions was assessed after 1, 5 and 15 min of exposure using the methyl thiazol tetrazolium (MTT) assay. Data were statistically analyzed using repeated measures ANOVA. Level of significance was set at 0.05. Results: The mean percentage of viable cells in all experimental groups was significantly different from the control and sterile saline groups at all the time points (P<0.0001). The mean percentage of viable cells significantly decreased over time in NaOCl, QMix, EDTA and MTAD groups, but no significant reduction was noted in CHX group. At all the time points the highest and the lowest cytotoxicity were seen in MTAD and normal saline groups, respectively. Cytotoxicity of the understudy materials from the highest to the lowest was as follows: MTAD>EDTA>QMax=NaOCl>CHX> sterile saline. Conclusion: Chlorhexidine had the lowest cytotoxicity compared to EDTA, MTAD, QMix and NaOCl and its cytotoxicity did not change over time compared to other solutions. PMID:27790258

  4. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  5. A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.

    PubMed

    Wang, Xiao-Nong; McGovern, Naomi; Gunawan, Merry; Richardson, Connor; Windebank, Martin; Siah, Tee-Wei; Lim, Hwee-Ying; Fink, Katja; Li, Jackson L Yao; Ng, Lai G; Ginhoux, Florent; Angeli, Veronique; Collin, Matthew; Haniffa, Muzlifah

    2014-04-01

    Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 μm beneath the dermoepidermal junction), Mφ were located deeper (40-60 μm), and CD3(+) lymphocytes were observed throughout (0-60 μm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 μm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

  6. Kinetics of 3-(4-methylbenzylidene)camphor in rats and humans after dermal application

    SciTech Connect

    Schauer, Ute M.D.; Voelkel, Wolfgang; Heusener, Alexander; Colnot, Thomas; Broschard, Thomas H.; Landenberg, Friedrich von; Dekant, Wolfgang . E-mail: dekant@toxi.uni-wuerzburg.de

    2006-10-15

    The toxicokinetics of 4-MBC after dermal administration were investigated in human subjects and in rats. Humans (3 male and 3 female subjects) were exposed to 4-MBC by topical application of a commercial sunscreen formulation containing 4% 4-MBC (w/w), covering 90% of the body surface and resulting in a mean dermal 4-MBC dose of 22 mg/kg bw. In rats, dermal 4-MBC doses of 400 and 2000 mg/kg bw were applied in a formulation using an occlusive patch for 24 h. Concentrations of 4-MBC and its metabolites were monitored over 96 h in plasma (rats and humans) and urine (humans). In human subjects, plasma levels of 4-MBC peaked at 200 pmol/ml in males and 100 pmol/ml in females 6 h after application and then decreased to reach the limit of detection after 24 h (females), respectively, 36 h (males). After dermal application of 4-MBC, peak plasma concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were 50-80 pmol/ml at 12 h and of 3-(4-carboxybenzylidene)camphor were 100-200 pmol/ml at 24 h. In male and female rats, peak plasma levels of 4-MBC were 200 (dose of 400 mg/kg bw) and 1 200 pmol/ml (dose of 2000 mg/kg bw). These levels remained constant for up to 24-48 h after dermal application. Peak plasma concentrations of 3-(4-carboxybenzylidene)-6-hydroxycamphor were 18,000 pmol/ml (males) and of 3-(4-carboxybenzylidene)camphor were 55,000 pmol/ml (females) between 48 and 72 h after application of the high dose of 4-MBC. In human subjects, only a small percentage of the dermally applied dose of 4-MBC was recovered in the form of metabolites in urine, partly as glucuronides. The obtained results suggest a more intensive biotransformation of 4-MBC in rats as compared to humans after dermal application and a poor absorption of 4-MBC through human skin.

  7. Comparative Host Response of 2 Human Acellular Dermal Matrices in a Primate Implant Model

    PubMed Central

    Sandor, Maryellen; Singh, Devinder; Silverman, Ronald P.; Xu, Hui; De Deyne, Patrick G.

    2014-01-01

    Objective: We examined the differences in capsule formation between 2 commercially available human acellular dermal matrices in a nonhuman primate model. Methods: Primates were implanted dorsally with a subcutaneously placed tissue expander and randomized into 3 groups, receiving skin coverage only, coverage with non-irradiated freeze-dried human acellular dermal matrix, or coverage with gamma-irradiated human acellular dermal matrix. After 9 weeks, soft tissue around the tissue expander was excised and evaluated qualitatively and quantitatively to assess extent of inflammation (CD68 antibodies and interleukin-6 levels), degradation and fibrosis (matrix metalloproteinase-1 and procollagen-1 staining), and mechanical (tensile) strength. Results: Histological evaluation of tissue around the tissue expander indicated differences in host response, suggesting capsule presence in the gamma-irradiated matrix group but not the freeze-dried matrix group. The extent of local inflammation was much higher in the gamma-irradiated matrix group which demonstrated mean (standard deviation) localized interleukin-6 concentration of 67.3 (53.6) vs 16.3 (6.7) pg/mg protein in the non-irradiated matrix group. There was robust degradation and fibrotic response in the gamma-irradiated matrix group versus the freeze-dried matrix group. Mechanical testing indicated mean (standard deviation) ultimate tensile strength of 12.0 (7.1) N in the gamma-irradiated matrix group versus 99.3 (48.8) N in the freeze-dried matrix group. Conclusions: Enclosure of a tissue expander with human acellular dermal matrix untreated by gamma irradiation led to minimal inflammation and minimal evidence of fibrosis/capsule around the tissue expander compared with robust capsule formation around the tissue expander that was covered by a gamma-irradiated human acellular dermal matrix. PMID:24570768

  8. Ultimate tensile failure loads of a human dermal allograft rotator cuff augmentation.

    PubMed

    Barber, F Alan; Herbert, Morley A; Boothby, Michael H

    2008-01-01

    The purpose of this study was to examine the failure mode of supraspinatus tendon repairs with and without human dermal allograft augmentation. Ten matched pairs of human cadaveric supraspinatus muscles and tendons were detached from their greater tuberosity insertions and then reattached with four simple sutures in 2 suture anchors as a control group. One shoulder from each matched pair was augmented with human dermal allograft secured to the humerus and the supraspinatus tendon using the same sutures and suture anchors. Additional interrupted mattress sutures secured the edges of the dermal allograft to the supraspinatus tendon. Each construct was preloaded at 10 N and then cyclically loaded between 10 N and 100 N for 10 cycles at 20 N/s followed by destructive testing at 33 mm/s. Force and displacement were recorded. The mean failure strengths for the control and augmented constructs were 273 +/- 116 N and 325 +/- 74 N, respectively (P = .047). No significant displacement occurred during the cyclic phase, and no anchors failed. These constructs failed by 2 different mechanisms: tendon-suture interface failure (8/10 non-augmented repairs and 6/10 augmented repairs) and suture breakage (2/10 non-augmented repairs and 4/10 augmented repairs). This examination of the failure characteristics and ultimate failure load of supraspinatus tendon tears augmented with GraftJacket (Wright Medical Technology, Arlington, TN) supported the study hypothesis that a human dermal allograft significantly increases the strength of a repaired tendon. The human dermal allograft can be expected to significantly increase the initial strength of a rotator cuff repair.

  9. Improved epidermal barrier formation in human skin models by chitosan modulated dermal matrices

    PubMed Central

    Mieremet, Arnout; Rietveld, Marion; Absalah, Samira; van Smeden, Jeroen

    2017-01-01

    Full thickness human skin models (FTMs) contain an epidermal and a dermal equivalent. The latter is composed of a collagen dermal matrix which harbours fibroblasts. Current epidermal barrier properties of FTMs do not fully resemble that of native human skin (NHS), which makes these human skin models less suitable for barrier related studies. To further enhance the resemblance of NHS for epidermal morphogenesis and barrier formation, we modulated the collagen dermal matrix with the biocompatible polymer chitosan. Herein, we report that these collagen-chitosan FTMs (CC-FTMs) possess a well-organized epidermis and maintain both the early and late differentiation programs as in FTMs. Distinctively, the epidermal cell activation is reduced in CC-FTMs to levels observed in NHS. Dermal-epidermal interactions are functional in both FTM types, based on the formation of the basement membrane. Evaluation of the barrier structure by the organization of the extracellular lipid matrix of the stratum corneum revealed an elongated repeat distance of the long periodicity phase. The ceramide composition exhibited a higher resemblance of the NHS, based on the carbon chain-length distribution and subclass profile. The inside-out barrier functionality indicated by the transepidermal water loss is significantly improved in the CC-FTMs. The expression of epidermal barrier lipid processing enzymes is marginally affected, although more restricted to a single granular layer. The novel CC-FTM resembles the NHS more closely, which makes them a promising tool for epidermal barrier related studies. PMID:28333992

  10. Improved epidermal barrier formation in human skin models by chitosan modulated dermal matrices.

    PubMed

    Mieremet, Arnout; Rietveld, Marion; Absalah, Samira; van Smeden, Jeroen; Bouwstra, Joke A; El Ghalbzouri, Abdoelwaheb

    2017-01-01

    Full thickness human skin models (FTMs) contain an epidermal and a dermal equivalent. The latter is composed of a collagen dermal matrix which harbours fibroblasts. Current epidermal barrier properties of FTMs do not fully resemble that of native human skin (NHS), which makes these human skin models less suitable for barrier related studies. To further enhance the resemblance of NHS for epidermal morphogenesis and barrier formation, we modulated the collagen dermal matrix with the biocompatible polymer chitosan. Herein, we report that these collagen-chitosan FTMs (CC-FTMs) possess a well-organized epidermis and maintain both the early and late differentiation programs as in FTMs. Distinctively, the epidermal cell activation is reduced in CC-FTMs to levels observed in NHS. Dermal-epidermal interactions are functional in both FTM types, based on the formation of the basement membrane. Evaluation of the barrier structure by the organization of the extracellular lipid matrix of the stratum corneum revealed an elongated repeat distance of the long periodicity phase. The ceramide composition exhibited a higher resemblance of the NHS, based on the carbon chain-length distribution and subclass profile. The inside-out barrier functionality indicated by the transepidermal water loss is significantly improved in the CC-FTMs. The expression of epidermal barrier lipid processing enzymes is marginally affected, although more restricted to a single granular layer. The novel CC-FTM resembles the NHS more closely, which makes them a promising tool for epidermal barrier related studies.

  11. Cytotoxic evaluation of biomechanically improved crosslinked ovine collagen on human dermal fibroblasts.

    PubMed

    Awang, M A; Firdaus, M A B; Busra, M B; Chowdhury, S R; Fadilah, N R; Wan Hamirul, W K; Reusmaazran, M Y; Aminuddin, M Y; Ruszymah, B H I

    2014-01-01

    Earlier studies in our laboratory demonstrated that collagen extracted from ovine tendon is biocompatible towards human dermal fibroblast. To be able to use this collagen as a scaffold in skin tissue engineering, a mechanically stronger scaffold is required that can withstand manipulation before transplantation. This study was conducted to improve the mechanical strength of this collagen sponge using chemical crosslinkers, and evaluate their effect on physical, chemical and biocompatible properties. Collagen sponge was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA). Tensile test, FTIR study and mercury porosimetry were used to evaluate mechanical properties, chemical property and porosity, respectively. MTT assay was performed to evaluate the cytotoxic effect of crosslinked collagen sponge on human dermal fibroblasts. The FTIR study confirmed the successful crosslinking of collagen sponge. Crosslinking with EDC and GA significantly increased the mechanical strength of collagen sponge, with GA being more superior. Crosslinking of collagen sponge significantly reduced the porosity and the effect was predominant in GA-crosslinked collagen sponge. The GA-crosslinked collagen showed significantly lower, 60% cell viability towards human dermal fibroblasts compared to that of EDC-crosslinked collagen, 80% and non-crosslinked collagen, 100%. Although the mechanical strength was better when using GA but the more toxic effect on dermal fibroblast makes EDC a more suitable crosslinker for future skin tissue engineering.

  12. Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures.

    PubMed

    Mammone, Thomas; Gan, David; Foyouzi-Youssefi, Reyhaneh

    2006-11-01

    Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

  13. Regenerative capacity of human dental pulp and apical papilla cells after treatment with a 3-antibiotic mixture.

    PubMed

    Phumpatrakom, Panupat; Srisuwan, Tanida

    2014-03-01

    A 3-antibiotic combination (3Mix) has been widely used in regenerative endodontics. Recent studies recommend that a safe concentration of 3Mix is in the range of 0.39 μg/mL and 1 mg/mL because higher concentrations may limit tissue regeneration. The aim of this study was to determine the regenerative capacity of isolated human dental pulp cells (DPCs) and apical papilla cells (APCs) after a 7-day treatment with selected doses of 3Mix. Primary human DPCs/APCs from the third passage were divided into control and experimental groups. In the control group, cells were cultured in regular complete media. In the experimental group, cells were cultured in complete media containing 0.39 μg/mL or 1 mg/mL of 3Mix for 7 days. After the treatment period, the media were changed, and the cells were further tested for proliferation and differentiation potential. For cell proliferation, a colorimetric qualification of 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was used on days 1, 3, 5, and 7. For differentiation potential, a dentinogenic differentiation medium was added into treated cells and cultured for 7, 14, and 21 days. Results were analyzed using quantitative alizarin red S staining and real-time reverse-transcription polymerase chain reaction. After 7 days of treatment, 100% cell death was discovered in the 1-mg/mL 3Mix group. The proliferative capacity of 0.39 μg/mL 3Mix-treated DPCs and APCs was significantly lower than that of untreated cells at all time points (P < .05). Mineralized nodule formation was found both in the 3Mix-treated and control groups, but it was significantly less in the 3Mix-treated groups at 7, 14, and 21 days (P < .01). Quantitative reverse-transcription polymerase chain reaction showed no statistically significant difference (95% confidence interval) in bone sialoprotein, alkaline phosphatase, and dentin matrix protein 1 gene expression in either 3Mix-treated DPCs or APCs compared with control groups. One milligram per

  14. microRNA miR-34a Regulates Cytodifferentiation and Targets Multi-signaling Pathways in Human Dental Papilla Cells

    PubMed Central

    Sun, Feifei; Tang, Yin; Ye, Ling; Fan, Yi; Klein, Ophir D.; Zhou, Xuedong; Zheng, Liwei

    2012-01-01

    Odontogenesis relies on the reciprocal signaling interactions between dental epithelium and neural crest-derived mesenchyme, which is regulated by several signaling pathways. Subtle changes in the activity of these major signaling pathways can have dramatic effects on tooth development. An important regulator of such subtle changes is the fine tuning function of microRNAs (miRNAs). However, the underlying mechanism by which miRNAs regulate tooth development remains elusive. This study determined the expression of miRNAs during cytodifferentiation in the human tooth germ and studied miR-34a as a regulator of dental papilla cell differentiation. Using microarrays, miRNA expression profiles were established at selected times during development (early bell stage or late bell stage) of the human fetal tooth germ. We identified 29 differentially expressed miRNAs from early bell stage/late bell stage comparisons. Out of 6 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. miR-34a was selected for further investigation because it has been previously reported to regulate organogenesis. miR-34a mimics and inhibitors were transfected into human fetal dental papilla cells, mRNA levels of predicted target genes were detected by quantitative real-time PCR, and levels of putative target proteins were examined by western blotting. ALP and DSPP expression were also tested by qPCR, western blotting, and immunofluorescence. Findings from these studies suggested that miR-34a may play important roles in dental papilla cell differentiation during human tooth development by targeting NOTCH and TGF-beta signaling. PMID:23226240

  15. TGFβ functionalized starPEG-heparin hydrogels modulate human dermal fibroblast growth and differentiation.

    PubMed

    Watarai, Akira; Schirmer, Lucas; Thönes, Stephan; Freudenberg, Uwe; Werner, Carsten; Simon, Jan C; Anderegg, Ulf

    2015-10-01

    Hydrogels are promising biomaterials that can adapt easily to complex tissue entities. Furthermore, chemical modifications enable these hydrogels to become an instructive biomaterial to a variety of cell types. Human dermal fibroblasts play a pivotal role during wound healing, especially for the synthesis of novel dermal tissue replacing the primary fibrin clot. Thus, the control of growth and differentiation of dermal fibroblasts is important to modulate wound healing. In here, we utilized a versatile starPEG-heparin hydrogel platform that can be independently adjusted with respect to mechanical and biochemical properties for cultivating human dermal fibroblasts. Cell-based remodeling of the artificial matrix was ensured by using matrix metalloprotease (MMP) cleavable crosslinker peptides. Attachment and proliferation of fibroblasts on starPEG-heparin hydrogels of differing stiffness, density of pro-adhesive RGD peptides and MMP cleavable peptide linkers were tested. Binding and release of human TGFβ1 as well as biological effect of the pre-adsorbed growth factor on fibroblast gene expression and myofibroblast differentiation were investigated. Hydrogels containing RGD peptides supported fibroblast attachment, spreading, proliferation matrix deposition and remodeling compared to hydrogels without any modifications. Reversibly conjugated TGFβ1 was demonstrated to be constantly released from starPEG-heparin hydrogels for several days and capable of inducing myofibroblast differentiation of fibroblasts as determined by induction of collagen type I, ED-A-Fibronectin expression and incorporation of alpha smooth muscle actin and palladin into F-actin stress fibers. Taken together, customized starPEG-heparin hydrogels could be of value to promote dermal wound healing by stimulating growth and differentiation of human dermal fibroblasts. The increasing number of people of advanced age within the population results in an increasing demand for the treatment of non

  16. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  17. Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla

    PubMed Central

    ZHANG, WEN; ZHANG, XIAOLEI; LING, JUNQI; WEI, XI; JIAN, YUTAO

    2016-01-01

    Stem cells from apical papilla (SCAP) possess clear osteo-/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo-/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo-/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector-mediated gene transfection was conducted with SCAP in order to construct blank vector-transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene-transfected SCAP (SCAP-VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP-VEGF was demonstrated to exhibit increased proliferation, and SCAP-BMP2-VEGF exhibited reduced proliferation during eight days observation. SCAP-BMP2-VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP-BMP2-VEGF group exhibited a significantly greater number of ALP-positive mineralized nodules than the other groups following 16 days culture in

  18. Evaluation of 3D-human skin equivalents for assessment of human dermal absorption of some brominated flame retardants.

    PubMed

    Abdallah, Mohamed Abou-Elwafa; Pawar, Gopal; Harrad, Stuart

    2015-11-01

    Ethical and technical difficulties inherent to studies in human tissues are impeding assessment of the dermal bioavailability of brominated flame retardants (BFRs). This is further complicated by increasing restrictions on the use of animals in toxicity testing, and the uncertainties associated with extrapolating data from animal studies to humans due to inter-species variations. To overcome these difficulties, we evaluate 3D-human skin equivalents (3D-HSE) as a novel in vitro alternative to human and animal testing for assessment of dermal absorption of BFRs. The percutaneous penetration of hexabromocyclododecanes (HBCD) and tetrabromobisphenol-A (TBBP-A) through two commercially available 3D-HSE models was studied and compared to data obtained for human ex vivo skin according to a standard protocol. No statistically significant differences were observed between the results obtained using 3D-HSE and human ex vivo skin at two exposure levels. The absorbed dose was low (less than 7%) and was significantly correlated with log Kow of the tested BFR. Permeability coefficient values showed increasing dermal resistance to the penetration of γ-HBCD>β-HBCD>α-HBCD>TBBPA. The estimated long lag times (>30 min) suggests that frequent hand washing may reduce human exposure to HBCDs and TBBPA via dermal contact.

  19. Evaluation of the Dermal Bioavailability of Aqueous Xylene in F344 Rats and Human Volunteers

    SciTech Connect

    Thrall, Karla D. ); Woodstock, Angie D. )

    2003-07-11

    Xylene is a clear, colorless liquid used as a solvent in the printing, rubber, and leather industries and is commonly found in paint thinners, paints, varnishes, and adhesives. Although humans are most likely to be exposed to xylene via inhalation, xylene is also found in well and surface water. Therefore, an assessment of the dermal contribution to total xylene uptake is useful for understanding human exposures. To evaluate the significance of these exposures, the dermal absorption of o-xylene was assessed in F344 male rats and human volunteers using a combination of real-time exhaled breath analysis and physiologically based pharmacokinetic (PBPK) modeling. Animals were exposed to o-xylene dermally. Immediately following the initiation of exposure, individual animals were placed in a glass off-gassing chamber and exhaled breath was monitored. Human volunteers participating in the study placed both legs into a stainless steel hydrotherapy tub containing an initial concentration of approximately 500 g/L o-xylene. Exhaled breath was continually analyzed from each volunteer before, during, and post-exposure to track absorption and subsequent elimination of the compound in real time. In both animal and human studies, a PBPK model was used to estimate the dermal permeability coefficient (Kp) to describe each set of exhaled breath data. Rat skin was found to be approximately 12 times more permeable to aqueous o-xylene than human skin. The estimated human and rat aqueous o-xylene Kp values were 0.005+/- 0.001 cm/hr and 0.058+/- 0.009 cm/hr, respectively.

  20. Recellularizing of human acellular dermal matrices imaged by high-definition optical coherence tomography.

    PubMed

    Boone, Marc A L M; Draye, Jean Pierre; Verween, Gunther; Aiti, Annalisa; Pirnay, Jean-Paul; Verbeken, Gilbert; De Vos, Daniel; Rose, Thomas; Jennes, Serge; Jemec, Gregor B E; Del Marmol, Veronique

    2015-05-01

    High-definition optical coherence tomography (HD-OCT) permits real-time 3D imaging of the impact of selected agents on human skin allografts. The real-time 3D HD-OCT assessment of (i) the impact on morphological and cellular characteristics of the processing of human acellular dermal matrices (HADMs) and (ii) repopulation of HADMs in vitro by human fibroblasts and remodelling of the extracellular matrix by these cells. Four different skin decellularization methods, Dispase II/Triton X-100, Dispase II/SDS (sodium dodecyl sulphate), NaCl/Triton X-100 and NaCl/SDS, were analysed by HD-OCT. HD-OCT features of epidermal removal, dermo-epidermal junction (DEJ) integrity, cellularity and dermal architecture were correlated with reflectance confocal microscopy (RCM), histopathology and immunohistochemistry. Human adult dermal fibroblasts were in vitro seeded on the NaCl/Triton X-100 processed HADMs, cultured up to 19 days and evaluated by HD-OCT in comparison with MTT proliferation test and histology. Epidermis was effectively removed by all treatments. DEJ was best preserved after NaCl/Triton X-100 treatment. Dispase II/SDS treatment seemed to remove all cellular debris in comparison with NaCl/Triton X-100 but disturbed the DEJ severely. The dermal micro-architectural structure and vascular spaces of (sub)papillary dermis were best preserved with the NaCl/Triton X-100. The impact on the 3D structure and vascular holes was detrimental with Dispase II/SDS. Elastic fibre fragmentation was only observed after Dispase II incubation. HD-OCT showed that NaCl/Triton X-100 processed matrices permitted in vitro repopulation by human dermal fibroblasts (confirmed by MTT test and histology) and underwent remodelling upon increasing incubation time. Care must be taken in choosing the appropriate processing steps to maintain selected properties of the extracellular matrix in HADMs. Processing HADMs with NaCl/Triton X-100 permits in vitro the proliferation and remodelling activity of

  1. Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis.

    PubMed

    Salerno, Simona; Messina, Antonietta; Giordano, Francesca; Bader, Augustinus; Drioli, Enrico; De Bartolo, Loredana

    2017-02-01

    Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT-PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Giampieri, Francesca; Afrin, Sadia; Mezzetti, Bruno; Quiles, Josè L; Bompadre, Stefano; Battino, Maurizio

    2017-01-21

    A protracted pro-inflammatory state is a major contributing factor in the development, progression and complication of the most common chronic pathologies. Fruit and vegetables represent the main sources of dietary antioxidants and their consumption can be considered an efficient tool to counteract inflammatory states. In this context an evaluation of the protective effects of strawberry extracts on inflammatory stress induced by E. coli LPS on human dermal fibroblast cells was performed in terms of viability assays, ROS and nitrite production and biomarkers of oxidative damage of the main biological macromolecules. The results demonstrated that strawberry extracts exerted an anti-inflammatory effect on LPS-treated cells, through an increase in cell viability, and the reduction of ROS and nitrite levels, and lipid, protein and DNA damage. This work showed for the first time the potential health benefits of strawberry extract against inflammatory and oxidative stress in LPS-treated human dermal fibroblast cells.

  3. Relationship between gustatory function and average number of taste buds per fungiform papilla measured by confocal laser scanning microscopy in humans.

    PubMed

    Saito, Takehisa; Ito, Tetsufumi; Ito, Yumi; Manabe, Yasuhiro; Sano, Kazuo

    2017-02-01

    The aim of this study was to elucidate the relationship between the gustatory function and average number of taste buds per fungiform papilla (FP) in humans. Systemically healthy volunteers (n = 211), pre-operative patients with chronic otitis media (n = 79), and postoperative patients, with or without a chorda tympani nerve (CTN) severed during middle ear surgery (n = 63), were included. Confocal laser scanning microscopy was employed to observe fungiform taste buds because it allows many FP to be observed non-invasively in a short period of time. Taste buds in an average of 10 FP in the midlateral region of the tongue were counted. In total, 3,849 FP were observed in 353 subjects. The gustatory function was measured by electrogustometry (EGM). An inverse relationship was found between the gustatory function and average number of fungiform taste buds per papilla. The healthy volunteers showed a lower EGM threshold (better gustatory function) and had more taste buds than did the patients with otitis media, and the patients with otitis media showed a lower EGM threshold and had more taste buds than did postoperative patients, reflecting the severity of damage to the CTN. It was concluded that the confocal laser scanning microscope is a very useful tool for using to observe a large number of taste buds non-invasively.

  4. Effect of Bromine Substitution on Human Dermal Absorption of Polybrominated Diphenyl Ethers.

    PubMed

    Abdallah, Mohamed Abou-Elwafa; Pawar, Gopal; Harrad, Stuart

    2015-09-15

    Human dermal absorption of eight mono- to deca-brominated diphenyl ethers (PBDEs) was investigated for the first time using EPISKIN human skin equivalent tissue. Using a standard in vitro protocol, EPISKIN tissues mounted in specially designed diffusion cells were exposed to the target PBDEs for 24 h. Estimated steady-state flux (Jss) and permeation coefficients (Papp) across the skin increased with decreasing bromine substitution from BDE-153 (Papp = 4.0 × 10(-4) cm/h) to BDE-1 (Papp = 1.1 × 10(-2) cm/h). This was accompanied by an increase in the time required to traverse the skin tissue into the receptor fluid (lag time) from 0.25 h for BDE-1 to 1.26 h for BDE-153. Papp values for the studied PBDEs were correlated significantly (P < 0.05) with physicochemical parameters like water solubility and log KOW. While less brominated congeners achieved faster dermal penetration, higher PBDEs displayed greater accumulation within the skin tissue. The PBDEs thus accumulated represent a contaminant depot from which they may be slowly released to the systemic circulation over a prolonged period. Maximal percutaneous penetration was observed for BDE-1 (∼ 30% of the applied 500 ng/cm(2) dose). Interestingly, BDE-183 and BDE-209 showed very low dermal absorption, exemplified by a failure to reach the steady state within the 24 h exposure period that was studied.

  5. Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

    PubMed

    Ghosh, Kaustabh; Ren, Xiang-Dong; Shu, Xiao Zheng; Prestwich, Glenn D; Clark, Richard A F

    2006-03-01

    Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

  6. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

    PubMed

    El Ghalbzouri, Abdoelwaheb; Commandeur, Suzan; Rietveld, Marion H; Mulder, Aat A; Willemze, Rein

    2009-01-01

    Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.

  7. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  8. In vitro dermal absorption of pyrethroid pesticides in human and rat skin

    SciTech Connect

    Hughes, Michael F.; Edwards, Brenda C.

    2010-07-15

    Dermal exposure to pyrethroid pesticides can occur during manufacture and application. This study examined the in vitro dermal absorption of pyrethroids using rat and human skin. Dermatomed skin from adult male Long Evans rats or human cadavers was mounted in flow-through diffusion cells, and radiolabeled bifenthrin, deltamethrin or cis-permethrin was applied in acetone to the skin. Fractions of receptor fluid were collected every 4 h. At 24 h, the skins were washed with soap and water to remove unabsorbed chemical. The skin was then solubilized. Two additional experiments were performed after washing the skin; the first was tape-stripping the skin and the second was the collection of receptor fluid for an additional 24 h. Receptor fluid, skin washes, tape strips and skin were analyzed for radioactivity. For rat skin, the wash removed 53-71% of the dose and 26-43% remained in the skin. The cumulative percentage of the dose at 24 h in the receptor fluid ranged from 1 to 5%. For human skin, the wash removed 71-83% of the dose and 14-25% remained in the skin. The cumulative percentage of the dose at 24 h in the receptor fluid was 1-2%. Tape-stripping removed 50-56% and 79-95% of the dose in rat and human skin, respectively, after the wash. From 24-48 h, 1-3% and about 1% of the dose diffused into the receptor fluid of rat and human skin, respectively. The pyrethroids bifenthrin, deltamethrin and cis-permethrin penetrated rat and human skin following dermal application in vitro. However, a skin wash removed 50% or more of the dose from rat and human skin. Rat skin was more permeable to the pyrethroids than human skin. Of the dose in skin, 50% or more was removed by tape-stripping, suggesting that permeation of pyrethroids into viable tissue could be impeded. The percentage of the dose absorbed into the receptor fluid was considerably less than the dose in rat and human skin. Therefore, consideration of the skin type used and fractions analyzed are important when using

  9. In vitro dermal absorption of pyrethroid pesticides in human and rat skin.

    PubMed

    Hughes, Michael F; Edwards, Brenda C

    2010-07-01

    Dermal exposure to pyrethroid pesticides can occur during manufacture and application. This study examined the in vitro dermal absorption of pyrethroids using rat and human skin. Dermatomed skin from adult male Long Evans rats or human cadavers was mounted in flow-through diffusion cells, and radiolabeled bifenthrin, deltamethrin or cis-permethrin was applied in acetone to the skin. Fractions of receptor fluid were collected every 4h. At 24h, the skins were washed with soap and water to remove unabsorbed chemical. The skin was then solubilized. Two additional experiments were performed after washing the skin; the first was tape-stripping the skin and the second was the collection of receptor fluid for an additional 24 h. Receptor fluid, skin washes, tape strips and skin were analyzed for radioactivity. For rat skin, the wash removed 53-71% of the dose and 26-43% remained in the skin. The cumulative percentage of the dose at 24 h in the receptor fluid ranged from 1 to 5%. For human skin, the wash removed 71-83% of the dose and 14-25% remained in the skin. The cumulative percentage of the dose at 24 h in the receptor fluid was 1-2%. Tape-stripping removed 50-56% and 79-95% of the dose in rat and human skin, respectively, after the wash. From 24-48 h, 1-3% and about 1% of the dose diffused into the receptor fluid of rat and human skin, respectively. The pyrethroids bifenthrin, deltamethrin and cis-permethrin penetrated rat and human skin following dermal application in vitro. However, a skin wash removed 50% or more of the dose from rat and human skin. Rat skin was more permeable to the pyrethroids than human skin. Of the dose in skin, 50% or more was removed by tape-stripping, suggesting that permeation of pyrethroids into viable tissue could be impeded. The percentage of the dose absorbed into the receptor fluid was considerably less than the dose in rat and human skin. Therefore, consideration of the skin type used and fractions analyzed are important when using in

  10. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    PubMed

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  11. Proliferation and odontogenic differentiation of BMP2 gene-transfected stem cells from human tooth apical papilla: An in vitro study

    PubMed Central

    ZHANG, WEN; ZHANG, XIAOLEI; LING, JUNQI; LIU, WEI; ZHANG, XINCHUN; MA, JINGLEI; ZHENG, JIANMAO

    2014-01-01

    Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue-derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral-mediated BMP2 gene-transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector-transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK-8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: i) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ii) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; iii) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro. PMID:25070743

  12. Proliferation and odontogenic differentiation of BMP2 gene‑transfected stem cells from human tooth apical papilla: an in vitro study.

    PubMed

    Zhang, Wen; Zhang, Xiaolei; Ling, Junqi; Liu, Wei; Zhang, Xinchun; Ma, Jinglei; Zheng, Jianmao

    2014-10-01

    Stem cells from the apical papilla (SCAP) have odontogenic potential, which plays a pivotal role in the root dentin development of permanent teeth. Human bone morphogenetic protein 2 (BMP2) is a well-known gene that participates in regulating the odontogenic differentiation of dental tissue‑derived stem cells. However, little is known regarding the effects of the BMP2 gene on the proliferation and odontogenic differentiation of SCAP. This study aimed to evaluate the odontogenic differentiation potential of lentiviral‑mediated BMP2 gene‑transfected human SCAP (SCAP/BMP2) in vitro. SCAP were isolated by enzymatic dissociation of human teeth apical papillae. The multipotential of SCAP was verified by their osteogenic and adipogenic differentiation characteristics. The phenotype of SCAP was evaluated by flow cytometry (FCM). The proliferation status of the blank vector‑transfected SCAP (SCAP/Vector) and SCAP/BMP2 was analyzed by a cell counting kit-8 (CCK‑8). Odontogenic genes, including alkaline phosphatase (ALP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) of the two groups of cells were evaluated by quantitative polymerase chain reaction (qPCR). ALP staining and alizarin red (AR) staining of the cells was performed on the 16th day after transfection. In vitro results of CCK-8, qPCR, ALP and AR staining demonstrated that: ⅰ) SCAP/BMP2 had a comparable proliferation rate to SCAP/Vector; ⅱ) SCAP/BMP2 presented significantly better potential to differentiate into odontoblasts compared to SCAP/Vector by upregulating ALP, OCN, DSPP and DMP1 genes; ⅲ) more ALP granules and mineralized deposits were formed by SCAP/BMP2 as compared to SCAP/Vector. The results suggested that lentiviral-mediated BMP2 gene transfection enhances the odontogenic differentiation capacity of human SCAP in vitro.

  13. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  14. Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

    PubMed

    Hill, R P; MacNeil, S; Haycock, J W

    2006-02-01

    Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

  15. Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture.

    PubMed

    Pandamooz, Sareh; Hadipour, Abbas; Akhavan-Niaki, Haleh; Pourghasem, Mohsen; Abedian, Zeinab; Ardekani, Ali Motevallizadeh; Golpour, Monireh; Hassan, Zuhair Mohammad; Mostafazadeh, Amrollah

    2012-01-01

    The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

  16. Human dermal stem/progenitor cell-derived conditioned medium ameliorates ultraviolet a-induced damage of normal human dermal fibroblasts.

    PubMed

    Shim, Joong Hyun; Park, Ju-Yearl; Lee, Mi-Gi; Kang, Hak Hee; Lee, Tae Ryong; Shin, Dong Wook

    2013-01-01

    Adult skin stem cells are considered an attractive cell resource for therapeutic potential in aged skin. We previously reported that multipotent human dermal stem/progenitor cells (hDSPCs) can be enriched from (normal human dermal fibroblasts (NHDFs) using collagen type IV. However, the beneficial effects of hDSPCs on aged skin remain to be elucidated. In the present study, we analyzed the growth factors secreted from hDSPCs in conditioned medium (CM) derived from hDSPCs (hDSPC-CM) and found that hDSPCs secreted higher levels of bFGF, IGFBP-1, IGFBP-2, HGF, VEGF and IGF-1 compared with non-hDSPCs. We then investigated whether hDSPC-CM has an effect on ultraviolet A (UVA)-irradiated NHDFs. Real-time RT-PCR analysis revealed that the treatment of UVA-irradiated NHDFs with hDSPC-CM significantly antagonized the UVA-induced up-regulation of the MMP1 and the UVA-induced down-regulation of the collagen types I, IV and V and TIMP1 mRNA expressions. Furthermore, a scratch wound healing assay showed that hDSPC-CM enhanced the migratory properties of UVA-irradiated NHDFs. hDSPC-CM also significantly reduced the number of the early and late apoptotic cell population in UVA-irradiated NHDFs. Taken together, these data suggest that hDSPC-CM can exert some beneficial effects on aged skin and may be used as a therapeutic agent to improve skin regeneration and wound healing.

  17. Morphological and Molecular Characterization of Human Dermal Lymphatic Collectors

    PubMed Central

    Buttler, Kerstin; Ströbel, Philipp; Becker, Jürgen; Aung, Thiha; Felmerer, Gunther; Wilting, Jörg

    2016-01-01

    Millions of patients suffer from lymphedema worldwide. Supporting the contractility of lymphatic collectors is an attractive target for pharmacological therapy of lymphedema. However, lymphatics have mostly been studied in animals, while the cellular and molecular characteristics of human lymphatic collectors are largely unknown. We studied epifascial lymphatic collectors of the thigh, which were isolated for autologous transplantations. Our immunohistological studies identify additional markers for LECs (vimentin, CCBE1). We show and confirm differences between initial and collecting lymphatics concerning the markers ESAM1, D2-40 and LYVE-1. Our transmission electron microscopic studies reveal two types of smooth muscle cells (SMCs) in the media of the collectors with dark and light cytoplasm. We observed vasa vasorum in the media of the largest collectors, as well as interstitial Cajal-like cells, which are highly ramified cells with long processes, caveolae, and lacking a basal lamina. They are in close contact with SMCs, which possess multiple caveolae at the contact sites. Immunohistologically we identified such cells with antibodies against vimentin and PDGFRα, but not CD34 and cKIT. With Next Generation Sequencing we searched for highly expressed genes in the media of lymphatic collectors, and found therapeutic targets, suitable for acceleration of lymphatic contractility, such as neuropeptide Y receptors 1, and 5; tachykinin receptors 1, and 2; purinergic receptors P2RX1, and 6, P2RY12, 13, and 14; 5-hydroxytryptamine receptors HTR2B, and 3C; and adrenoceptors α2A,B,C. Our studies represent the first comprehensive characterization of human epifascial lymphatic collectors, as a prerequisite for diagnosis and therapy. PMID:27764183

  18. Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin.

    PubMed

    Llames, Sara G; Del Rio, Marcela; Larcher, Fernando; García, Eva; García, Marta; Escamez, María José; Jorcano, Jose L; Holguín, Purificación; Meana, Alvaro

    2004-02-15

    Keratinocyte cultures have been used for the treatment of severe burn patients. Here, we describe a new cultured bioengineered skin based on (1) keratinocytes and fibroblasts obtained from a single skin biopsy and (2) a dermal matrix based on human plasma. A high expansion capacity achieved by keratinocytes grown on this plasma-based matrix is reported. In addition, the results of successful preclinical and clinical tests are presented. Keratinocytes and fibroblasts were obtained by a double enzymatic digestion (trypsin and collagenase, respectively). In this setting, human fibroblasts are embedded in a clotted plasma-based matrix that serves as a three-dimensional scaffold. Human keratinocytes are seeded on the plasma-based scaffold to form the epidermal component of the skin construct. Regeneration performance of the plasma-based bioengineered skin was tested on immunodeficient mice as a preclinical approach. Finally, this skin equivalent was grafted on two severely burned patients. Keratinocytes seeded on the plasma-based scaffold grew to confluence, allowing a 1,000-fold cultured-area expansion after 24 to 26 days of culture. Experimental transplantation of human keratinocytes expanded on the engineered plasma scaffold yielded optimum epidermal architecture and phenotype, including the expression of structural intracellular proteins and basement-membrane components. In addition, we report here the successful engraftment and stable skin regeneration in two severely burned patients at 1 and 2 years follow-up. Our data demonstrate that this new dermal equivalent allows for (1) generation of large bioengineered skin surfaces, (2) restoration of both the epidermal and dermal skin compartments, and (3) functional epidermal stem-cell preservation.

  19. A composite dermal filler comprising cross-linked hyaluronic acid and human collagen for tissue reconstruction.

    PubMed

    Kim, Z-Hun; Lee, Yongjun; Kim, Sun-Mi; Kim, Hojin; Yun, Chang-Koo; Choi, Yong-Soo

    2015-03-01

    In this study, we developed a composite filler comprising cross-linked hyaluronic acid (HA) and human collagen (COL) derived from the human umbilical cord with the aim of improving its biocompatibility and longevity compared with commercially available fillers. After HA/ COL composite fillers were made in two different ratios (10:1 and 5:1), the physical properties of the fillers were evaluated. The interior morphologies and in vivo weight change of these hydrogels were also characterized at 1-16 weeks after injection into mice. To evaluate their biocompatibility and durability in vivo, we injected the composite fillers into nude mice subcutaneously. The variations of injected gel weight were measured and compared with the commercial dermal fillers (Restylane and TheraFill). The composites showed improved or similar physical properties (complex viscosity of 19-22 × 10(5) cP, and injection force of 10- 12 N) over the commercial dermal fillers. Sixteen weeks following the injection, the ratio of remaining composite filler weight to initial weight (75.5 ± 16.9%; 10:1) was shown to be greater than that of the commercial fillers (43.2 ± 8.1%, Restylane; 12.3 ± 5.3%, TheraFill). In addition, immunohistochemical analysis with angiogenesis-related markers such as isolectin and vWF revealed newly formed blood vessels and cellular influx into the composite filler, which were not observed in the other fillers. These results clearly suggest that the HA/COL composite filler is a superior candidate for soft tissue reconstruction. The filler we developed may be a suitable candidate as an injectable dermal filler for tissue augmentation in humans.

  20. Adverse effects of citrate/gold nanoparticles on human dermal fibroblasts.

    PubMed

    Pernodet, Nadine; Fang, Xiaohua; Sun, Yuan; Bakhtina, Asya; Ramakrishnan, Aditi; Sokolov, Jonathan; Ulman, Abraham; Rafailovich, Miriam

    2006-06-01

    Nanoscale engineering is one of the most dynamically growing areas at the interface between electronics, physics, biology, and medicine. As there are no safety regulations yet, concerns about future health problems are rising. We investigated the effects of citrate/gold nanoparticles at different concentrations and exposure times on human dermal fibroblasts. We found that, as a result of intracellular nanoparticle presence, actin stress fibers disappeared, thereby inducing major adverse effects on cell viability. Thus, properties such as cell spreading and adhesion, cell growth, and protein synthesis to form the extracellular matrix were altered dramatically. These results suggest that the internal cell activities have been damaged.

  1. Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage.

    PubMed

    von Montfort, Claudia; Alili, Lirija; Teuber-Hanselmann, Sarah; Brenneisen, Peter

    2015-01-01

    Recently, it has been published that cerium (Ce) oxide nanoparticles (CNP; nanoceria) are able to downregulate tumor invasion in cancer cell lines. Redox-active CNP exhibit both selective pro-oxidative and antioxidative properties, the first being responsible for impairment of tumor growth and invasion. A non-toxic and even protective effect of CNP in human dermal fibroblasts (HDF) has already been observed. However, the effect on important parameters such as cell death, proliferation and redox state of the cells needs further clarification. Here, we present that nanoceria prevent HDF from reactive oxygen species (ROS)-induced cell death and stimulate proliferation due to the antioxidative property of these particles.

  2. A prospective, randomized evaluation of acellular human dermal matrix augmentation for arthroscopic rotator cuff repair.

    PubMed

    Barber, F Alan; Burns, Joseph P; Deutsch, Allen; Labbé, Marc R; Litchfield, Robert B

    2012-01-01

    To prospectively evaluate the safety and effectiveness of arthroscopic acellular human dermal matrix augmentation of large rotator cuff tear repairs. A prospective, institutional review board-approved, multicenter series of patients undergoing arthroscopic repair of 2-tendon rotator cuff tears measuring greater than 3 cm were randomized by sealed envelopes opened at the time of surgery to arthroscopic single-row rotator cuff repair with GraftJacket acellular human dermal matrix (Wright Medical Technology, Arlington, TN) augmentation (group 1) or without augmentation (group 2). Preoperative and postoperative functional outcome assessments were obtained by use of the American Shoulder and Elbow Surgeons (ASES), Constant, and University of California, Los Angeles scales. Gadolinium-enhanced magnetic resonance imaging (MRI) evaluation of these repairs was obtained at a mean of 14.5 months (range, 12 to 24 months). Adverse events were recorded. There were 22 patients in group 1 and 20 in group 2 with a mean age of 56 years. The mean follow-up was 24 months (range, 12 to 38 months). The ASES score improved from 48.5 to 98.9 in group 1 and from 46.0 to 94.8 in group 2. The scores in group 1 were statistically better than those in group 2 (P = .035). The Constant score improved from 41.0 to 91.9 in group 1 and from 45.8 to 85.3 in group 2. The scores in group 1 were statistically better than those in group 2 (P = .008). The University of California, Los Angeles score improved from 13.3 to 28.2 in group 1 and from 15.9 to 28.3 in group 2 (P = .43). Gadolinium-enhanced MRI scans showed intact cuffs in 85% of repairs in group 1 and 40% in group 2 (P < .01). No adverse events were attributed to the presence of the matrix grafts. Acellular human dermal matrix augmentation of large (>3 cm) cuff tears involving 2 tendons showed better ASES and Constant scores and more frequent intact cuffs as determined by gadolinium-enhanced MRI. Intact repairs were found in 85% of the augmented

  3. Human cadaveric dermal matrix for management of challenging surgical defects on the scalp.

    PubMed

    Stebbins, William G; Hanke, C William; Petersen, Jeffrey

    2011-03-01

    Biologic scaffolds have shown promise in patients unable to tolerate prolonged surgical closure or extensive wound care, but there has been little research in the field of Mohs micrographic surgery (MMS) on human cadaveric dermis in this capacity. To evaluate the utility of human cadaveric dermis as a means of decreasing operative time, minimizing postoperative wound care, and improving aesthetic outcomes in selected patients with deep surgical defects, including those with exposed bone. Fourteen patients (8 men, 6 women) with deep postoperative defects after MMS were treated with a cadaveric dermal allograft as part or all of their postoperative wound management. Allograft placement was well tolerated, with high satisfaction levels relating to minimal postoperative wound care and aesthetic outcome. Significantly shorter operative times were noted in all patients than with primary closure or grafting. In patients with significant comorbidities, inability to tolerate extended surgical repairs, or inability to perform extensive wound care, human cadaveric dermal allografts can decrease operative time and minimize wound care complexity while providing an excellent aesthetic outcome in many cases. Shorter healing times than expected were also noted in a number of patients. The authors have indicated no significant interest with commercial supporters. © 2011 by the American Society for Dermatologic Surgery, Inc.

  4. Gene expression in response to cyclic mechanical stretch in primary human dermal fibroblasts.

    PubMed

    Reichenbach, Maria; Reimann, Kerstin; Reuter, Hendrik

    2014-12-01

    The human dermal skin is permanently exposed to mechanical stress, for instance during facial expression, which might cause wrinkles with age. Cyclic mechanical stretching of cells results in cellular and cytoskeleton alignment perpendicular to the stretch direction regulating cellular response. With gene expression profiling it was aimed to identify the differentially expressed genes associated with the regulation of the cytoskeleton to investigate the stretch-induced cell alignment mechanism. Here, the transcription activity of the genome in response to cyclic mechanical stress was measured using DNA microarray technology with Agilent SurePrint G3 Human GE 8x60k Microarrays, based on the overall measurement of the mRNA. Gene expression was measured at the beginning of the alignment process showing first reoriented cells after 5 h stretching and at the end after 24 h, where nearly all cells are aligned. Gene expression data of control vs. stretched primary human dermal fibroblasts after 5 h and 24 h demonstrated the regulation of differentially expressed genes associated with metabolism, differentiation and morphology and were deposited at http://www.ncbi.nlm.nih.gov/geo with the accession number GSE58389.

  5. Lactobacillus sakei lipoteichoic acid inhibits MMP-1 induced by UVA in normal dermal fibroblasts of human.

    PubMed

    You, Ga-Eun; Jung, Bong-Jun; Kim, Hye-Rim; Kim, Han-Geun; Kim, Tae-Rahk; Chung, Dae-Kyun

    2013-10-28

    Human skin is continuously exposed to ultraviolet (UV)-induced photoaging. UVA increases the activity of MMP-1 in dermal fibroblasts through mitogen-activated protein kinase (MAPK), p38, signaling. The irradiation of keratinocytes by UVA results in the secretion of the inflammatory cytokine, tumor necrosis factor-α (TNF-α), and the stimulation of MMP-1 in normal human dermal fibroblasts (NHDFs). Lipoteichoic acid (LTA) is a component of the cell wall of gram-positive Lactobacillus spp. of bacteria. LTA is well known as an anti-inflammation molecule. LTA of the bacterium Lactobacillus plantarum has an anti-photoaging effect, but the potential anti-photoaging effect of the other bacteria has not been examined to date. The current study showed that L. sakei LTA (sLTA) has an immune modulating effect in human monocyte cells. Our object was whether inhibitory effects of sLTA on MMP-1 are caused from reducing the MAPK signal in NHDFs. It inhibits MMP-1 and MAPK signaling induced by UVA in NHDFs. We also confirmed effects of sLTA suppressing TNF-α inducing MMP-1 in NHDFs.

  6. The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.

    PubMed

    Kim, J-A; Ahn, B-N; Kong, C-S; Kim, S-K

    2013-05-01

    Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  7. Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro

    SciTech Connect

    Zhang, Jing; Wang, Zhihua; Jiang, Yong; Niu, Zhongying; Fu, Lei; Luo, Zhirong; Cooper, Paul R.; Smith, Anthony J.; He, Wenxi

    2015-03-15

    The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs. - Highlights: • NFIC promotes the proliferation of SCAPs in vitro. • NFIC promotes osteo/odontogenic differentiation of SCAPs in vitro. • Knockdown of NFIC inhibits odontogenic differentiation in SCAPs.

  8. Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro.

    PubMed

    Zhang, Jing; Wang, Zhihua; Jiang, Yong; Niu, Zhongying; Fu, Lei; Luo, Zhirong; Cooper, Paul R; Smith, Anthony J; He, Wenxi

    2015-03-15

    The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs.

  9. Scarring occurs at a critical depth of skin injury: precise measurement in a graduated dermal scratch in human volunteers.

    PubMed

    Dunkin, Christopher S J; Pleat, Jonathon M; Gillespie, Patrick H; Tyler, Michael P H; Roberts, Anthony H N; McGrouther, D Angus

    2007-05-01

    The association between scarring and the depth of dermal injury or burn is clinically recognized but not quantified. The authors tested the hypothesis that there is a critical depth beyond which a fibrous scar develops. A novel jig produced a wound that was deep dermal at one end and superficial dermal at the other. Pilot studies in cadaveric and ex vivo breast skin confirmed the depth of injury. Healthy volunteers had a standardized dermal wound made on the lateral aspect of the hip. Digital photography recorded the surface appearance of wound healing and scar development. High-frequency ultrasound demonstrated the depth of the healing wound and subsequent scar in vivo. One hundred thirteen human subjects participated in the clinical study. Mean length of follow up was 28.6 +/- 13.2 weeks. The deep dermal end of the wound healed with a visible scar and the superficial end had no visible residual mark after week 18. The initial length of injury was 51.3 +/- 0.6 mm, which reduced to a scar of 34.9 +/- 1.0 mm at 36 weeks (corresponding areas were 196.6 +/- 7.5 mm and 92.7 +/- 9.4 mm). High-frequency ultrasound analysis showed a gradual reduction in scar thickness at the deep end and no detectable scar at the shallow end. The transition point between scar and no scar marked the threshold depth for scarring. This was calculated as 0.56 +/- 0.03 mm, or 33.1 percent of normal hip skin thickness. The dermal scratch provides a well-tolerated, standardized, and reproducible wound model for investigating the healing response to dermal injury of different depths. There is a threshold depth of dermal injury at which scarring develops.

  10. Human Dermal Mast Cells Contain and Release Tumor Necrosis Factor α, which Induces Endothelial Leukocyte Adhesion Molecule 1

    NASA Astrophysics Data System (ADS)

    Walsh, Laurence J.; Trinchieri, Giorgio; Waldorf, Heidi A.; Whitaker, Diana; Murphy, George F.

    1991-05-01

    Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-α within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-α protein and TNF-α mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-α. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion.

  11. CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Robichaud, Patrick; Voorhees, John J; Fisher, Gary J

    2011-08-01

    Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

  12. Contaminated soils (III): in vitro dermal absorption of ethylene glycol and nonylphenol in human skin.

    PubMed

    Moody, Richard P; Joncas, Julie; Richardson, Mark; Petrovic, Sanya; Chu, Ih

    2010-01-01

    Dermal absorption of contaminants from soils at federal contaminated sites in Canada was investigated using one hydrophile, (14)C-ethylene glycol (EG), and one lipophile, (14)C-nonylphenol (NP). In vitro dermal absorption of EG and NP was examined in dermatomed (0.4-0.5 mm) human skin using Bronaugh Teflon flow-through cells with Hanks HEPES buffered (pH 7.4) receiver solution with 4% bovine serum albumin (BSA). Tests were conducted under occlusive conditions with and without a commercial gardening soil spiked with EG or NP applied to skin at a soil load of 5 mg/cm(2). With percent absorption in skin depot included, a total of 9.9 + or - 6.28% (n = 6) and 34.8 + or - 8.47% (n = 6) absorption of EG with and without soil, respectively, and 20.6 + or - 5.56% (n = 7) and 41.1 + or - 6.46% (n = 7) of NP, with and without soil, respectively, were obtained. For tests without soil a reverse pattern was observed with significantly lower percent absorption into the receiver than depot with the lipophile NP, but significantly higher percent absorption in receiver versus depot for the hydrophile EG. This pattern was different in tests with soil, and caution needs to be exercised when extrapolating data from in vitro tests conducted without soil in human health risk assessments at contaminated sites.

  13. Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

    PubMed

    Lee, Geum-Young; Park, Kang-Gyun; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2016-03-01

    Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml.

  14. A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria.

    PubMed

    Kicinska, Anna; Augustynek, Bartlomiej; Kulawiak, Bogusz; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam; Bednarczyk, Piotr

    2016-12-01

    Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca(2+) and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and β-subunits (predominantly the β3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca(2+)-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  15. Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts

    PubMed Central

    Gothai, Sivapragasam; Arulselvan, Palanisamy; Tan, Woan Sean; Fakurazi, Sharida

    2016-01-01

    Background/Aim: Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for the treatment of cuts, wounds and burns. Moringa oleifera (MO) is an herb used as a traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of MO leaves extract are completely unknown. Materials and Methods: In the current study, ethyl acetate fraction of MO leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate) in human normal dermal fibroblast cells. Results: Results revealed that lower concentration (12.5 µg/ml, 25 µg/ml, and 50 µg/ml) of ethyl acetate fraction of MO leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: This study suggested that ethyl acetate fraction of MO leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use. PMID:27069722

  16. Macrophage inhibitory cytokine MIC-1 is upregulated by short-wavelength light in cultured normal human dermal fibroblasts.

    PubMed

    Akiyama, Masashi; Okano, Keiko; Fukada, Yoshitaka; Okano, Toshiyuki

    2009-03-04

    To better understand dermal response to visible light, we used DNA microarray analysis to search genes induced by blue or near-UV light in normal human dermal fibroblasts. Of about 12800 transcripts analyzed, near-UV light most prominently upregulated the transcript level of Mic-1, a gene encoding a TGF-beta superfamily protein. Quantitative RT-PCR and immunoblot analyses revealed that mRNA and protein levels of Mic-1 were upregulated by both short-wavelength light but not by green or red light. These results suggest that the human dermis is a site for macrophage inhibitory cytokine-1 (MIC-1) production and that visible light activates a dermal transcription cascade. Considering the role of MIC-1 in immune regulation and appetite control, photic MIC-1 regulation is of physiological importance.

  17. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging: A Mini-Review.

    PubMed

    Quan, Taihao; Fisher, Gary J

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin diseases, such as delayed wound healing and skin cancer development. This mini-review describes cellular mechanisms that give rise to self-perpetuating, collagen fibril fragmentation that creates an age-associated dermal microenvironment, which contributes to decline of human skin function.

  18. Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate.

    PubMed

    Abuarqoub, Duaa; Awidi, Abdalla; Abuharfeil, Nizar

    2015-10-01

    Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts.

    PubMed

    Malpass, Gloria E; Arimilli, Subhashini; Prasad, G L; Howlett, Allyn C

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1h or 5h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5h), which was increased by nicotine but suppressed by other components of STE. Within 2h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research.

  20. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    PubMed Central

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G. L.; Howlett, Allyn C.

    2015-01-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. PMID:24927667

  1. Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

    PubMed

    Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

    2016-05-01

    Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.

  2. Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.

    PubMed

    Rnjak-Kovacina, Jelena; Wise, Steven G; Li, Zhe; Maitz, Peter K M; Young, Cara J; Wang, Yiwei; Weiss, Anthony S

    2011-10-01

    We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Young's moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery.

    PubMed

    Römgens, Anne M; Bader, Dan L; Bouwstra, Joke A; Baaijens, Frank P T; Oomens, Cees W J

    2015-10-01

    Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm(2)/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Dermal absorption and skin damage following hydrofluoric acid exposure in an ex vivo human skin model.

    PubMed

    Dennerlein, Kathrin; Kiesewetter, Franklin; Kilo, Sonja; Jäger, Thomas; Göen, Thomas; Korinth, Gintautas; Drexler, Hans

    2016-04-25

    The wide industrial use of hydrofluoric acid (HF) poses a high risk for accidental dermal exposure. Despite local and systemic hazards associated with HF, information on percutaneous penetration and tissue damage is rare. In the present ex vivo study, the dermal absorption of HF (detected in terms of fluoride ions) was quantified and the skin damaging potential as a function of concentration and exposure duration was assessed. Percutaneous penetration of HF (c=5, 30, and 50%) at 3 exposure durations (3, 5, and 10 min) was investigated in a static diffusion cell model using freshly excised human skin. Alterations of skin were histologically evaluated. HF rapidly penetrated through skin under formation of a considerable intradermal reservoir (∼ 13-67% of total absorbed fluoride). Histologically, epidermal alterations were detected already after exposure to 5% HF for 3 min. The degree of skin damage increased with rising concentration and exposure duration leading to coagulation necrosis. For HF concentrations of ≥ 30%, skin damage progressed into deeper skin layers. Topically applied HF concentration was the principal parameter determining HF induced skin effects. The intradermal HF retention capacity associated with progression and prolongation of HF induced skin effects must be considered in the review of skin decontamination procedures. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. p38 mitogen-activated protein kinase activation by ultraviolet A radiation in human dermal fibroblasts.

    PubMed

    Le Panse, Rozen; Dubertret, Louis; Coulomb, Bernard

    2003-08-01

    UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen-activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH2-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.

  6. Ultraviolet A induces transport of compatible organic osmolytes in human dermal fibroblasts.

    PubMed

    Warskulat, Ulrich; Brookmann, Stefanie; Felsner, Ingo; Brenden, Heidi; Grether-Beck, Susanne; Häussinger, Dieter

    2008-12-01

    Compatible organic osmolytes, such as betaine, myo-inositol and taurine, are involved in cell protection. Human dermal fibroblasts accumulate these osmolytes and express mRNA specific for their transporting systems betaine-/gamma-amino-n-butyric acid (GABA) transporter (BGT-1), sodium-dependent myo-inositol transporter (SMIT) and taurine transporter (TAUT). Taurine uptake was about sixfold higher than that of betaine and myo-inositol. Compared with normoosmotic (305 mOsm/l) control, hyperosmotic exposure (405 mOsm/l) led to a twofold induction of osmolyte uptake. Ultraviolet A (UVA) upregulated osmolyte transporter mRNA levels and increased osmolyte uptake. Taurine inhibited UVA-induced interleukin-6 (Il-6) mRNA expression by 40%. Furthermore, Il-6 accumulation in the supernatants of UVA-irradiated dermal fibroblasts was much slower when cells were preincubated with taurine. These data indicate that taurine accumulation seems to be part of the fibroblast response to UVA radiation and may protect against UVA-induced Il-6 overexpression.

  7. UVA radiation impairs phenotypic and functional maturation of human dermal dendritic cells.

    PubMed

    Furio, Laetitia; Berthier-Vergnes, Odile; Ducarre, Blandine; Schmitt, Daniel; Peguet-Navarro, Josette

    2005-11-01

    There is now strong evidence that the ultraviolet A (UVA) part of the solar spectrum contributes to the development of skin cancers. Its effect on the skin immune system, however, has not been fully investigated. Here, we analyzed the effects of UVA radiation on dermal dendritic cells (DDC), which, in addition, provided further characterization of these cells. Dermal sheets were obtained from normal human skin and irradiated, or not, with UVA at 2 or 12 J per cm2. After a 2 d incubation, the phenotype of emigrant cells was analyzed by double immunostaining and flow cytometry. Results showed that migratory DDC were best characterized by CD1c expression and that only few cells co-expressed the Langerhans cell marker Langerin. Whereas the DC extracted from the dermis displayed an immature phenotype, emigrant DDC showed increased expression of HLA-DR and acquired co-stimulation and maturation markers. We showed here that UVA significantly decreased the number of viable emigrant DDC, a process related to increased apoptosis. Furthermore, UVA irradiation impaired the phenotypic and functional maturation of migrating DDC into potent antigen-presenting cells, in a concentration-dependent manner. The results provide further evidence that UVA are immunosuppressive and suggest an additional mechanism by which solar radiation impairs immune response.

  8. UVB-Induced Senescence of Human Dermal Fibroblasts Involves Impairment of Proteasome and Enhanced Autophagic Activity.

    PubMed

    Cavinato, Maria; Koziel, Rafal; Romani, Nikolaus; Weinmüllner, Regina; Jenewein, Brigitte; Hermann, Martin; Dubrac, Sandrine; Ratzinger, Gudrun; Grillari, Johannes; Schmuth, Matthias; Jansen-Dürr, Pidder

    2017-05-01

    In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

    PubMed Central

    Lai, Pei-Lun; Lin, Hsuan; Chen, Shang-Fu; Yang, Shang-Chih; Hung, Kuo-Hsuan; Chang, Ching-Fang; Chang, Hsiang-Yi; Lu, Frank Leigh; Lee, Yi-Hsuan; Liu, Yu-Chuan; Huang, Hsiao-Chun; Lu, Jean

    2017-01-01

    Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine. PMID:28303927

  10. CYCLOSPORIN A AFFECTS THE PROLIFERATION PROCESS IN NORMAL HUMAN DERMAL FIBROBLASTS.

    PubMed

    Janikowska, Grazyna; Janikowsk, Tomasz; Pyka, Alina; Wilczok, Adam; Mazurek, Urszula

    2016-01-01

    Cyclosporin A is an immunosuppressant drug that is used not only in solid transplant rejection, but also in moderate and severe forms of psoriasis, pyoderma, lupus or arthritis. Serious side effects of the drug such as skin cancer or gingival hyperplasia probably start with the latent proliferation process. Little is known about the influence of cyclosporin A on molecular signaling in epidermal tissue. Thus, the aim of this study was to estimate the influence of cyclosporin A on the process of proliferation in normal human dermal fibroblasts. Fibroblasts were cultured in a liquid growth medium in standard conditions. Cyclosporin A was added to the culture after the confluence state. Survival and proliferation tests on human dermal fibroblast cells were performed. Total RNA was extracted from fibroblasts, based on which cDNA and cRNA were synthesized. The obtained cRNA was hybridized with the expression microarray HGU-133A_2.0. Statistical analysis of 2734 mRNAs was performed by the use of GeneSpring 13.0 software and only results with p < 0.05 were accepted. Analysis of variance with Tukey post hoc test with Benjamini-Hochberg correction for all three (8, 24, 48 h) culture stages (with and without cyclosporin A) was performed to lower the number of statistically significant results from 679 to 66, and less. Between statistically and biologically significant mRNAs down-regulated were EGRJ, BUBIB, MKI67, CDK1, TTK, E2F8, TPX2, however, the INSIG1, FOSL1, HMOX1 were up-regulated. The experiment data revealed that cyclosporin A up-regulated FOSL1 in the first 24 h, afterwards down-regulating its expression. The HMOX1 gene was up-regulated in the first stage of the experiment (CsA 8 h), however, after the next 16 h of culture time its expression was down-regulated (CsA 24 h), to finally increased in the later time period. The results indicate that cyclosporin A had a significant effect on proliferation in normal human dermal fibroblasts through the changes in the

  11. Specific Age-Associated DNA Methylation Changes in Human Dermal Fibroblasts

    PubMed Central

    Lin, Qiong; Bork, Simone; Goergens, Maria; Joussen, Sylvia; Pallua, Norbert; Ho, Anthony D.; Zenke, Martin; Wagner, Wolfgang

    2011-01-01

    Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner. PMID:21347436

  12. Tetracycline suppresses ATP gamma S-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human dermal microvascular endothelial cells.

    PubMed

    Bender, Anna; Zapolanski, Tamar; Watkins, Shannon; Khosraviani, Ava; Seiffert, Kristina; Ding, Wanhong; Wagner, John A; Granstein, Richard D

    2008-09-01

    Tetracyclines (TCN) have powerful anti-inflammatory properties in addition to their anti-microbial effects. These anti-inflammatory effects are thought to play a role in inhibiting cutaneous inflammation in patients with rosacea and acne; however, the mechanism(s) of this action remains poorly understood. We have previously shown that adenosine-5'-triphosphate (ATP)gamma S, a hydrolysis-resistant ATP analogue, augments secretion of pro-inflammatory messengers by a human dermal microvascular endothelial cell line (HMEC-1). ATP released by the sympathetic nerves during stress may stimulate release of pro-inflammatory chemokines by dermal vessel endothelial cells, resulting in recruitment of inflammatory cells and exacerbation of inflammatory skin disease. Here we demonstrate that TCN inhibits ATP gamma S-induced release of pro-inflammatory mediators by HMEC-1 cells and primary human dermal microvascular endothelial cells. TCN dose-dependently inhibited ATP gamma S-induced augmentation of CXCL8 (interleukin-8) and CXCL1 (growth-regulated oncogene-alpha) production by HMEC-1 cells and primary human dermal endothelial cells in vitro. TCN and ATP gamma S did not affect HMEC-1 cell viability as determined by trypan-blue exclusion and cell counts. Inhibition of production of inflammatory mediators by endothelial cells may be one mechanism by which TCN improves inflammatory skin diseases. The ability to inhibit release of inflammatory mediators induced in HMEC-1 cells by purinergic agonists may be a useful way to screen for potential therapeutic agents for cutaneous inflammation.

  13. Antioxidant effects of the sarsaparilla via scavenging of reactive oxygen species and induction of antioxidant enzymes in human dermal fibroblasts.

    PubMed

    Park, Gunhyuk; Kim, Tae-mi; Kim, Jeong Hee; Oh, Myung Sook

    2014-07-01

    Ultraviolet (UV) radiation from sunlight causes distinct changes in collagenous skin tissues as a result of the breakdown of collagen, a major component of the extracellular matrix. UV irradiation downregulates reactive oxygen species (ROS)-elimination pathways, thereby promoting the production of ROS, which are implicated in skin aging. Smilax glabra Roxb (sarsaparilla) has been used in folk medicine because of its many effects. However, no study on the protective effects of sarsaparilla root (SR) on human dermal fibroblasts has been reported previously. Here, we investigated the protective effect of SR against oxidative stress in dermal fibroblasts. SR significantly inhibited oxidative damage and skin-aging factor via mitogen-activated protein kinase signaling pathways. Also, SR decreased Ca(2+) and ROS, mitochondrial membrane potential, dysfunction, and increased glutathione, NAD(P)H dehydrogenase and heme oxygenase-1. These results demonstrate that SR can protect dermal fibroblasts against UVB-induced skin aging via antioxidant effects.

  14. S-arylcysteine-keratin adducts as biomarkers of human dermal exposure to aromatic hydrocarbons.

    PubMed

    Kang-Sickel, Juei-Chuan C; Fox, Donii D; Nam, Tae-Gyu; Jayaraj, Karupiah; Ball, Louise M; French, John E; Klapper, David G; Gold, Avram; Nylander-French, Leena A

    2008-04-01

    To measure biomarkers of skin exposure to ubiquitous industrial and environmental aromatic hydrocarbons, we sought to develop an ELISA to quantitate protein adducts of metabolites of benzene and naphthalene in the skin of exposed individuals. We hypothesized that electrophilic arene oxides formed by CYP isoforms expressed in the human skin react with nucleophilic sites on keratin, the most abundant protein in the stratum corneum that is synthesized de novo during keratinocyte maturation and differentiation. The sulfhydryl groups of cysteines in the head region of the keratin proteins 1 (K1) and 10 (K10) are likely targets. The following synthetic S-arylcysteines were incorporated into 10-mer head sequences of K1 [GGGRFSS( S-aryl-C)GG] and K10 [GGGG( S-aryl-C)GGGGG] to form the predicted immunogenic epitopes for antibody production for ELISA: S-phenylcysteine-K1 (SPK1), S-phenylcysteine-K10 (SPK10), S-(1-naphthyl)cysteine-K1 (1NK1), S-(1-naphthyl)cysteine-K10 (1NK10), S-(2-naphthyl)cysteine-K1 (2NK1), and S-(2-naphthyl)cysteine-K10 (2NK10). Analysis by ELISA was chosen based on its high throughput and sensitivity, and low cost. The synthetic modified oligopeptides, available in quantity, served both as immunogens and as chemical standards for quantitative ELISA. Polyclonal rabbit antibodies produced against the naphthyl-modified keratins reacted with their respective antigens with threshold sensitivities of 15-31 ng/mL and high specificity over a linear range up to 500 ng/mL. Anti- S-phenylcysteine antibodies were not sufficiently specific or sensitive toward the target antigens for use in ELISA under our experimental conditions. In dermal tape-strip samples collected from 13 individuals exposed to naphthalene-containing jet fuel, naphthyl-conjugated peptides were detected at levels from 0.343 +/- 0.274 to 2.34 +/- 1.61 pmol adduct/microg keratin but were undetectable in unexposed volunteers. This is the first report of adducts of naphthalene (or of any polycyclic

  15. Genotoxicity assessment of reactive and disperse textile dyes using human dermal equivalent (3D cell culture system).

    PubMed

    Leme, Daniela Morais; Primo, Fernando Lucas; Gobo, Graciely Gomides; da Costa, Cleber Rafael Vieira; Tedesco, Antonio Claudio; de Oliveira, Danielle Palma

    2015-01-01

    Thousands of dyes are marketed daily for different purposes, including textile dyeing. However, there are several studies reporting attributing to dyes deleterious human effects such as DNA damage. Humans may be exposed to toxic dyes through either ingestion of contaminated waters or dermal contact with colored garments. With respect to dermal exposure, human skin equivalents are promising tools to assess in vitro genotoxicity of dermally applied chemicals using a three-dimensional (3D) model to mimic tissue behavior. This study investigated the sensitivity of an in-house human dermal equivalent (DE) for detecting genotoxicity of textile dyes. Two azo (reactive green 19 [RG19] and disperse red 1[DR1]) dyes and one anthraquinone (reactive blue 2 [RB2]) dye were analyzed. RG19 was genotoxic for DE in a dose-responsive manner, whereas RB2 and DR1 were nongenotoxic under the conditions tested. These findings are not in agreement with previous genotoxicological assessment of these dyes carried out using two-dimensional (2D) cell cultures, which showed that DR1 was genotoxic in human hepatoma cells (HepG2) and RG19 was nongenotoxic for normal human dermal fibroblasts (NHDF). These discrepant results probably may be due to differences between metabolic activities of each cell type (organ-specific genotoxicity, HepG2 and fibroblasts) and the test setup systems used in each study (fibroblasts cultured at 2D and three-dimensional [3D] culture systems). Genotoxicological assessment of textile dyes in context of organ-specific genotoxicity and using in vitro models that more closely resemble in vivo tissue architecture and physiology may provide more reliable estimates of genotoxic potential of these chemicals.

  16. GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

    PubMed

    Uehara, Eriko; Hokazono, Hideki; Hida, Mariko; Sasaki, Takako; Yoshioka, Hidekatsu; Matsuo, Noritaka

    2017-06-01

    The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-β-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

  17. The effect of moesin overexpression on ageing of human dermal microvascular endothelial cells.

    PubMed

    Lee, Ju Hee; Hong, In Ae; Oh, Sang Ho; Kwon, Yeon Sook; Cho, Soo Hyun; Lee, Kwang Hoon

    2009-11-01

    Senescence of microvascular endothelial cells is known to play an important role in the pathophysiology of vascular diseases related to ageing, but the accurate mechanism or related genes are not known. Moesin, a cytoskeletal protein and the most potent candidate as an ageing-related protein, showed obvious changes in expression when compared before and after ageing. In this study, a lentivirus was used to overexpress moesin in endothelial cells. The expression of cell cycle mediators such as p16, cyclin D1 and cdk4, which can be the markers of ageing, was compared by RNA and was shown to be suppressed in moesin overexpressed endothelial cells. In conclusion, it can be said that the expression of moesin delays senescence of human dermal microvascular endothelial cells and this fundamental discovery can be used as a basis for understanding the mechanism of ageing and age-related diseases.

  18. Inhibitory effects of (-)-loliolide on cellular senescence in human dermal fibroblasts.

    PubMed

    Yang, Hyo Hyun; Hwangbo, Kyoung; Zheng, Ming Shan; Cho, Jung Hee; Son, Jong-Keun; Kim, Hwa Young; Baek, Suk Hwan; Choi, Hyung Chul; Park, So Young; Kim, Jae-Ryong

    2015-01-01

    Cellular senescence influences tumor suppression and progress, tissue repair and regeneration, tissue and organismal aging, and age-related diseases. Aging intervention might be an advantageous target for prevention and treatment of diverse age-related diseases. In this study, we investigated whether (-)-loliolide purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). (-)-Loliolide diminished senescence-associated β-galactosidase activity (SA-β-gal), the level of p21 protein, and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. (-)-Loliolide also attenuated SA-β-gal activity in HDFs under replicative senescence. These findings imply that (-)-loliolide rescues cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that ameliorate tissue aging or age-associated diseases.

  19. Inhibitory effects of juglanin on cellular senescence in human dermal fibroblasts.

    PubMed

    Yang, Hyo Hyun; Hwangbo, Kyoung; Zheng, Ming Shan; Son, Jong-Keun; Kim, Hwa Young; Baek, Suk Hwan; Choi, Hyung Chul; Park, So Young; Kim, Jae-Ryong

    2014-07-01

    Cellular senescence contributes to tissue and organismal aging, tumor suppression and progress, tissue repair and regeneration, and age-related diseases. Thus, aging intervention might be a promising target for treatment and prevention of diverse age-related diseases. In the present study, we investigated whether juglanin purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). Juglanin decreased senescence-associated β-galactosidase activity (SA-β-gal) and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. Juglanin also repressed SA-β-gal activity in HDFs under replicative senescence. These results suggest that juglanin represses cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that alleviate tissue aging or age-related diseases.

  20. Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

    PubMed Central

    Romero, Ibeth; Téllez, Jair; Suárez, Yazmín; Cardona, Maria; Figueroa, Roger; Zelazny, Adrian; Gore Saravia, Nancy

    2010-01-01

    Background The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients. Methods The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts. Results 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates. Conclusion Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis. PMID:20856851

  1. Differential cytotoxic effects of sodium meta-arsenite on human cancer cells, dental papilla stem cells and somatic cells correlate with telomeric properties and gene expression.

    PubMed

    Jeon, Byeong-Gyun; Kumar, B Mohana; Kang, Eun-Ju; Maeng, Geun-Ho; Lee, Yeon-Mi; Hah, Young-Sool; Ock, Sun-A; Kwack, Dae-Oh; Park, Bong-Wook; Rho, Gyu-Jin

    2011-12-01

    We investigated the effects of sodium meta-arsenite (NaAsO(2)) on human cancer cells (MDA-MB-231, MCF-7 and U-87 MG), dental papilla tissue stem cells (DPSCs) and somatic cells [MRC-5 fetal fibroblasts and adult muscle cells (MCs)] by examining telomeric properties, endogenous reverse transcriptase (RT) activity and the expression of tumorigenesis-linked genes. Half maximal inhibitory concentration (IC(50)) values were higher in DPSCs and MCs, possessing longer telomere lengths when compared to cancer cells. Levels of telomerase and RT activity, and the expression of protein 53 (p53), B-cell lymphoma 2 (BCL2), nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB), transforming growth factor beta (TGFβ) and vascular endothelial growth factor (VEGF) were significantly lower in cancer cells following sodium meta-arsenite treatment, whereas the effect was absent or marginally detected in DPSCs and somatic cells. Collectively, sodium meta-arsenite effectively induced cellular cytotoxicity by inhibiting telomerase and RT activity, and down-regulating transcript levels in cancer cells with shorter telomere lengths, whereas more tolerance was evident in DPSCs and somatic cells possessing longer telomere lengths.

  2. The effects of acellular amniotic membrane matrix on osteogenic differentiation and ERK1/2 signaling in human dental apical papilla cells.

    PubMed

    Chen, Yi-Jane; Chung, Min-Chun; Jane Yao, Chung-Chen; Huang, Chien-Hsun; Chang, Hao-Hueng; Jeng, Jiiang-Huei; Young, Tai-Horng

    2012-01-01

    The amniotic membrane (AM) has been widely used in the field of tissue engineering because of the favorable biological properties for scaffolding material. However, little is known about the effects of an acellular AM matrix on the osteogenic differentiation of mesenchymal stem cells. In this study, it was found that both basement membrane side and collagenous stroma side of the acellular AM matrix were capable of providing a preferential environment for driving the osteogenic differentiation of human dental apical papilla cells (APCs) with proven stem cell characteristics. Acellular AM matrix potentiated the induction effect of osteogenic supplements (OS) such as ascorbic acid, β-glycerophosphate, and dexamethasone and enhanced the osteogenic differentiation of APCs, as seen by increased core-binding factor alpha 1 (Cbfa-1) phosphorylation, alkaline phosphatase activity, mRNA expression of osteogenic marker genes, and mineralized matrix deposition. Even in the absence of soluble OS, acellular AM matrix also could exert the substrate-induced effect on initiating APCs' differentiation. Especially, the collagenous stroma side was more effective than the basement membrane side. Moreover, the AM-induced effect was significantly inhibited by U0126, an inhibitor of extracellular signaling-regulated kinase 1/2 (ERK1/2) signaling. Taken together, the osteogenic differentiation promoting effect on APCs is AM-specific, which provides potential applications of acellular AM matrix in bone/tooth tissue engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Brugia malayi infective larvae fail to activate Langerhans cells and dermal dendritic cells in human skin.

    PubMed

    Cotton, R N; McDonald-Fleming, R; Boyd, A; Spates, K; Nutman, T B; Tolouei Semnani, R

    2015-02-01

    Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.

  4. Development and manufacture of an investigational human living dermal equivalent (ICX-SKN).

    PubMed

    Flasza, Marzena; Kemp, Paul; Shering, David; Qiao, Jizeng; Marshall, Damian; Bokta, Ausha; Johnson, Penny A

    2007-11-01

    To design and manufacture an investigational living skin graft replacement (ICX-SKN) that is able to incorporate into the host, providing healing by primary intent without the need for a second intervention. The ICX-SKN skin graft replacement has been designed as an allogeneic dermal substitute comprising an extracellular matrix composed largely of human collagen and human dermal fibroblast cells (HDFs). ICX-SKN is first formed by casting a provisional matrix of fibrin, into which HDFs are seeded. Through a process of maturation, HDFs are induced to lay down collagen and other extracellular matrix materials and, as the construct matures, the original fibrin is largely replaced by collagen, which provides tensile strength and flexibility to the construct. In order to design a product and manufacturing system that lends itself to large-scale production the process was developed as a discontinuous process consisting of four stages: 1. batch casting and maturation of the initial construct (pSKN), 2. freeze-drying of pSKN to produce a second intermediate (dSKN), 3. sterilization by gamma-irradiation of dSKN to produce a third intermediate (sSKN), and finally, 4. repopulation of sSKN by fresh HDFs to produce the final product, ICX-SKN skin graft replacement. Preliminary characterization of ICX-SKN and its application in a preclinical model are described. The 7-week maturation period resulted in a construct (pSKN) with robust handling properties, which was composed mainly of human collagen I. Following development of a process for freeze-drying and subsequent sterilization, the matrix was successfully repopulated with fresh HDFs. In addition, it was demonstrated that human keratinocytes attached and differentiated on the matrix. Application of human keratinocytes to the repopulated constructs (ICX-SKN) resulted in expression of markers of basement membranes that was largely dependent on the presence of living HDFs on the constructs. ICX-SKN graft replacements applied to

  5. DERMAL, ORAL AND INHALATION PHARMACOKINETICS OF METHYL TERTIARY-BUTYL ETHER (MTBE) IN HUMAN VOLUNTEERS

    EPA Science Inventory


    Methyl tertiary butyl ether (MTBE), a gasoline additive used to increase octane and reduce carbon monoxide emissions and ozone precursors, has contaminated drinking water and can lead to exposure by oral, inhalation, and dermal routes. To determine its dermal, oral, and inhal...

  6. DERMAL, ORAL, AND INHALATION PHARMACOKINETICS OF METHYL TERTIARY BUTYL ETHER (MTBE) IN HUMAN VOLUNTEERS

    EPA Science Inventory

    Methyl tertiary butyl ether (MTBE), a gasoline additive, used to increase octane and reduce carbon monoxide emissions and ozone precursors has contaminated drinking water leading to exposure by oral, inhalation, and dermal routes. To determine its dermal, oral, and inhalation ki...

  7. DERMAL, ORAL AND INHALATION PHARMACOKINETICS OF METHYL TERTIARY-BUTYL ETHER (MTBE) IN HUMAN VOLUNTEERS

    EPA Science Inventory


    Methyl tertiary butyl ether (MTBE), a gasoline additive used to increase octane and reduce carbon monoxide emissions and ozone precursors, has contaminated drinking water and can lead to exposure by oral, inhalation, and dermal routes. To determine its dermal, oral, and inhal...

  8. DERMAL, ORAL, AND INHALATION PHARMACOKINETICS OF METHYL TERTIARY BUTYL ETHER (MTBE) IN HUMAN VOLUNTEERS

    EPA Science Inventory

    Methyl tertiary butyl ether (MTBE), a gasoline additive, used to increase octane and reduce carbon monoxide emissions and ozone precursors has contaminated drinking water leading to exposure by oral, inhalation, and dermal routes. To determine its dermal, oral, and inhalation ki...

  9. Lidocaine Impairs Proliferative and Biosynthetic Functions of Aged Human Dermal Fibroblasts.

    PubMed

    Bentov, Itay; Damodarasamy, Mamatha; Spiekerman, Charles; Reed, May J

    2016-09-01

    The aged are at increased risk of postoperative wound healing complications. Because local anesthetics are infiltrated commonly into the dermis of surgical wounds, we sought to determine whether local anesthetics adversely affect proliferative and biosynthetic functions of dermal fibroblasts. We also evaluated the effect of local anesthetics on insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1), growth factors that are important regulators of wound healing. Human dermal fibroblasts (HFB) from aged and young donors were exposed to local anesthetic agents at clinically relevant concentrations. We screened the effects of lidocaine, bupivacaine, mepivacaine, and ropivacaine on proliferation of HFB. Lidocaine was most detrimental to proliferation in HFB. We then evaluated the effect of lidocaine on expression and function of the growth factors, IGF-1 and TGF-β1. Lastly, concurrent exposure to lidocaine and IGF-1 or TGF-β1 was evaluated for their effects on proliferation and expression of dermal collagens, respectively. Lidocaine and mepivacaine inhibited proliferation in aged HFB (for lidocaine 88% of control, 95% confidence interval [CI], 80%-98%, P = .009 and for mepivacaine 90% of control, 95% CI, 81%-99%, P = .032) but not in young HFB. Ropivacaine and bupivacaine did not inhibit proliferation. Because of the clinical utility of lidocaine relative to mepivacaine, we focused on lidocaine. Lidocaine decreased proliferation in aged HFB, which was abrogated by IGF-1. Lidocaine inhibited transcripts for IGF-1 and insulin-like growth factor-1 receptor (IGF1R) in fibroblasts from aged donors (IGF-1, log2 fold-change -1.25 [42% of control, 95% CI, 19%-92%, P = .035] and IGF1R, log2 fold-change -1.00 [50% of control, 95% CI, 31%-81%, P = .014]). In contrast, lidocaine did not affect the expression of IGF-1 or IGF1R transcripts in the young HFB. Transcripts for collagen III were decreased after lidocaine exposure in aged and young HFB (log2

  10. Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts

    PubMed Central

    Purohit, Trupta; Qin, Zhaoping; Quan, Chunji; Lin, Zhenhua

    2017-01-01

    The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-β signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-β signaling. CCN2 acts as a downstream mediator of TGF-β/Smad signaling and acting synergistically with TGF-β to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-β/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin. PMID:28267785

  11. Effect of UVA fluence rate on indicators of oxidative stress in human dermal fibroblasts.

    PubMed

    Hoerter, James D; Ward, Christopher S; Bale, Kyle D; Gizachew, Admasu N; Graham, Rachelle; Reynolds, Jaclyn; Ward, Melanie E; Choi, Chesca; Kagabo, Jean-Leonard; Sauer, Michael; Kuipers, Tara; Hotchkiss, Timothy; Banner, Nate; Chellson, Renee A; Ohaeri, Theresa; Gant, Langston; Vanderhill, Leah

    2008-02-19

    During the course of a day human skin is exposed to solar UV radiation that fluctuates in fluence rate within the UVA (290-315 nm) and UVB (315-400 nm) spectrum. Variables affecting the fluence rate reaching skin cells include differences in UVA and UVB penetrating ability, presence or absence of sunscreens, atmospheric conditions, and season and geographical location where the exposure occurs. Our study determined the effect of UVA fluence rate in solar-simulated (SSR) and tanning-bed radiation (TBR) on four indicators of oxidative stress---protein oxidation, glutathione, heme oxygenase-1, and reactive oxygen species--in human dermal fibroblasts after receiving equivalent UVA and UVB doses. Our results show that the higher UVA fluence rate in TBR increases the level of all four indicators of oxidative stress. In sequential exposures when cells are exposed first to SSR, the lower UVA fluence rate in SSR induces a protective response that protects against oxidative stress following a second exposure to a higher UVA fluence rate. Our studies underscore the important role of UVA fluence rate in determining how human skin cells respond to a given dose of radiation containing both UVA and UVB radiation.

  12. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    PubMed

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  13. Characterization and tissue incorporation of cross-linked human acellular dermal matrix.

    PubMed

    Lee, Ju Hee; Kim, Hyung Goo; Lee, Won Jai

    2015-03-01

    Here, we describe a novel human acellular dermal matrix (ADM) cross-linked using electron beam irradiation. Structural and biomechanical characteristics of the human ADM were assessed by infrared spectrometry and uni-axial tensile testing. Electron beam irradiation affects collagen secondary structure, which can be detected in the amide I spectral region (1660 cm(-1) and 1690 cm(-1)). At doses exceeding 25 kGy, cross-linking of the collagen matrix results in a denser, more stratified appearance and parallel arrangement, with significantly increased tensile strength and elastic modulus. In a micropig model, the implanted ADM elicits rapid host cell infiltration and extracellular matrix deposition; however, the delayed remodeling resulted in long-term structural integrity. Furthermore, mean densities of collagen and elastin, expression of extracellular matrix proteins, and microvessel formation within the implanted ADM increased significantly, whereas the thickness of the implanted ADM did not decrease during the course of the study. Compared with normal adjacent tissue, type I collagen mRNA levels in the ADM increased 12-fold at 3 months after implantation, and transforming growth factor-β mRNA levels increased 3.3-fold at 2 months. Matrix metalloproteinase (MMP)-1 and MMP-9 mRNA levels were also elevated. Collectively, these results demonstrate that the structural and biomechanical properties of this novel cross-linked human ADM are adequate for use as a biologic tissue substitute. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Dermal uptake of phthalates from clothing: Comparison of model to human participant results.

    PubMed

    Morrison, G C; Weschler, C J; Bekö, G

    2016-11-11

    In this research, we extend a model of transdermal uptake of phthalates to include a layer of clothing. When compared with experimental results, this model better estimates dermal uptake of diethylphthalate and di-n-butylphthalate (DnBP) than a previous model. The model predictions are consistent with the observation that previously exposed clothing can increase dermal uptake over that observed in bare-skin participants for the same exposure air concentrations. The model predicts that dermal uptake from clothing of DnBP is a substantial fraction of total uptake from all sources of exposure. For compounds that have high dermal permeability coefficients, dermal uptake is increased for (i) thinner clothing, (ii) a narrower gap between clothing and skin, and (iii) longer time intervals between laundering and wearing. Enhanced dermal uptake is most pronounced for compounds with clothing-air partition coefficients between 10(4) and 10(7) . In the absence of direct measurements of cotton cloth-air partition coefficients, dermal exposure may be predicted using equilibrium data for compounds in equilibrium with cellulose and water, in combination with computational methods of predicting partition coefficients.

  15. Diverticulum With Papillae: Does Position of Papilla Affect Technical Success?

    PubMed

    Parlak, Erkan; Suna, Nuretdin; Kuzu, Ufuk B; Taşkiran, İsmail; Yildiz, Hakan; Torun, Serkan; Yüksel, Mahmut; Çiçek, Bahattin; Dişibeyaz, Selçuk; Şahin, Burhan

    2015-10-01

    The presence of peripapillary diverticulum (PPD) can cause some biliary diseases, especially common bile duct stones, and also, literally, can change the technique of endoscopic retrograde cholangiopancreatography (ERCP) and affect the complication ratio of this procedure. In this study, we investigate the effect of localization and position of the papilla according to the diverticulum on the success of therapeutic ERCP procedures. The study was conducted prospectively in the patients with naive papillae, who underwent ERCP for a period of 16 months. In all patients, the position of papillae according to the diverticulum (the periphery of the diverticulum is thought as the clock circumference, and the position of papillae is defined as the dials of clock), the success rate of biliary cannulation, total procedure time, overall treatment success rate of ERCP, and the complications are investigated. During this period, 222 (18.5%) of the 1205 enrolled patients who underwent ERCP had PPD. Of the patients with PPD, 123 (55.4%) were female and 99 (44.6%) were male, and the median age was 68.9±10.1 years. According to the position of the papilla by the diverticulum, 90 (40.5%) patients have it on 7 o'clock position, 64 (28.8%) patients have on 6 o'clock position, 63 (28.3%) patients have on 5 o'clock position, and 5 (2.3%) patients have on 1 o'clock position. In the cases of the papilla on 1 o'clock position according to the diverticulum, cannulation procedures were found to be more difficult than other patients (P<0.05). The presence of the diverticulum did not affect the success of therapeutic procedures and did not increase the ratio of complications. In the presence of PPD, additional cannulation techniques may be required for the procedure. Particularly, the aid of percutaneous techniques may be needed for the papilla on 1 o'clock position.

  16. Investigation of dermal toxicity of ionic liquids in monolayer-cultured skin cells and 3D reconstructed human skin models.

    PubMed

    Hwang, Jee-Hyun; Park, Hyeonji; Choi, Dal Woong; Nam, Ki Taek; Lim, Kyung-Min

    2017-09-25

    Ionic liquids have gained increasing attention in the chemical industry as potential green substitutes for traditional solvents. However, little is known about toxicity of ionic liquids on the skin, a major exposure portal to toxic substances. Here, we evaluated dermal toxicity of ionic liquids using human keratinocyte and fibroblast cell line, 3D reconstructed human epidermis, and full-thickness model to investigate underlying mechanisms. Cytotoxicity of ionic liquids was evaluated for representative anions, [TFSI], [PF6], [BF4], and [DCA], as well as for cations, [EMIM], [BMPY], [TBA] and [Zn], in human keratinocyte cell line, HaCaT, and human dermal fibroblasts. In our results, significant cytotoxicity was induced by ionic liquids with [TFSI] in both cell lines. Notably, cytotoxicity of [TFSI] containing ionic liquids was comparable to xylene, a toxic conventional organic solvent. Fluorescent and flow cytometric analysis revealed that [TFSI]-exposed cells underwent necrotic cell death. Reactive oxygen species (ROS) was increased while the amount of glutathione was decreased by [TFSI] in dose-dependent manner, which was reversed by antioxidant, N-acetylcysteine. In 3D reconstructed human epidermis and full-thickness model, a single application of [TFSI] induced toxicity although it was minimal and largely limited to epidermal layer. Collectively, these results demonstrated potential dermal toxicity of ionic liquids. Copyright © 2017. Published by Elsevier Ltd.

  17. Ultraviolet-B Protective Effect of Flavonoids from Eugenia caryophylata on Human Dermal Fibroblast Cells

    PubMed Central

    Patwardhan, Juilee; Bhatt, Purvi

    2015-01-01

    Background: The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. Objective: To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast cells. Materials and Methods: Protective ability of flavonoid-enriched (FE) fraction of clove was studied against UV-B induced cytotoxicity, anti-oxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2 antioxidant response element (Nrf2 ARE) pathway. Results: FE fraction showed a significant antioxidant potential. Pretreatment of cells with FE fraction (10–40 μg/ml) reversed the effects of UV-B induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: The present study demonstrated for the first time that the FE fraction from clove could confer UV-B protection probably through the Nrf2-ARE pathway, which included the down-regulation of Nrf2 and HO-1. These findings suggested that the flavonoids from clove could potentially be considered as UV-B protectants and can be explored further for its topical application to the area of the skin requiring protection. SUMMARY Pretreatment of human dermal fibroblast with flavonoid-enriched fraction of Eugenia caryophylata attenuated effects of ultraviolet-B radiationsIt also conferred protection through nuclear factor E2-related factor 2-antioxidant response pathway and increased tolerance of cells against oxidative stress

  18. Biomonitoring as a tool in the human health risk characterization of dermal exposure.

    PubMed

    Boogaard, P J

    2008-04-01

    Dermal exposure is an important factor in risk characterization. In occupational settings it becomes relatively more important because of the continuous reduction in inhalation exposure. In the public health arena, dermal exposure may also form a significant contribution to the total exposure. Dermal exposure, however, is difficult to assess directly because it is determined by a host of factors, which are difficult to quantify. As a consequence, dermal exposure is often estimated by application of models for external exposure. In combination with modeled or measured data for percutaneous penetration, these provide an estimate for the internal exposure that is directly related to the systemic effects. The advantages and drawbacks of EASE (Estimation and Assessment of Substance Exposure) and RISKOFDERM (Risk Assessment of Occupational Dermal Exposure), two models for external exposure that are mentioned in the Technical Guidance Document for the European Union risk assessments performed under the Existing Substances Regulation (EEC/793/93), are discussed. Although new chemicals regulation (REACh, 1907/2006/EC) is now in place in the European Union, the principles applied under the previous legislation do not change and the same models will continue to be used. The results obtained with these models for styrene, 2-butoxyethanol, and 1-methoxy-2-propanol in specific exposure scenarios are compared with an alternative method that uses biomonitoring data to assess dermal exposure. Actual external exposure measurements combined with measured or modeled percutaneous penetration data give acceptable results in risk assessment of dermal exposure, but modeled data of external dermal exposure should only be used if no other data are available. However, if available, biomonitoring should be considered the method of choice to assess (dermal) exposure.

  19. Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in human dermal fibroblasts

    PubMed Central

    Prasad, Raju Y.; Chastain, Paul D.; Nikolaishvili-Feinberg, Nana; Smeester, Lisa M.; Kaufmann, William K.; Fry, Rebecca C.

    2013-01-01

    The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 were examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA combed fibers. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway. PMID:22770119

  20. Photoprotective Effects of Cycloheterophyllin against UVA-Induced Damage and Oxidative Stress in Human Dermal Fibroblasts.

    PubMed

    Huang, Cheng-Hua; Li, Hsin-Ju; Wu, Nan-Lin; Hsiao, Chien-Yu; Lin, Chun-Nan; Chang, Hsun-Hsien; Hung, Chi-Feng

    2016-01-01

    Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H2O2) generation and down-regulate H2O2-induced MAPK phosphorylation. In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging.

  1. Complete artificial saliva alters expression of proinflammatory cytokines in human dermal fibroblasts.

    PubMed

    Malpass, Gloria E; Arimilli, Subhashini; Prasad, Gaddamanugu L; Howlett, Allyn C

    2013-07-01

    Complete artificial saliva (CAS) is a saliva substitute often used as a vehicle for test articles, including smokeless tobacco products. In the course of a study employing normal adult human dermal fibroblasts (HDFa) as a model in vitro, we discovered that CAS as a vehicle introduced a significant change in the expression of proinflammatory cytokines. To determine the effects of CAS on gene expression, real-time quantitative reverse-transcriptase PCR gene array analysis was used. Results indicate that robust changes in the expression of the proinflammatory cytokine interleukin 8 (IL8) and the vascular cell adhesion molecule 1 (VCAM1) occur within 5h of exposure to CAS. To determine whether CAS also alters cytokine release into the culture media, cytometric bead array assays for human inflammatory cytokines were performed. Analysis shows that CAS induced the release of IL8 and IL6. This study focused on determining which components in CAS were responsible for the proinflammatory response in HDFa. The following components were investigated: α-amylase, lysozyme, acid phosphatase, and urea. Results demonstrated that enzymatically active α-amylase induced gene expression for proinflammatory cytokines IL8, IL6, tumor necrosis factor-α, and IL1α and for VCAM1. Therefore, it is important to carefully evaluate the "vehicle effects" of CAS and its components in in vitro toxicology research.

  2. UV-protective effects of phytoecdysteroids from Microsorum grossum extracts on human dermal fibroblasts.

    PubMed

    Ho, Raimana; Teai, Taivini; Meybeck, Alain; Raharivelomanana, Phila

    2015-01-01

    Microsorum grossum (Polypodiaceae), locally called metuapua'a, is one of the most frequently used fern species in Polynesian traditional medicine. Fronds or rhizomes of this species are common ingredients of popular medicine recipes to cure various ailments. M. grossum frond and rhizome extracts contain, as their main bioactive components, phytoecdysteroids such as 20-hydroxyecdysone, known to have many interesting biological activities and considered to be adaptogenic compounds [1]. The skin-active effect of M. grossum extract was investigated in two ways on human dermal fibroblasts: a transcriptomic study with c-DNA array for gene expression modulation and a Stress Induced Premature Senescence (SIPS) test. The total extract of M. grossum up-regulates Heme Oxygenase 1 (HO1), an enzyme which protects cells from oxidative stress and which is responsible for skin photoimmunoprotection. The present paper also reports that premature senescence of human skin induced by repeated UV irradiations can be prevented by an ecdysteroid fraction of M. grossum. Our data indicate that extracts of M. grossum could protect skin against oxidative stresses and suggest that they could be used as innovative active cosmetic ingredients.

  3. Human keratinocyte growth and differentiation on acellular porcine dermal matrix in relation to wound healing potential.

    PubMed

    Zajicek, Robert; Mandys, Vaclav; Mestak, Ondrej; Sevcik, Jan; Königova, Radana; Matouskova, Eva

    2012-01-01

    A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

  4. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    PubMed

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  5. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells.

    PubMed

    Miyoshi, Keiko; Horiguchi, Taigo; Tanimura, Ayako; Hagita, Hiroko; Noma, Takafumi

    2015-01-01

    Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  6. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    PubMed Central

    Miyoshi, Keiko; Horiguchi, Taigo; Tanimura, Ayako; Hagita, Hiroko; Noma, Takafumi

    2015-01-01

    Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming. PMID:26339586

  7. Photoprotective Effects of Cycloheterophyllin against UVA-Induced Damage and Oxidative Stress in Human Dermal Fibroblasts

    PubMed Central

    Huang, Cheng-Hua; Li, Hsin-Ju; Wu, Nan-Lin; Hsiao, Chien-Yu; Lin, Chun-Nan; Chang, Hsun-Hsien; Hung, Chi-Feng

    2016-01-01

    Ultraviolet (UV) radiation, particularly ultraviolet A (UVA), is known to play a major role in photoaging of the human skin. Many studies have demonstrated that UV exposure causes the skin cells to generate reactive oxygen species and activates the mitogen-activated protein kinase (MAPK) pathway. Previous studies have also demonstrated that cycloheterophyllin has an antioxidant effect and can effectively scavenge free radicals. Extending the aforementioned investigations, in this study, human dermal fibroblasts were used to investigate the protective effect of cycloheterophyllin against UV-induced damage. We found that cycloheterophyllin not only significantly increased cell viability, but also attenuated the phosphorylation of MAPK after UVA exposure. Furthermore, cycloheterophyllin could reduce hydrogen peroxide (H2O2) generation and down-regulate H2O2-induced MAPK phosphorylation. In the in vivo studies, the topical application of cycloheterophyllin before UVA irradiation significantly decreased trans-epidermal water loss (TEWL), erythema, and blood flow rate. These results indicate that cycloheterophyllin is a photoprotective agent that inhibits UVA-induced oxidative stress and damage, and could be used in the research on and prevention of skin photoaging. PMID:27583973

  8. Apigenin prevents ultraviolet-B radiation induced cyclobutane pyrimidine dimers formation in human dermal fibroblasts.

    PubMed

    Britto, S Mary; Shanthakumari, D; Agilan, B; Radhiga, T; Kanimozhi, G; Prasad, N Rajendra

    2017-09-01

    Exposure to solar ultraviolet-B (UVB) radiation leads to the formation of cyclobutane pyrimidine dimers (CPDs). We investigated the protective effect of apigenin against UVB-induced CPDs formation in human dermal fibroblasts cells (HDFa). For this purpose, HDFa cells were treated with apigenin (15μM) prior to UVB irradiation (20mJ/cm(2)); DNA damage and subsequent molecular end points were observed. Exposure to UVB radiation increased significant CPDs formation in HDFa cells and the frequencies of CPDs were reduced by treatment with apigenin (15μM). UVB-induced CPDs downregulates the expression of nucleotide excision repair (NER) genes such as xeroderma pigmentosum complementation group C, B, G and F (XPC, XPB, XPG and XPF), transcription factor II human (TFIIH) and excision repair cross-complementation group 1 (ERCC1) in HDFa cells. Conversely, apigenin treatment restored UVB-induced loss of NER proteins in HDFa cells, which indicates its preventive effect against CPDs formation. Besides, single low dose UVB-exposure induced nuclear fragmentation, apoptotic frequency and apoptotic proteins expression (Bax and Caspase-3) have been prevented by the apigenin pretreatment. Furthermore, apigenin exhibits strong UV absorbance property and showed 10.08 SPF value. Thus, apigenin can protect skin cells against UVB-induced CPDs formation probably through its sunscreen effect. Hence, apigenin can be considered as an effective protective agent against UV induced skin damages. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts

    PubMed Central

    Budiyanto, Arief; Soebono, Hardyanto

    2016-01-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm-2) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined. PMID:27401663

  10. Protecting effect of phytoncide solution, on normal human dermal fibroblasts against reactive oxygen species.

    PubMed

    Fujimori, Hiroaki; Hisama, Masayoshi; Shibayama, Hiroharu; Iwaki, Masahiro

    2009-01-01

    Four types of phytoncide solutions (A-Type, AB-Type, D-Type and G-Type) was evaluated for reduction of cell damage induced by oxidative stress, ultraviolet A (UVA), ultraviolet B (UVB), hydroxyperoxide (H2O2) and t-butyl-hydroperoxide (t-BHP); stimulation of collagen synthesis against UVA irradiation; and inhibition of matrix metalloproteinase-1 (MMP-1) activity induced by UVA in human normal dermal fibroblasts and human reconstituted skin model. The A-Type, AB-Type, D-Type and G-Type of phytoncide solutions pretreatment resulted in significant protection against cell damage induced by UVB, UVA, H2O2 and t-BHP. The amount of type I collagen following UVA irradiation was increased by treatment with phytoncide solutions in a concentration-dependent manner. On the other hand, phytoncide solutions also suppressed the excess MMP-1 irradiated UVA in a concentration-dependent manner. These effects of G-type solution were superior to those of other types solutions.

  11. Effects of silver nanoparticles on human dermal fibroblasts and epidermal keratinocytes.

    PubMed

    Galandáková, A; Franková, J; Ambrožová, N; Habartová, K; Pivodová, V; Zálešák, B; Šafářová, K; Smékalová, M; Ulrichová, J

    2016-09-01

    Biomedical application of silver nanoparticles (AgNPs) has been rapidly increasing. Owing to their strong antimicrobial activity, AgNPs are used in dermatology in the treatment of wounds and burns. However, recent evidence for their cytotoxicity gives rise to safety concerns. This study was undertaken as a part of an ongoing programme in our laboratory to develop a topical agent for wound healing. Here, we investigated the potential toxicity of AgNPs using normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) with the aim of comparing the effects of AgNPs and ionic silver (Ag-I). Besides the effect of AgNPs and Ag-I on cell viability, the inflammatory response and DNA damage in AgNPs and Ag-I-treated cells were examined. The results showed that Ag-I were significantly more toxic than AgNPs both on NHDF and NHEK. Non-cytotoxic concentrations of AgNPs and Ag-I did not induce DNA strand breaks and did not affect inflammatory markers, except for a transient increase in interleukin 6 levels in Ag-I-treated NHDF. The results showed that AgNPs are more suitable for the intended application as a topical agent for wound healing up to the concentration 25 µg/mL.

  12. Muse Cells Derived from Dermal Tissues Can Differentiate into Melanocytes.

    PubMed

    Tian, Ting; Zhang, Ru-Zhi; Yang, Yu-Hua; Liu, Qi; Li, Di; Pan, Xiao-Ru

    2017-02-07

    The objective of the authors has been to obtain multilineage-differentiating stress-enduring cells (Muse cells) from primary cultures of dermal fibroblasts, identify their pluripotency, and detect their ability to differentiate into melanocytes. The distribution of SSEA-3-positive cells in human scalp skin was assessed by immunohistochemistry, and the distribution of Oct4, Sox2, Nanog, and SSEA-3-positive cells was determined by immunofluorescence staining. The expression levels of Sox2, Oct4, hKlf4, and Nanog mRNAs and proteins in Muse cells were determined by reverse transcription polymerase chain reaction (RT-PCR) analyses and Western blots, respectively. These Muse cells differentiated into melanocytes in differentiation medium. The SSEA-3-positive cells were scattered in the basement membrane zone and the dermis, with comparatively more in the sebaceous glands, vascular and sweat glands, as well as the outer root sheath of hair follicles, the dermal papillae, and the hair bulbs. Muse cells, which have the ability to self-renew, were obtained from scalp dermal fibroblasts by flow cytometry sorting with an anti-SSEA-3 antibody. The results of RT-PCR, Western blot, and immunofluorescence staining showed that the expression levels of Oct4, Nanog, Sox2, and Klf4 mRNAs and proteins in Muse cells were significantly different from their parental dermal fibroblasts. Muse cells differentiated into melanocytes when cultured in melanocyte differentiation medium, and the Muse cell-derived melanocytes expressed the melanocyte-specific marker HMB45. Muse cells could be obtained by flow cytometry from primary cultures of scalp dermal fibroblasts, which possessed the ability of pluripotency and self-renewal, and could differentiate into melanocytes in vitro.

  13. Human skin gene expression: Natural (trans) resveratrol versus five resveratrol analogs for dermal applications.

    PubMed

    Lephart, Edwin D; Andrus, Merritt B

    2017-09-01

    Resveratrol (RV) is a polyphenolic compound naturally produced by plants. Polyphenolic compounds incorporated into medicinal products are beneficial but, RV is rapidly metabolized with an associated decline in biological activity. This study tested RV as the standard and compared five structurally modified RV analogs: butyrate, isobutyrate, palmitoate, acetate, and diacetate (to improve functionality) at 1% concentration(s) for 24 h in epiderm full thickness cultures by gene array/qPCR mRNA analysis. When silent mating type information regulation 2 homolog 1, extracellular elements (collagen1A1, 3A1, 4A1; elastin, tissue inhibitor of matrix metalloproteinase 1, fibrillin 1 laminin beta1 and matrix metalloproteinase 9), anti-aging and aging genes, inflammatory biomarkers (interleukin-1A [IL1A], IL1R2, IL-6 and IL-8), nerve growth factor, and the antioxidants (proliferating cell nuclear antigen, catalase, superoxide dismutase and metallothionein 1H/2H) were evaluated, ranking each from highest-to-lowest for gene expression: butyrate > isobutyrate > diacetate > acetate > palmitoate. This study showed that the butyrate and isobutyrate analogs are more biologically active compared to resveratrol and have potential use in topical applications to improve dermal and other health applications. Impact statement Resveratrol has been reported to have a wide variety of health benefits but its rapid metabolism especially after oral ingestion results in very low bioavailability. Notably, the first human skin gene expression study of resveratrol was not published until 2014. The purpose of this study was to determine if increased stability and biological activity could be obtained by modifying the chemical structure of natural (trans) resveratrol and quantifying human gene expression by qPCR of skin biomarkers that enhance dermal health. Five resveratrol analogs were synthesized that increased their lipophilic index to enhance tissue penetration and augment

  14. Contaminated soils (I): In vitro dermal absorption of benzo[a]pyrene in human skin.

    PubMed

    Moody, Richard P; Joncas, Julie; Richardson, Mark; Chu, Ih

    2007-11-01

    Dermal absorption of the lipophile and potential carcinogen benzo[a]pyrene (BaP) in soils from contaminated sites was simulated in vitro using human skin exposed to 14C-BaP-spiked soil. This study is the first in a series of tests at Health Canada with several soil contaminants spanning a wide range of lipophilicity conducted with viable human skin. Breast skin was obtained fresh from a local hospital and dermatomed to a thickness of 0.4-0.5 mm. Teflon Bronaugh diffusion cells were perfused with HEPES buffered Hanks saline (pH 7.4) with 4% bovine serum albumin (BSA) and fractions were collected at 6-h intervals for up to 24 h exposure either to 14C-BaP applied in acetone or spiked in a commercial gardening soil. As skin depot 14C levels were still high at 24 h, the study was repeated for up to 42 h to examine skin depot bioavailability. Skin was washed with soapy water at 24 h in both the 24- and 42-h studies. Exposure to 14C-BaP both with and without soil was conducted in triplicate with skin specimens from at least 4 patients. In the 24-h exposure tests including the skin depot there was 15 and 56% absorption with and without soil, respectively. The lower total percent absorption from the spiked soil applied to skin resulted from lower depot absorption of 8% with and 45% without soil. Data for 42-h studies were similar and revealed no significant decrease in skin depot levels. Including the 42-h depots there was 16 and 50% absorption with and without soil, respectively, with respective depots of 7 and 39%. As there was no significant difference between the 24- and 42-h depots both with and without soil, the data suggest the depot for BaP was not bioavailable for at least the additional 18-h post soap wash exposure. The bioavailability of BaP is discussed in relation to previous in vitro and in vivo studies in perspective with dermal exposure to contaminated soils.

  15. Healing rates for challenging rotator cuff tears utilizing an acellular human dermal reinforcement graft

    PubMed Central

    Agrawal, Vivek

    2012-01-01

    Purpose: This study presents a retrospective case series of the clinical and structural outcomes (1.5 T MRI) of arthroscopic rotator cuff repair with acellular human dermal graft reinforcement performed by a single surgeon in patients with large, massive, and previously repaired rotator cuff tears. Materials and Methods: Fourteen patients with mean anterior to posterior tear size 3.87 ± 0.99 cm (median 4 cm, range 2.5–6 cm) were enrolled in the study and were evaluated for structural integrity using a high-field (1.5 T) MRI at an average of 16.8 months after surgery. The Constant-Murley scores, the Flexilevel Scale of Shoulder Function (Flex SF), scapular plane abduction, and strength were analyzed. Results: MRI results showed that the rotator cuff repair was intact in 85.7% (12/14) of the patients studied. Two patients had a Sugaya Type IV recurrent tear (2 of 14; 14.3%), which were both less than 1 cm. The Constant score increased from a preoperative mean of 49.72 (range 13–74) to a postoperative mean of 81.07 (range 45–92) (P value = 0.009). Flexilevel Scale of Shoulder Function (Flex SF) Score normalized to a 100-point scale improved from a preoperative mean of 53.69 to a postoperative mean of 79.71 (P value = 0.003). The Pain Score improved from a preoperative mean of 7.73 to a postoperative mean of 13.57 (P value = 0.008). Scapular plane abduction improved from a preoperative mean of 113.64° to a postoperative mean of 166.43° (P value = 0.010). The strength subset score improved from a preoperative mean of 1.73 kg to a postoperative mean of 7.52 kg (P value = 0.006). Conclusions: This study presents a safe and effective technique that may help improve the healing rates of large, massive, and revision rotator cuff tears with the use of an acellular human dermal allograft. This technique demonstrated favorable structural healing rates and statistically improved functional outcomes in the near term. Level of Evidence: 4. Retrospective case series. PMID

  16. Application of dermal microdialysis for the determination of bioavailability of clobetasol propionate applied to the skin of human subjects.

    PubMed

    Au, W L; Skinner, M F; Benfeldt, E; Verbeeck, R K; Kanfer, I

    2012-01-01

    Dermal microdialysis was used to assess the bioavailability of a topical corticosteroid, clobetasol propionate, following application onto the skin of human subjects. The penetration of clobetasol propionate from a 4% m/v ethanolic solution applied onto 4 sites on one forearm of healthy human volunteers was studied. A lipid emulsion, Intralipid®, was used as the perfusate and linear microdialysis probes with a 2-kDa cutoff were inserted intradermally at the designated sites. The results indicated that Intralipid could be used as a suitable perfusate for in vivo microdialysis of this lipophilic drug of interest. Furthermore, the study clearly demonstrated the application of dermal microdialysis as a valuable tool to assess the bioavailability/bioequivalence of clobetasol propionate penetration into the skin following topical application.

  17. 18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts.

    PubMed

    Hung, Chi-Feng; Hsiao, Chien-Yu; Hsieh, Wen-Hao; Li, Hsin-Ju; Tsai, Yi-Ju; Lin, Chun-Nan; Chang, Hsun-Hsien; Wu, Nan-Lin

    2017-01-01

    Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18β-glycyrrhetinic acid (18β-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18β-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18β-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18β-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18β-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18β-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18β-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects.

  18. Periplanosides A-C: new insect-derived dihydroisocoumarin glucosides from Periplaneta americana stimulating collagen production in human dermal fibroblasts.

    PubMed

    Yang, Yong-Xun; Luo, Qi; Hou, Bo; Yan, Yong-Ming; Wang, Yue-Hu; Tang, Jian-Jun; Dong, Xiao-Ping; Ma, Xiu-Ying; Yang, Tong-Hua; Zuo, Zhi-Li; Cheng, Yong-Xian

    2015-01-01

    Three new dihydroisocoumarin glucosides, termed periplanosides A-C (1-3), a known analog, pericanaside (4), and the other twenty known compounds were isolated from the insect Periplaneta americana. Their structures including absolute configurations were determined by comprehensive spectroscopic analyses and computational methods. Biological evaluation showed that compound 2 could stimulate collagen production by 31.2% in human dermal fibroblasts-adult (HDFa) at the concentration of 30 μM, indicating its significance in skin repair and ulcer.

  19. The lazaroid tirilazad is a new inhibitor of direct and indirect UVA-induced lipid peroxidation in human dermal fibroblasts.

    PubMed

    Dissemond, J; Schneider, L A; Wlaschek, M; Brauns, T C; Goos, M; Scharffetter-Kochanek, K

    2003-12-01

    Lipid peroxidation caused by oxidative stress within the tissue leads to destruction and dysfunction of cellular membranes. Human dermal fibroblasts in the skin are subject to constant photooxidative stress caused mainly by deeply penetrating UVA irradiation. Therefore, the membrane damage caused by this photooxidative stress may be a major promoter of photoaging and photocarcinogenic processes initiated and promoted by long-term UVA exposure of the skin. The oxidative destruction is counterbalanced by a complex network of enzymatic and nonenzymatic antioxidants creating the skin's line of defence against UVA-induced reactive oxygen species. The lazaroid tirilazad represents a new synthetic group of antioxidants with structural molecular similarity to glucocorticosteroids. We investigated the antioxidative capacity of tirilazad by determining its effects on the levels of malondialdehyde (MDA), as a marker of lipid peroxidation, induced directly or indirectly by UVA in human dermal fibroblasts. In a time- and dose-dependent kinetic, we demonstrated that fibroblasts incubated with tirilazad are well protected against subsequent UVA irradiation and show no increase in MDA levels similar to the unirradiated controls. This was also observed when lipid peroxidation was caused chemically by incubation of human dermal fibroblasts with 200 micro M Fe(3+)-citrate and 1 m M ascorbyl phosphate as a model of indirect UVA-induced skin damage. Lysates of fibroblasts treated this way showed a tenfold increase in MDA levels, whereas preincubation with tirilazad resulted in a significantly lower increase in MDA levels. Furthermore, in a comparison with the well-established radical scavenger Trolox, an alpha-tocopherol analogue, tirilazad offered better protection to the membranes. Our results demonstrate for the first time that the lazaroid tirilazad is an effective inhibitor of direct and indirect UVA-induced increases in MDA as a marker of lipid peroxidation in human dermal

  20. Induction of hepatocyte growth factor production in human dermal fibroblasts by caffeic acid derivatives.

    PubMed

    Kurisu, Manami; Nakasone, Rie; Miyamae, Yusaku; Matsuura, Daisuke; Kanatani, Hirotoshi; Yano, Shingo; Shigemori, Hideyuki

    2013-01-01

    Hepatocyte growth factor (HGF) has mitogenic, motogenic, and morphogenic activities in epithelial cells. Induction of HGF production may be involved in organ regeneration, wound healing and embryogenesis. In this study, we examined the effects of caffeic acid derivatives including 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) on HGF production in Neonatal Normal Human Dermal Fibroblasts (NHDF). Both 4,5-di-O-caffeoylquinic acid (1) and acteoside (2) significantly induced HGF production dose-dependent manner. To know the important substructure for HGF production activity, we next investigated the effect of the partial structure of these caffeic acid derivatives. From the results, caffeic acid (3) showed strong activity on the promotion of HGF production, while hydroxytyrosol (4) and quinic acid (5) didn't show any activity. Our findings suggest that the caffeoyl moiety of caffeic acid derivatives is essential for accelerated production of HGF. The compound which has the caffeoyl moiety may be useful for the treatment of some intractable organ disease.

  1. The influence of substrate stiffness gradients on primary human dermal fibroblasts.

    PubMed

    Hopp, Isabel; Michelmore, Andrew; Smith, Louise E; Robinson, David E; Bachhuka, Akash; Mierczynska, Agnieszka; Vasilev, Krasimir

    2013-07-01

    Materials mechanical properties are known to be an important regulator of cellular processes such as proliferation, differentiation and migration, and have seen increasing attention in recent years. At present, there are only few approaches where the mechanical properties of thin films can be controllably varied across an entire surface. In this work, we present a technique for controlled generation of gradients of surface elastic moduli involving a weak polyelectrolyte multilayer (PEM) system of approximately 100 nm thickness and time dependent immersion in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a crosslinking agent. Uniform surface chemistry across the gradient and wettability was provided by the addition of a 10 nm thick plasma polymer layer deposited from vapour of either allylamine or acrylic acid. We used the resultant stiffness gradients (0.5-110 MPa in hydrated state) to investigate the adhesion, morphology and proliferation on human dermal fibroblasts (HDFs). We show that substrate mechanical properties strongly influence HDF cell fate. We also found that in the experimental range of surface properties used in this study, the surface stiffness was a stronger driving force to cells fate compared to chemistry and wettability.

  2. Anti-Photoaging Effects of Angelica acutiloba Root Ethanol Extract in Human Dermal Fibroblasts

    PubMed Central

    Park, Min Ah; Sim, Mi Ja; Kim, Young Chul

    2017-01-01

    The effects that ultraviolet rays elicit on collagen synthesis and degradation are the most common causes of wrinkle formation and photo-aging in skin. The objectives of this study were to evaluate the effects of Angelica acutiloba root ethanol extract (AAEE) to promote collagen synthesis and inhibit collagen degradation in human dermal fibroblasts. By examining total polyphenol and flavonoid contents, electron donating ability, radical scavenging activity, and superoxide dismutase-like activity, we found that AAEE exhibited fairly good antioxidant activity. Treatment with AAEE significantly increased type I procollagen production by cultured fibroblasts, as well as reduced ultraviolet-induced matrix metalloproteinase-1 (MMP-1) expression and MMP-2 activity in a dose-dependent manner (p < 0.05). In addition, AAEE significantly increased TIMP-1 mRNA expression (p < 0.05), although without an associated dose-dependent increase in TIMP-1 protein expression. In summary, we suggest that AAEE may be a potentially effective agent for the prevention or alleviation of skin-wrinkle formation induced by ultraviolet rays. PMID:28503261

  3. Cordyceps militaris Extract Protects Human Dermal Fibroblasts against Oxidative Stress-Induced Apoptosis and Premature Senescence

    PubMed Central

    Park, Jun Myoung; Lee, Jong Seok; Lee, Ki Rim; Ha, Suk-Jin; Hong, Eock Kee

    2014-01-01

    Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS. PMID:25230212

  4. Glycerolized Reticular Dermis as a New Human Acellular Dermal Matrix: An Exploratory Study

    PubMed Central

    Ferrando, Pietro Maria; Balmativola, Davide; Cambieri, Irene; Scalzo, Maria Stella; Bergallo, Massimiliano; Annaratone, Laura; Casarin, Stefania; Fumagalli, Mara; Stella, Maurizio; Sapino, Anna; Castagnoli, Carlotta

    2016-01-01

    Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco’s Modified Eagle’s Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties. PMID:26918526

  5. Hydroxychloroquine modulates metabolic activity and proliferation and induces autophagic cell death of human dermal fibroblasts.

    PubMed

    Ramser, Bettina; Kokot, Agatha; Metze, Dieter; Weiss, Nina; Luger, Thomas A; Böhm, Markus

    2009-10-01

    Hydroxychloroquine (HCQ) is a commonly used therapeutic agent in skin disorders. Some reports also suggest that HCQ can be useful in fibroblastic diseases of the skin. Here, we investigated the effects of HCQ in human dermal fibroblasts (HDFs). HCQ significantly reduced the metabolic activity and suppressed cell proliferation (IC(50) = approximately 30 microM) of HDFs. The antiproliferative effect of HCQ was associated with decreased activation of the extracellular signal-regulated kinases 1/2 but not with inhibition of the mammalian target of the rapamycin pathway or with dephosphorylation of Akt. HCQ induced a distinct type of cell death in HDFs, characterized by surface exposure of phosphatidylserine but a lack of morphological signs of apoptosis and absence of DNA fragmentation. The HCQ-treated HDFs instead showed autophagic vacuoles with double membranes and digested organelle content. These vacuoles showed light-chain 3 immunostaining, in accordance with increased protein amounts of this autophagy marker. Induction of autophagic cell death by HCQ was also paralleled by increased expression of Beclin-1, a key regulator of autophagy. Our findings indicate that HDFs are target cells of HCQ and form a rationale on the basis of which the in vivo effects of antimalarials can be studied in patients with aberrant fibroblast function.

  6. Investigate correlation between mechanical property and aging biomarker in passaged human dermal fibroblasts.

    PubMed

    Kim, Kyung Sook; Park, Hun-Kuk; Lee, Jin-Woo; Kim, Young Il; Shin, Min Kyung

    2015-04-01

    Skin aging is associated with changes in both the mechanical properties of the skin and extracellular matrix (ECM) components. In this study, we investigated the relationships between mechanical property and aging biomarkers in passaged human dermal fibroblasts (HDFs). The stiffness of HDFs from passages 5-20 was assessed by atomic force microscopy. The ECM components including collagen, elastin, and fibrillin-1 and that of signaling molecules (SIRTs) were determined from each passage of cells. The stiffness of HDFs increased linearly from passages 5-15 and then became saturated: the average stiffness was 0.356 N/m at passages 5 and 1.186 N/m at passages 15, respectively. Expression of all aging biomarkers, including pro-collagen I and VII, elastin, fibrillin-1, and SIRT1 and SIRT6, were down-regulated by passaging. In particular, a change in the level of procollagen Type I was significantly associated with early aging, while a change in the level of fibrillin-1 was associated with late aging. All biomarkers except elastin showed a strong correlation with the cellular stiffness of HDFs. © 2015 Wiley Periodicals, Inc.

  7. Cordyceps militaris extract protects human dermal fibroblasts against oxidative stress-induced apoptosis and premature senescence.

    PubMed

    Park, Jun Myoung; Lee, Jong Seok; Lee, Ki Rim; Ha, Suk-Jin; Hong, Eock Kee

    2014-09-16

    Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

  8. Analysis of Gene Expression in Human Dermal Fibroblasts Treated with Senescence-Modulating COX Inhibitors

    PubMed Central

    Han, Jeong A.; Kim, Jong-Il

    2017-01-01

    We have previously reported that NS-398, a cyclooxygenase-2 (COX-2)–selective inhibitor, inhibited replicative cellular senescence in human dermal fibroblasts and skin aging in hairless mice. In contrast, celecoxib, another COX-2–selective inhibitor, and aspirin, a non-selective COX inhibitor, accelerated the senescence and aging. To figure out causal factors for the senescence-modulating effect of the inhibitors, we here performed cDNA microarray experiment and subsequent Gene Set Enrichment Analysis. The data showed that several senescence-related gene sets were regulated by the inhibitor treatment. NS-398 up-regulated gene sets involved in the tumor necrosis factor β receptor pathway and the fructose and mannose metabolism, whereas it down-regulated a gene set involved in protein secretion. Celecoxib up-regulated gene sets involved in G2M checkpoint and E2F targets. Aspirin up-regulated the gene set involved in protein secretion, and down-regulated gene sets involved in RNA transcription. These results suggest that COX inhibitors modulate cellular senescence by different mechanisms and will provide useful information to understand senescence-modulating mechanisms of COX inhibitors. PMID:28638310

  9. Adverse effects of titanium dioxide nanoparticles on human dermal fibroblasts and how to protect cells.

    PubMed

    Pan, Zhi; Lee, Wilson; Slutsky, Lenny; Clark, Richard A F; Pernodet, Nadine; Rafailovich, Miriam H

    2009-04-01

    The effects of exposure of human dermal fibroblasts to rutile and anatase TiO(2) nanoparticles are reported. These particles can impair cell function, with the latter being more potent at producing damage. The exposure to nanoparticles decreases cell area, cell proliferation, mobility, and ability to contract collagen. Individual particles are shown to penetrate easily through the cell membrane in the absence of endocytosis, while some endocytosis is observed for larger particle clusters. Once inside, the particles are sequestered in vesicles, which continue to fill up with increasing incubation time till they rupture. Particles coated with a dense grafted polymer brush are also tested, and, using flow cytometry, are shown to prevent adherence to the cell membrane and hence penetration of the cell, which effectively decreases reactive oxygen species (ROS) formation and protects cells, even in the absence of light exposure. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating can potentially play an important role in protecting cells and tissue from damage.

  10. Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

    PubMed

    Pasi, Francesca; Sanna, Samuele; Paolini, Alessandro; Alquati, Marco; Lascialfari, Alessandro; Corti, Maurizio Enrico; Liberto, Riccardo Di; Cialdai, Francesca; Monici, Monica; Nano, Rosanna

    2016-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

  11. Surgical repair of hip abductors. A new technique using Graft Jacket allograft acellular human dermal matrix.

    PubMed

    Rao, Biyyam M; Kamal, Tamer T; Vafaye, John; Taylor, Lee

    2012-10-01

    Avulsion of the abductors from the hip can be an infrequent but debilitating complication after total hip arthroplasty performed through a trans-gluteal approach. This can result in intractable pain, limp, Trendelenberg lurch and instability of the hip. There have been various methods described for repairing or reconstruction of this abductor muscle complex including direct trans-osseous repair, muscle transfers, muscle and tendon sling, bone tendon allograft reconstruction and endoscopic repair techniques. In a prospective study at our institution we evaluated the results of a surgical technique in 12 patients using a trans-osseous repair of gluteus medius and minimus insertions augmented by a Graft Jacket allograft acellular human dermal matrix (Graft Jacket; Wright Medical Technology, Arlington, TN) over the anterior and anterolateral aspects of the greater trochanter. Diagnosis of hip abductor avulsions was made by evaluation of the history of presenting complaint, clinical examination and confirmed by ultrasound or MRI scans. Evaluation of results included pain scoring, gait evaluation, Trendelenberg test, and the Harris hip score. There was a significant improvement in pain (VAS mean values 8.25 to 2.33; p value < 0.0001), limp and gait along with abductor strength. The Trendelenberg test became negative in all but one. At the mean follow up of 22 months Harris hip scores improved from 34.05 to 81.26 (p value <0.0001). Overall this procedure appears to be safe and associated with high patient satisfaction, without the morbidity of any tendon or muscle transfers.

  12. Automated Delineation of Dermal-Epidermal Junction In Reflectance Confocal Microscopy Image Stacks Of Human Skin

    PubMed Central

    Park, Brian

    2014-01-01

    Reflectance confocal microscopy (RCM) images skin non-invasively, with optical sectioning and nuclear-level resolution comparable to that of pathology. Based on assessment of the dermal-epidermal junction (DEJ) and morphologic features in its vicinity, skin cancer can be diagnosed in vivo with high sensitivity and specificity. However, the current visual, qualitative approach for reading images leads to subjective variability in diagnosis. We hypothesize that machine learning-based algorithms may enable a more quantitative, objective approach. Testing and validation was performed with two algorithms that can automatically delineate the DEJ in RCM stacks of normal human skin. The test set was composed of 15 fair and 15 dark skin stacks (30 subjects) with expert labellings. In dark skin, in which the contrast is high due to melanin, the algorithm produced an average error of 7.9±6.4μm. In fair skin, the algorithm delineated the DEJ as a transition zone, with average error of 8.3±5.8μm for the epidermis-to-transition zone boundary and 7.6±5.6μm for the transition zone-to-dermis. Our results suggest that automated algorithms may quantitatively guide the delineation of the DEJ, to assist in objective reading of RCM images. Further development of such algorithms may guide assessment of abnormal morphological features at the DEJ. PMID:25184959

  13. Automated delineation of dermal-epidermal junction in reflectance confocal microscopy image stacks of human skin.

    PubMed

    Kurugol, Sila; Kose, Kivanc; Park, Brian; Dy, Jennifer G; Brooks, Dana H; Rajadhyaksha, Milind

    2015-03-01

    Reflectance confocal microscopy (RCM) images skin noninvasively, with optical sectioning and nuclear-level resolution comparable with that of pathology. On the basis of the assessment of the dermal-epidermal junction (DEJ) and morphologic features in its vicinity, skin cancer can be diagnosed in vivo with high sensitivity and specificity. However, the current visual, qualitative approach for reading images leads to subjective variability in diagnosis. We hypothesize that machine learning-based algorithms may enable a more quantitative, objective approach. Testing and validation were performed with two algorithms that can automatically delineate the DEJ in RCM stacks of normal human skin. The test set was composed of 15 fair- and 15 dark-skin stacks (30 subjects) with expert labelings. In dark skin, in which the contrast is high owing to melanin, the algorithm produced an average error of 7.9±6.4 μm. In fair skin, the algorithm delineated the DEJ as a transition zone, with average error of 8.3±5.8 μm for the epidermis-to-transition zone boundary and 7.6±5.6 μm for the transition zone-to-dermis. Our results suggest that automated algorithms may quantitatively guide the delineation of the DEJ, to assist in objective reading of RCM images. Further development of such algorithms may guide assessment of abnormal morphological features at the DEJ.

  14. Effects of photodynamic treatment on contraction of collagen gels by cultured human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Szeimies, Rolf-Markus; Bosserhoff, Anja K.; Hein, Ruediger; Dylla, Armin; Baeumler, Wolfgang; Landthaler, Michael

    1995-03-01

    Usually cell viability (CV) after PDT is observed in methods based on metabolic parameters (MTT assay) or dye exclusion tests [trypan-blue (TBT)]. Although these tests are accurate in the determination of cell death, functional impairment can not be easily detected. We examined the effects of PDT on human dermal fibroblasts (DF) to determine whether collagen gel contraction (CGC) can be inhibited and whether it correlates well with CV as measured by the TBT. Five-aminolevulinic acid (5-ALA) (1, 5, 10 (mu) g/ml) was added to DF cultured in dishes. Twenty-four hours later, a collagen type I solution was added and irradiation with a tunable dye laser (630 nm, 100 mW/cm2, 80 J/cm2) was performed. After 24 h, diameters of contracted gels and TBT were measured. As a result, PDT with 5-ALA inhibited CGC and affected CV as a function of dose level. Consequently, CGC data allows a better estimation of the threshold 5-ALA dose affecting the cells.

  15. Wound healing potential of Spirulina platensis extracts on human dermal fibroblast cells

    PubMed Central

    Syarina, Pauzi Nur Aimi; Karthivashan, Govindarajan; Abas, Faridah; Arulselvan, Palanisamy; Fakurazi, Sharida

    2015-01-01

    Blue-green alga (Spirulina platensis) is a well renowned nutri-supplement due to its high nutritional and medicinal properties. The aim of this study was to examine the wound healing efficiency of Spirulina platensis at various solvent extracts using in vitro scratch assay on human dermal fibroblast cells (HDF). Various gradient solvent extracts (50 μg/ml of methanolic, ethanolic and aqueous extracts) from Spirulina platensis were treated on HDF cells to acquire its wound healing properties through scratch assay and in this investigation we have used allantoin, as a positive control to compare efficacy among the phytoextracts. Interestingly, aqueous extract were found to stimulate proliferation and migration of HDF cells at given concentrations and enhanced closure rate of wound area within 24 hours after treatment. Methanolic and ethanolic extracts have shown proliferative effect, however these extracts did not aid in the migration and closure of wound area when compared to aqueous extract. Based on phytochemical profile of the plant extracts analyzed by LC-MS/MS, it was shown that compounds supposedly involved in accelerating wound healing are cinnamic acid, narigenin, kaempferol, temsirolimus, phosphatidylserine isomeric derivatives and sulphoquinovosyl diacylglycerol. Our findings concluded that blue-green algae may pose potential biomedical application to treat various chronic wounds especially in diabetes mellitus patients. PMID:27004048

  16. Effects of ethyl-α-d-glucoside on human dermal fibroblasts.

    PubMed

    Bogaki, Takayuki; Mitani, Keiichi; Oura, Yuki; Ozeki, Kenji

    2017-09-01

    Ethyl α-d-glucoside (α-EG) is a glycoside present in sake, Japanese rice wine. Previous studies have reported that α-EG suppresses skin roughness after ultraviolet B irradiation, transepidermal water loss, and hepatic function disorder, and has a skin moisturizing effect. In this study, 0.48 μM of α-EG was found to increase the proliferation of normal human dermal fibroblasts (NHDF) by 121.0%, and the amount of collagen I produced by NHDF increased by 159.6% at an α-EG concentration of 0.048 μM, compared to those in cells cultured without α-EG. In NHDF cultured in α-EG-supplemented medium, the expression of fibroblast growth factor I and VII mRNA increased by 148.8 and 153.1%, at an α-EG concentration of 4.8 and 0.048 μM, respectively, as measured by a quantitative reverse transcription-polymerase chain reaction. Transcript levels of type I collagen genes, COL1A1 and COL1A2, increased by 152.4 and 129.7%, respectively, and that of a type III collagen gene, COL3A1, increased by 131.8% at an α-EG concentration of 0.48 μM. These findings supported the possibility that α-EG was involved in the maintenance and improvement of skin homeostasis and moisturizing functions.

  17. Photocytotoxicity in human dermal fibroblasts elicited by permanent makeup inks containing titanium dioxide.

    PubMed

    Wamer, Wayne G; Yin, Jun-Jie

    2011-01-01

    Titanium dioxide (TiO2) is a pigment widely used in decorative tattoo and permanent makeup inks. However, little is known about the risks associated with its presence in these products. We have developed an in vitro assay to identify inks containing TiO2 that are cytotoxic and/or photocytotoxic. The presence of TiO2 in ten permanent makeup inks was established by X-ray fluorescence. Using X-ray diffraction, we found that seven inks contained predominately TiO2 (anatase), the more photocatalytically active crystalline form of TiO2. The remaining inks contained predominately TiO2 (rutile). To identify cytotoxic and/or photocytotoxic inks, human dermal fibroblasts were incubated for 18 h in media containing inks or pigments isolated from inks. Fibroblasts were then irradiated with 10 J/cm2 UVA radiation combined with 45 J/cm2 visible light for determining photocytotoxicity, or kept in the dark for determining cytotoxicity. Toxicity was assessed as inhibition of colony formation. No inks were cytotoxic. However eight inks, and the pigments isolated from these inks, were photocytotoxic. Using ESR, we found that most pigments from photocytotoxic inks generated hydroxyl radicals when photoexcited with UV radiation. Therefore, the possibility of photocytotoxicity should be considered when evaluating the safety of permanent makeup inks containing TiO2.

  18. Bioactivation, protein haptenation, and toxicity of sulfamethoxazole and dapsone in normal human dermal fibroblasts

    SciTech Connect

    Bhaiya, Payal; Roychowdhury, Sanjoy; Vyas, Piyush M.; Doll, Mark A.; Hein, David W.; Svensson, Craig K. . E-mail: craig-svensson@uiowa.edu

    2006-09-01

    Cutaneous drug reactions (CDRs) associated with sulfonamides are believed to be mediated through the formation of reactive metabolites that result in cellular toxicity and protein haptenation. We evaluated the bioactivation and toxicity of sulfamethoxazole (SMX) and dapsone (DDS) in normal human dermal fibroblasts (NHDF). Incubation of cells with DDS or its metabolite (D-NOH) resulted in protein haptenation readily detected by confocal microscopy and ELISA. While the metabolite of SMX (S-NOH) haptenated intracellular proteins, adducts were not evident in incubations with SMX. Cells expressed abundant N-acetyltransferase-1 (NAT1) mRNA and activity, but little NAT2 mRNA or activity. Neither NAT1 nor NAT2 protein was detected. Incubation of NHDF with S-NOH or D-NOH increased reactive oxygen species formation and reduced glutathione content. NHDF were less susceptible to the cytotoxic effect of S-NOH and D-NOH than are keratinocytes. Our studies provide the novel observation that NHDF are able to acetylate both arylamine compounds and bioactivate the sulfone DDS, giving rise to haptenated proteins. The reactive metabolites of SMX and DDS also provoke oxidative stress in these cells in a time- and concentration-dependent fashion. Further work is needed to determine the role of the observed toxicity in mediating CDRs observed with these agents.

  19. Selective staining of actin in live human dermal fibroblast cells using quantum dots

    NASA Astrophysics Data System (ADS)

    Adurkar, Udita S.; Agrawal, Amit; Nie, Shuming

    2005-03-01

    Semiconductor quantum dots (QD) are nanometer size fluorophores with improved brightness, resistance against photobleaching and narrow emission bands. These properties make QDs ideal for ultrasensitive imaging of biomolecules in living cells, in multiplexed format. By conjugating QDs with a delivery agent such as TAT peptide and a target-recognition element such as an antibody, we have delivered and imaged target-specific fluorescent probes in living cells. In this work, we demonstrate staining of actin filaments in living Human Dermal Fibroblast (HDF) cells using QD probes functionalized with monoclonal actin antibody. Actin probes were developed by coupling streptavidin coated QDs (λem = 605 nm, QDC Corp.) to biotinylated monoclonal β-actin antibody. Antibody molecules on QDs were conjugated with the TAT peptide. Finally, HDF cells were incubated with the QD-actin antibody-TAT construct. As expected, the characteristic fine streaks of actin filaments were observed in the cells and on the periphery of the cells, similar to phalloidin staining of actin filaments in fixed cells. Using a similar approach, one may image cellular components, proteins or nucleic acids, in a living cell, in real time.

  20. Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

    PubMed

    Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

    2017-09-01

    Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Ultraviolet a induces endoplasmic reticulum stress response in human dermal fibroblasts.

    PubMed

    Komori, Ryota; Taniguchi, Mai; Ichikawa, Yoshiaki; Uemura, Aya; Oku, Masaya; Wakabayashi, Sadao; Higuchi, Kazuhiko; Yoshida, Hiderou

    2012-01-01

    The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism against the accumulation of unfolded proteins in the ER (ER stress) that consists of three response pathways (the ATF6, IRE1 and PERK pathways) in mammals. These pathways regulate the transcription of ER-related genes through specific cis-acting elements, ERSE, UPRE and AARE, respectively. Because the mammalian ER stress response is markedly activated in professional secretory cells, its main function was thought to be to upregulate the capacity of protein folding in the ER in accordance with the increased synthesis of secretory proteins. Here, we found that ultraviolet A (UVA) irradiation induced the conversion of an ER-localized sensor pATF6α(P) to an active transcription factor pATF6α(N) in normal human dermal fibroblasts (NHDFs). UVA also induced IRE1-mediated splicing of XBP1 mRNA as well as PERK-mediated phosphorylation of an α subunit of eukaryotic initiation factor 2. Consistent with these observations, we found that UVA increased transcription from ERSE, UPRE and AARE elements. From these results, we concluded that UVA irradiation activates all branches of the mammalian ER stress response in NHDFs. This suggests that the mammalian ER stress response is activated by not only intrinsic stress but also environmental stress.

  2. Photoprotection of human dermal fibroblasts against ultraviolet light by antioxidant combinations present in tomato.

    PubMed

    Fernández-García, Elisabet

    2014-02-01

    In the current study, we evaluated and compared, for the first time in a cell model, the effect of lycopene alone or in association with various antioxidants present in tomato such as α-tocopherol or naringenin, on their capacity to protect against oxidative stress generated in human dermal fibroblasts (hdf) exposed to ultraviolet-A (UVA) light. UVA irradiation of hdf led to a reduced cell viability in a dose dependent manner. Similar effects were observed when cells were exposed to lycopene. This reduction was suppressed in the presence of naringenin but not with α-tocopherol. Reactive oxygen species production was strongly induced by UVA irradiation. Only co-incubation with naringenin (highest level) was able to inhibit this effect. The combination of lycopene : naringenin further increased the stability of the carotenoid. Heme-oxygenase 1 (HO-1) expression was induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in the study. Indeed, lycopene (1 μM) led to a further 2.5-fold rise in the UVA-induced HO-1 expression. However, this effect was suppressed by concomitant addition of naringenin. In our study, naringenin prevents oxidative degradation of lycopene. These results strengthen the hypothesis that combinations of dietary antioxidants present in tomato other than lycopene alone could play a role in the health effects of tomato as evidenced by epidemiological studies.

  3. Influence of silica particle internalization on adhesion and migration of human dermal fibroblasts.

    PubMed

    Zhang, Yuying; Hu, Ling; Yu, Dahai; Gao, Changyou

    2010-11-01

    In spite of significant advantages exhibiting in the applications of silicon dioxide particles in biological and medicine fields, their adverse effects still remain a big concern. Herein monodisperse spherical SiO(2) particles with diameters of 80 nm and 500 nm were used to study their interactions with human dermal fibroblasts. Both the particles were readily internalized into the fibroblasts within a short time. The 500 nm particles were taken up in a larger amount through macropinocytosis and clathrin-mediated endocytosis pathways, whereas uptake of the 80 nm SiO(2) particles was mediated corporately by macropinocytosis, clathrin-mediated and caveolae-mediated endocytosis. The particles mainly dispersed in the cytoplasm or resided within the lysosomal vesicles, but could not enter into the cell nucleus within 24 h culture in vitro. Treatment with the 80 nm SiO(2) particles caused apparently decrease of cell viability and also weakened the mitochondrial membrane potential. Further experiments demonstrated that the cell adhesion and migration were greatly affected by uptake of the SiO(2) particles regardless of their size. RT-PCR results indicated down regulation of the mRNA expression of adhesion relevant genes, i.e. fibronectin, laminin and focal adhesion kinase (FAK). Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  4. Wound healing potential of Spirulina platensis extracts on human dermal fibroblast cells.

    PubMed

    Syarina, Pauzi Nur Aimi; Karthivashan, Govindarajan; Abas, Faridah; Arulselvan, Palanisamy; Fakurazi, Sharida

    2015-01-01

    Blue-green alga (Spirulina platensis) is a well renowned nutri-supplement due to its high nutritional and medicinal properties. The aim of this study was to examine the wound healing efficiency of Spirulina platensis at various solvent extracts using in vitro scratch assay on human dermal fibroblast cells (HDF). Various gradient solvent extracts (50 μg/ml of methanolic, ethanolic and aqueous extracts) from Spirulina platensis were treated on HDF cells to acquire its wound healing properties through scratch assay and in this investigation we have used allantoin, as a positive control to compare efficacy among the phytoextracts. Interestingly, aqueous extract were found to stimulate proliferation and migration of HDF cells at given concentrations and enhanced closure rate of wound area within 24 hours after treatment. Methanolic and ethanolic extracts have shown proliferative effect, however these extracts did not aid in the migration and closure of wound area when compared to aqueous extract. Based on phytochemical profile of the plant extracts analyzed by LC-MS/MS, it was shown that compounds supposedly involved in accelerating wound healing are cinnamic acid, narigenin, kaempferol, temsirolimus, phosphatidylserine isomeric derivatives and sulphoquinovosyl diacylglycerol. Our findings concluded that blue-green algae may pose potential biomedical application to treat various chronic wounds especially in diabetes mellitus patients.

  5. Strawberry-Based Cosmetic Formulations Protect Human Dermal Fibroblasts against UVA-Induced Damage.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Afrin, Sadia; Reboredo-Rodriguez, Patricia; Cianciosi, Danila; Mezzetti, Bruno; Quiles, Josè L; Bompadre, Stefano; Battino, Maurizio; Giampieri, Francesca

    2017-06-14

    Extreme exposure of skin to Ultraviolet A (UVA)-radiation may induce a dysregulated production of reactive oxygen species (ROS) which can interact with cellular biomolecules leading to oxidative stress, inflammation, DNA damage, and alteration of cellular molecular pathways, responsible for skin photoaging, hyperplasia, erythema, and cancer. For these reasons, the use of dietary natural bioactive compounds with remarkable antioxidant activity could be a strategic tool to counteract these UVA-radiation-caused deleterious effects. Thus, the purpose of the present work was to test the efficacy of strawberry (50 μg/mL)-based formulations supplemented with Coenzyme Q10 (100 μg/mL) and sun protection factor 10 in human dermal fibroblasts irradiated with UVA-radiation. The apoptosis rate, the amount of intracellular reactive oxygen species (ROS) production, the expression of proteins involved in antioxidant and inflammatory response, and mitochondrial functionality were evaluated. The results showed that the synergic topical use of strawberry and Coenzyme Q10 provided a significant (p < 0.05) photoprotective effect, reducing cell death and ROS, increasing antioxidant defense, lowering inflammatory markers, and improving mitochondrial functionality. The obtained results suggest the use of strawberry-based formulations as an innovative, natural, and useful tool for the prevention of UVA exposure-induced skin diseases in order to decrease or substitute the amount of synthetic sunscreen agents.

  6. Strawberry-Based Cosmetic Formulations Protect Human Dermal Fibroblasts against UVA-Induced Damage

    PubMed Central

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y.; Afrin, Sadia; Reboredo-Rodriguez, Patricia; Cianciosi, Danila; Mezzetti, Bruno; Quiles, Josè L.; Bompadre, Stefano; Battino, Maurizio; Giampieri, Francesca

    2017-01-01

    Extreme exposure of skin to Ultraviolet A (UVA)-radiation may induce a dysregulated production of reactive oxygen species (ROS) which can interact with cellular biomolecules leading to oxidative stress, inflammation, DNA damage, and alteration of cellular molecular pathways, responsible for skin photoaging, hyperplasia, erythema, and cancer. For these reasons, the use of dietary natural bioactive compounds with remarkable antioxidant activity could be a strategic tool to counteract these UVA-radiation-caused deleterious effects. Thus, the purpose of the present work was to test the efficacy of strawberry (50 μg/mL)-based formulations supplemented with Coenzyme Q10 (100 μg/mL) and sun protection factor 10 in human dermal fibroblasts irradiated with UVA-radiation. The apoptosis rate, the amount of intracellular reactive oxygen species (ROS) production, the expression of proteins involved in antioxidant and inflammatory response, and mitochondrial functionality were evaluated. The results showed that the synergic topical use of strawberry and Coenzyme Q10 provided a significant (p < 0.05) photoprotective effect, reducing cell death and ROS, increasing antioxidant defense, lowering inflammatory markers, and improving mitochondrial functionality. The obtained results suggest the use of strawberry-based formulations as an innovative, natural, and useful tool for the prevention of UVA exposure-induced skin diseases in order to decrease or substitute the amount of synthetic sunscreen agents. PMID:28613256

  7. Human dermal fibroblast proliferation activity of usimine-C from Antarctic lichen Ramalina terebrata.

    PubMed

    Lee, Sung Gu; Koh, Hye Yeon; Oh, Hyuncheol; Han, Se Jong; Kim, Il-Chan; Lee, Hong Kum; Yim, Joung Han

    2010-04-01

    Type I collagen is the major structural protein in dermis and its presence is used to monitor skin cell proliferation and aging. Recently, novel usimine compounds have been found in the Antarctic lichen Ramalina terebrata. In the present study, usimine-C induced cell proliferation of human dermal fibroblast, CCD-986SK, up to 1.6-fold after treating with 90 microg/ml for 48 h. Type I procollagen synthesis was significantly increased 1.3-fold, 3-fold, and 5-fold after treating with 0.14, 0.72, and 3.6 microg usimine-C/ml for 24 h, respectively, whereas no significant increase in type I procollagen was observed after treating with usimine-A or -B. Usimines are usnic acid derivatives. Considering that the difference among the derivatives is a side chain, the proliferation activity may be related to this side chain, triggering an internal signal for type I procollagen expression. Further studies still remain to clarify the signaling pathways for the type I procollagen induction, which is activated by usimine-C.

  8. Analysis of Gene Expression in Human Dermal Fibroblasts Treated with Senescence-Modulating COX Inhibitors.

    PubMed

    Han, Jeong A; Kim, Jong-Il

    2017-06-01

    We have previously reported that NS-398, a cyclooxygenase-2 (COX-2)-selective inhibitor, inhibited replicative cellular senescence in human dermal fibroblasts and skin aging in hairless mice. In contrast, celecoxib, another COX-2-selective inhibitor, and aspirin, a non-selective COX inhibitor, accelerated the senescence and aging. To figure out causal factors for the senescence-modulating effect of the inhibitors, we here performed cDNA microarray experiment and subsequent Gene Set Enrichment Analysis. The data showed that several senescence-related gene sets were regulated by the inhibitor treatment. NS-398 up-regulated gene sets involved in the tumor necrosis factor β receptor pathway and the fructose and mannose metabolism, whereas it down-regulated a gene set involved in protein secretion. Celecoxib up-regulated gene sets involved in G2M checkpoint and E2F targets. Aspirin up-regulated the gene set involved in protein secretion, and down-regulated gene sets involved in RNA transcription. These results suggest that COX inhibitors modulate cellular senescence by different mechanisms and will provide useful information to understand senescence-modulating mechanisms of COX inhibitors.

  9. Chondrogenic potential of human dermal fibroblasts in a contractile, soft, self-assembling, peptide hydrogel.

    PubMed

    Bussmann, Bianca M; Reiche, Sven; Marí-Buyé, Núria; Castells-Sala, Cristina; Meisel, Hans Jörg; Semino, Carlos E

    2016-02-01

    The present paper describes a simple approach to obtain three-dimensional (3D) cartilage constructs using human normal dermal fibroblasts (hNDFs) cultured in a self-assembling peptide nanofibre scaffold. During the first days of culture, the 3D constructs underwent morphological changes consisting of a substantial contraction process that ended in a small compact structure. During this process the system became sensitive to induction with standard chondrogenic medium, evidenced by the expression of specific markers of mature cartilage. First, it was detected that the samples become highly stained with toluidine blue dye over time (40-50 days), indicating that the system produced significantly high amounts of glycosaminoglycans. By quantitative PCR, it was confirmed that the system significantly upregulated the expression of the proteoglycan aggrecan, a good indicator of cartilage commitment. Moreover, collagen type II was upregulated at protein level, confirming that the system differentiated to a chondrocyte-like construct. Additionally, during the first days of culture in control medium analysed hNDFs proliferation capacity in this 3D system was analysed. This platform could be used in the future to obtain an autologous source of cells from a simple patient skin biopsy, which could be easily translated into a low-cost and effective regenerative therapy.

  10. RNA-Mediated Reprogramming of Primary Adult Human Dermal Fibroblasts into c-kit(+) Cardiac Progenitor Cells.

    PubMed

    Pratico, Elizabeth D; Feger, Bryan J; Watson, Michael J; Sullenger, Bruce A; Bowles, Dawn E; Milano, Carmelo A; Nair, Smita K

    2015-11-15

    Cardiovascular disease is the leading cause of death in the United States. Heart failure is a common, costly, and potentially fatal condition that is inadequately managed by pharmaceuticals. Cardiac repair therapies are promising alternative options. A potential cardiac repair therapy involves reprogramming human fibroblasts toward an induced cardiac progenitor-like state. We developed a clinically useful and safer reprogramming method by nonintegrative delivery of a cocktail of cardiac transcription factor-encoding mRNAs into autologous human dermal fibroblasts obtained from skin biopsies. Using this method, adult and neonatal dermal fibroblasts were reprogrammed into cardiac progenitor cells (CPCs) that expressed c-kit, Isl-1, and Nkx2.5. Furthermore, these reprogrammed CPCs differentiated into cardiomyocytes (CMs) in vitro as judged by increased expression of cardiac troponin T, α-sarcomeric actinin, RyR2, and SERCA2 and displayed enhanced caffeine-sensitive calcium release. The ability to reprogram patient-derived dermal fibroblasts into c-kit(+) CPCs and differentiate them into functional CMs provides clinicians with a potential new source of CPCs for cardiac repair from a renewable source and an alternative therapy in the treatment of heart failure.

  11. In vivo quantification of human dermal skin aging using SHG and autofluorescence

    NASA Astrophysics Data System (ADS)

    Puschmann, Stefan; Rahn, Christian-Dennis; Wenck, Horst; Gallinat, Stefan; Fischer, Frank

    2012-03-01

    There are visible changes during skin aging. In the extracellular matrix these changes referred to as intrinsic aging (skin areas not exposed to sunlight) and extrinsic aging can be measured using various methods, such as subjective clinical evaluation, histology and molecular analysis. In this study we developed a new parameter for the non-invasive quantitative determination of dermal skin aging utilizing a five-dimensional intravital tomography (5D-IVT). This device, also known as 5D - multi-photon laser scanning microscopy, is a powerful tool to investigate (photo)aging-associated alterations in vivo. Structural alterations in the dermis of extrinsically aged (chronically sun-exposed) and intrinsically aged (sun-protected) human skin were recorded utilizing the collagen-specific second harmonic generation (SHG) signal and the elastin-specific autofluorescence (AF) signal. Recording took place in young and elderly volunteers. The resulting images were processed in order to gain the elastin percentage and the collagen percentage per image. Then, the elastin - to - collagen ratio (ELCOR) was calculated. With respect to volar forearm skin, the ELCOR significantly increased with age. In elderly volunteers, the ELCOR value calculated for the chronically sun-exposed temple area was significantly augmented compared with the sun-protected upper arm area. Based on 5D-IVT we introduce the ELCOR as a new means to quantify age-associated alterations in the extracellular matrix of in vivo human skin. This novel parameter is compared to the currently used "SHG to AF aging index" of the dermis (SAAID).

  12. Levels of bisphenol-A in different paper products in Guangzhou, China, and assessment of human exposure via dermal contact.

    PubMed

    Fan, Ruifang; Zeng, Biyan; Liu, Xiaosu; Chen, Chao; Zhuang, Qinwei; Wang, Yongjun; Hu, Mingli; Lv, Yanshan; Li, Junnan; Zhou, Yuanxiu; Lin, Zhi Yuan William

    2015-03-01

    Bisphenol A (BPA) is a chemical widely used both in plastics production as a food and beverage container and in thermal papers as a color developer. Dietary consumption is the main route of human exposure to BPA, but dermal absorption through handling different papers might be underestimated or ignored. In this study, BPA in different paper products, including different types of papers, receipts and Chinese currencies, were investigated. BPA was detected in receipts (n = 87) and Chinese currencies (n = 46) with concentrations of 0.17-2.675 × 10(4) μg per g paper and 0.09-288.55 μg per g paper, respectively. Except for parchment papers (n = 3), copy papers (n = 3) and food contact papers (n = 3), BPA was measured in all of the other paper products, with levels of 0.01-6.67 μg per g paper. BPA transferred from thermal papers to common papers increased with the increasing contact pressure. Compared with that in water, the migration speed of BPA was doubled in the synthetic sweat. Washing hands could reduce BPA dermal exposure, and washing hands with lotion was the most efficient way. However, about 19-47% of BPA was still found on hands after different washing methods. Dermal absorption via handling of receipts and papers was estimated to be 36.45 ng per day for the general population and 1.54 × 10(-3) to 248.73 μg per day for a cashier. These values are below the maximum doses recommended by the U.S. Environmental Protection Agency and the European Food Safety Authority. However, due to its uncertain adverse effects on human beings, long-term BPA exposure through dermal absorption should be paid more attention, particularly for some occupational populations.

  13. Antiaging effects of the mixture of Panax ginseng and Crataegus pinnatifida in human dermal fibroblasts and healthy human skin.

    PubMed

    Hwang, Eunson; Park, Sang-Yong; Yin, Chang Shik; Kim, Hee-Taek; Kim, Yong Min; Yi, Tae Hoo

    2017-01-01

    Human skin undergoes distinct changes throughout the aging process, based on both intrinsic and extrinsic factors. In a process called photoaging, UVB irradiation leads to upregulation of matrix metalloproteinase-1, which then causes collagen degradation and premature aging. Mixtures of medicinal plants have traditionally been used as drugs in oriental medicine. Based on the previously reported antioxidant properties of Panax ginseng Meyer and Crataegus pinnatifida, we hypothesized that the mixture of P. ginseng Meyer and C. pinnatifida (GC) would have protective effects against skin aging. Anti-aging activity was examined both in human dermal fibroblasts under UVB irradiation by using Western blot analysis and in healthy human skin by examining noninvasive measurements. In vitro studies showed that GC improved procollagen type I expression and diminished matrix metalloproteinase-1 secretion. Based on noninvasive measurements, skin roughness values, including total roughness (R1), maximum roughness (R2), smoothness depth and average roughness (R3), and global photodamage scores were improved by GC application. Moreover, GC ameliorated the high values of smoothness depth (R4), which means that GC reduced loss of skin moisture. These results suggest that GC can prevent aging by inhibiting wrinkle formation and increasing moisture in the human skin.

  14. Denver Papillae Protocol for Objective Analysis of Fungiform Papillae.

    PubMed

    Nuessle, Tiffany M; Garneau, Nicole L; Sloan, Meghan M; Santorico, Stephanie A

    2015-06-08

    The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller & Reedy's 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller & Reedy's original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller & Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

  15. Ultrastructure of Lingual Papillae in Common Chimpanzee (Pan troglodytes) Foetus, Newborn and Adult Specimens.

    PubMed

    Pastor, J F; Barbosa, M; De Paz, F J; San José, I; Levanti, M; Potau, J M; Vega, J A; Cabo, R

    2017-10-01

    Among primates, the two recognized species of chimpanzees (common chimpanzee, Pan troglodytes; pygmy chimpanzee, Pan paniscus) are considered to be the most similar to humans. Importantly, in mammals, the food intake behaviour largely determines the tongue morphology, including the type, proportion and distribution of gustatory and non-gustatory tongue papillae. The lingual papillae form during its development and mature in post-natal life depending on the different feeding. In this study, we have used scanning electron microscopy to analyse the age-related changes in the lingual papillae of foetal, newborn and adult P. troglodytes. Four main types of lingual papillae, denominated filiform, fungiform, foliate and vallate, and one subtype of filiform papillae called conical papillae, were found. The main age-related changes observed in all kinds of papillae were a progressive keratinization and morphological complexity along the lifespan. During the foetal period, there was scarce keratinization, which progressively increases in young animals to adulthood. The number of filiform increased with ageing, and both filiform and fungiform papillae in adult tongues are divided into pseudopapillae. On the other hand, the vallate papillae vary from smooth simple surfaces in foetal tongues to irregular surfaces with grooves and pseudopapillae (microscopic papilla-shaped formations within the papilla itself) in adults. These results describe for the first time the age-related variations in the three-dimensional aspect of lingual papillae of the chimpanzee tongue and provide new data to characterize more precisely these structures in the human closest specie. © 2017 Blackwell Verlag GmbH.

  16. Coculture of stem cells from apical papilla and human umbilical vein endothelial cell under hypoxia increases the formation of three-dimensional vessel-like structures in vitro.

    PubMed

    Yuan, Changyong; Wang, Penglai; Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Tong, Edith H Y; Jin, Lijian; Zhang, Chengfei

    2015-03-01

    The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity before implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVECs) under hypoxia promotes the formation of endothelial tubules and a blood vessel network. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, whereas SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α and VEGF, and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture Petri dishes were detected

  17. Coculture of Stem Cells from Apical Papilla and Human Umbilical Vein Endothelial Cell Under Hypoxia Increases the Formation of Three-Dimensional Vessel-Like Structures in Vitro

    PubMed Central

    Yuan, Changyong; Wang, Penglai; Zhu, Lifang; Dissanayaka, Waruna Lakmal; Green, David William; Tong, Edith H.Y.; Jin, Lijian

    2015-01-01

    The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity before implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVECs) under hypoxia promotes the formation of endothelial tubules and a blood vessel network. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, whereas SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α and VEGF, and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture Petri dishes were detected

  18. Dental Apical Papilla as Therapy for Spinal Cord Injury.

    PubMed

    De Berdt, P; Vanacker, J; Ucakar, B; Elens, L; Diogenes, A; Leprince, J G; Deumens, R; des Rieux, A

    2015-11-01

    Stem cells of the apical papilla (SCAP) represent great promise regarding treatment of neural tissue damage, such as spinal cord injury (SCI). They derive from the neural crest, express numerous neurogenic markers, and mediate neurite outgrowth and axonal targeting. The goal of the present work was to investigate for the first time their potential to promote motor recovery after SCI in a rat hemisection model when delivered in their original stem cell niche-that is, by transplantation of the human apical papilla tissue itself into the lesion. Control groups consisted of animals subjected to laminectomy only (shams) and to lesion either untreated or injected with a fibrin hydrogel with or without human SCAP. Basso-Beattie-Bresnahan locomotor scores at 1 and 3 d postsurgery confirmed early functional decline in all SCI groups. This significant impairment was reversed, as seen in CatWalk analyses, after transplantation of apical papilla into the injured spinal cord wound, whereas the other groups demonstrated persistent functional impairment. Moreover, tactile allodynia did not develop as an unwanted side effect in any of the groups, even though the SCAP hydrogel group showed higher expression of the microglial marker Iba-1, which has been frequently associated with allodynia. Notably, the apical papilla transplant group presented with reduced Iba-1 expression level. Masson trichrome and human mitochondria staining showed the preservation of the apical papilla integrity and the presence of numerous human cells, while human cells could no longer be detected in the SCAP hydrogel group at the 6-wk postsurgery time point. Altogether, our data suggest that the transplantation of a human apical papilla at the lesion site improves gait in spinally injured rats and reduces glial reactivity. It also underlines the potential interest for the application of delivering SCAP in their original niche, as compared with use of a fibrin hydrogel.

  19. Complication Rates With Human Acellular Dermal Matrices: Retrospective Review of 211 Consecutive Breast Reconstructions

    PubMed Central

    Carman, Claire M.; Tobin, Chase; Chase, Serena A.; Rossmeier, Kerri A.

    2016-01-01

    Background: Human acellular dermal matrix (HADM) is commonly used to provide coverage and support for breast reconstruction. The primary purpose of this study was to evaluate the complication rates associated with breast reconstruction procedures when performed in conjunction with multiple types of HADM in a consecutive series. Methods: After receiving institutional review board approval, medical records from a single surgeon were retrospectively reviewed for 126 consecutive patients (170 breasts and 211 procedures) who received a breast reconstruction or revision with implantation of HADM between 2012 and 2014. Patient demographics, surgical technique, and the complication profile of 4 major types of HADM were evaluated by procedure. Complication data were primarily evaluated for infection, seroma formation, necrosis, and other complications requiring additional surgery. Results: The total complication rate was 19.4%. The complication rates were not statistically different between all 4 types of HADM: Alloderm (n = 143); Alloderm RTU (n = 19); FlexHD (n = 18); hMatrix (n = 32) (P > 0.05). Smokers and large-breasted women (≥500 g) had a significantly higher complication rate than the rest of the population (P < 0.01 and P < 0.03, respectively). The complication rates associated with all other patient cohorts analyzed (age, body mass index, comorbid conditions, cancer diagnosis, prepectoral technique) showed no influence on complication rates (P > 0.05). Conclusions: In characteristically similar cohorts, there was no statistically significant difference in complication rates based on type of HADM; however, certain risk factors and anatomy should be considered before HADM-assisted breast reconstruction. PMID:27975023

  20. Complication Rates With Human Acellular Dermal Matrices: Retrospective Review of 211 Consecutive Breast Reconstructions.

    PubMed

    Schnarrs, Robert H; Carman, Claire M; Tobin, Chase; Chase, Serena A; Rossmeier, Kerri A

    2016-11-01

    Human acellular dermal matrix (HADM) is commonly used to provide coverage and support for breast reconstruction. The primary purpose of this study was to evaluate the complication rates associated with breast reconstruction procedures when performed in conjunction with multiple types of HADM in a consecutive series. After receiving institutional review board approval, medical records from a single surgeon were retrospectively reviewed for 126 consecutive patients (170 breasts and 211 procedures) who received a breast reconstruction or revision with implantation of HADM between 2012 and 2014. Patient demographics, surgical technique, and the complication profile of 4 major types of HADM were evaluated by procedure. Complication data were primarily evaluated for infection, seroma formation, necrosis, and other complications requiring additional surgery. The total complication rate was 19.4%. The complication rates were not statistically different between all 4 types of HADM: Alloderm (n = 143); Alloderm RTU (n = 19); FlexHD (n = 18); hMatrix (n = 32) (P > 0.05). Smokers and large-breasted women (≥500 g) had a significantly higher complication rate than the rest of the population (P < 0.01 and P < 0.03, respectively). The complication rates associated with all other patient cohorts analyzed (age, body mass index, comorbid conditions, cancer diagnosis, prepectoral technique) showed no influence on complication rates (P > 0.05). In characteristically similar cohorts, there was no statistically significant difference in complication rates based on type of HADM; however, certain risk factors and anatomy should be considered before HADM-assisted breast reconstruction.

  1. Pretreatment of Ferulic Acid Protects Human Dermal Fibroblasts against Ultraviolet A Irradiation

    PubMed Central

    Hahn, Hyung Jin; Kim, Ki Bbeum; Bae, Seunghee; Choi, Byung Gon; An, Sungkwan

    2016-01-01

    Background Approximately 90%~99% of ultraviolet A (UVA) ray reaches the Earth's surface. The deeply penetrating UVA rays induce the formation of reactive oxygen species (ROS), which results in oxidative stress such as photoproducts, senescence, and cell death. Thus, UVA is considered a primary factor that promotes skin aging. Objective Researchers investigated whether pretreatment with ferulic acid protects human dermal fibroblasts (HDFs) against UVA-induced cell damages. Methods HDF proliferation was analyzed using the water-soluble tetrazolium salt assay. Cell cycle distribution and intracellular ROS levels were assessed by flow cytometric analysis. Senescence was evaluated using a senescence-associated β-galactosidase assay, while Gadd45α promoter activity was analyzed through a luciferase assay. The expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), xeroderma pigmentosum complementation group A and C, matrix metalloproteinase 1 and 3, as well as p21 and p16 were measured using quantitative real-time polymerase chain reaction. Results Inhibition of proliferation and cell cycle arrest were detected in cells that were irradiated with UVA only. Pretreatment with ferulic acid significantly increased the proliferation and cell cycle progression in HDFs. Moreover, ferulic acid pretreatment produced antioxidant effects such as reduced DCF intensity, and affected SOD1 and CAT mRNA expression. These effects were also demonstrated in the analysis of cell senescence, promoter activity, expression of senescent markers, and DNA repair. Conclusion These results demonstrate that ferulic acid exerts protective effects on UVA-induced cell damages via anti-oxidant and stress-inducible cellular mechanisms in HDFs. PMID:27904274

  2. Chondrogenesis of Human Infrapatellar Fat Pad Stem Cells on Acellular Dermal Matrix

    PubMed Central

    Ye, Ken; Traianedes, Kathy; Choong, Peter F. M.; Myers, Damian E.

    2016-01-01

    Acellular dermal matrix (ADM) has been in clinical use for decades in numerous surgical applications. The ability for ADM to promote cellular repopulation, revascularisation and tissue regeneration is well documented. Adipose stem cells have the ability to differentiate into mesenchymal tissue types, including bone and cartilage. The aim of this study was to investigate the potential interaction between ADM and adipose stem cells in vitro using TGFβ3 and BMP6. Human infrapatellar fat pad-derived adipose stem cells (IPFP-ASC) were cultured with ADM derived from rat dermis in chondrogenic (TGFβ3 and BMP6) medium in vitro for 2 and 4 weeks. Histology, qPCR, and immunohistochemistry were performed to assess for markers of chondrogenesis (collagen Type II, SOX9 and proteoglycans). At 4 weeks, cell-scaffold constructs displayed cellular changes consistent with chondrogenesis, with evidence of stratification of cell layers and development of a hyaline-like cartilage layer superficially, which stained positively for collagen Type II and proteoglycans. Significant cell–matrix interaction was seen between the cartilage layer and the ADM itself with seamless integration between each layer. Real time qPCR showed significantly increased COL2A1, SOX9, and ACAN gene expression over 4 weeks when compared to control. COL1A2 gene expression remained unchanged over 4 weeks. We believe that the principles that make ADM versatile and successful for tissue regeneration are applicable to cartilage regeneration. This study demonstrates in vitro the ability for IPFP-ASCs to undergo chondrogenesis, infiltrate, and interact with ADM. These outcomes serve as a platform for in vivo modelling of ADM for cartilage repair. PMID:26858950

  3. Cryoprotective effect of low-molecular-weight hyaluronan on human dermal fibroblast monolayers.

    PubMed

    Ujihira, Masanobu; Iwama, Akira; Aoki, Makie; Aoki, Kanako; Omaki, Sayaka; Goto, Erika; Mabuchi, Kiyoshi

    2010-01-01

    The purpose of this study was to assess the availability of low-molecular-weight (low-MW) hyaluronan (HA) as a cryoprotectant for cellular cryopreservation. To clarify whether low-MW HA is cryoprotective, we evaluated the effect of HA concentration (0-5% w/w) in a cryoprotectant solution on cell membrane integrity after freeze-thaw. A test sample was created using human dermal fibroblast monolayers incubated in a culture dish for 24 h (37 degrees C, 5% CO2). Sodium hyaluronate (MW 3 x 10(4)-5 x 10(4)) dissolved in medium served as the cryoprotectant solution. Samples were immersed in the solution for 2 h at 0-4 degrees C. They were frozen at a cooling rate of 3 degrees C/min from 4 to -80 degrees C, cooled further to below -185 degrees C, and then thawed. Cell membrane integrity after thawing was evaluated using a trypan blue exclusion assay. The sample and freezing procedures were repeated in subsequent experiments, while the conditions of the solution immersion with respect to the sample varied. Next, to clarify whether the cryoprotective action of HA is intra- or extracellular, we performed three experiments. The first studied the dependence of membrane integrity after freeze-thaw on preliminary incubation time (0.75-24 h at 37 degrees C) with a sample immersed in the solution (5% w/w HA). In the second, membrane integrity of thawed samples that were initially frozen in a medium instead of solution, by removing extracellular HA following a preliminary 6-h incubation period, were evaluated. Thirdly, we investigated cellular uptake of fluorescein isothiocyanate-labeled HA (MW 10(5), 1% w/w) after a preliminary 6-h incubation period under fluorescent microscopy (without freeze-thaw). The results show that HA had a cryoprotective effect, and that this cryoprotective action was intracellular. Therefore, low- MW HA proves to be a promising cellular cryoprotectant.

  4. Bog blueberry anthocyanins alleviate photoaging in ultraviolet-B irradiation-induced human dermal fibroblasts.

    PubMed

    Bae, Ji-Young; Lim, Soon Sung; Kim, Sun Ju; Choi, Jung-Suk; Park, Jinseu; Ju, Sung Mi; Han, Seoung Jun; Kang, Il-Jun; Kang, Young-Hee

    2009-06-01

    Fruits of bog blueberry (Vaccinium uliginosum L.) are rich in anthocyanins that contribute pigmentation. Anthocyanins have received much attention as agents with potentials preventing chronic diseases. This study investigated the capacity of anthocyanin-rich extract from bog blueberry (ATH-BBe) to inhibit photoaging in UV-B-irradiated human dermal fibroblasts. BBe anthocyanins were detected as cyanidin-3-glucoside, petunidin-3-glucoside, malvidin-3-glucoside, and delphinidin3-glucoside. ATH-BBe attenuated UV-B-induced toxicity accompanying reactive oxygen species (ROS) production and the resultant DNA damage responsible for activation of p53 and Bad. Preincubation of ATH-BBe markedly suppressed collagen degradation via blunting production of collagenolytic matrix metalloproteinases (MMP). Additionally, ATH-BBe enhanced UV-B-downregulated procollagen expression at transcriptional levels. We next attempted to explore whether ATH-BBe mitigated the MMP-promoted collagen degradation through blocking nuclear factor kappaB (NF-kappaB) activation and MAPK-signaling cascades. UV-B radiation enhanced nuclear translocation of NF-kappaB, which was reversed by treatment with ATH-BBe. The UV-B irradiation rapidly activated apoptosis signal-regulating kinase-1 (ASK-1)-signaling cascades of JNK and p38 mitogen-activated protein kinase (p38 MAPK), whereas ATH-BBe hampered phosphorylation of c-Jun, p53, and signal transducers and activators of transcription-1 (STAT-1) linked to these MAPK signaling pathways. ATH-BBe diminished UV-B augmented-release of inflammatory interleukin (IL)-6 and IL-8. These results demonstrate that ATH-BBe dampens UV-B-triggered collagen destruction and inflammatory responses through modulating NF-kappaB-responsive and MAPK-dependent pathways. Therefore, anthocyanins from edible bog blueberry may be protective against UV-induced skin photoaging.

  5. Influence of artificial sebum on the dermal absorption of chemicals in excised human skin: A proof-of-concept study.

    PubMed

    Schneider, Désirée; Dennerlein, Kathrin; Göen, Thomas; Schaller, Karl Heinz; Drexler, Hans; Korinth, Gintautas

    2016-06-01

    In an initial diffusion cell study, the influence of artificial sebum on dermal penetration and intradermal reservoir of ethanol and toluene was investigated in comparison with the effects of a skin cream (o/w- and w/o-emulsion) and untreated (control) skin. Human skin was exposed to neat ethanol and toluene for 4h, respectively. During the experiments, the penetration of the compounds was assessed in the receptor fluid. The amounts of the test compounds in the skin were determined at the end of exposure. In the control experiments, 42% of the total resorbed ethanol amounts were found in the intradermal reservoir after 4h, whereas 82% of the toluene amounts were found in the skin compartments. The treatment with artificial sebum showed no significant differences in dermal absorption of both test compounds compared to control skin. In contrast, the treatment with skin cream increased the percutaneous penetration (p<0.001) and the intradermal reservoir of ethanol ~2-fold but not of toluene. In all exposure scenarios, a relevant intradermal reservoir was formed. The results indicate that sebum does not influence the percutaneous penetration and the intradermal reservoir of epidermally applied chemicals, whereas the application of skin creams may increase the dermal penetration of the compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

    PubMed

    Bae, Seunghee; An, In-Sook; An, Sungkwan

    2015-09-01

    Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

  7. Investigation of the effect of hydration on dermal collagen in ex vivo human skin tissue using second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Samatham, Ravikant; Wang, Nicholas K.; Jacques, Steven L.

    2016-02-01

    Effect of hydration on the dermal collagen structure in human skin was investigated using second harmonic generation microscopy. Dog ears from the Mohs micrographic surgery department were procured for the study. Skin samples with subject aged between 58-90 years old were used in the study. Three dimensional Multiphoton (Two-photon and backward SHG) control data was acquired from the skin samples. After the control measurement, the skin tissue was either soaked in deionized water for 2 hours (Hydration) or kept at room temperature for 2 hours (Desiccation), and SHG data was acquired. The data was normalized for changes in laser power and detector gain. The collagen signal per unit volume from the dermis was calculated. The desiccated skin tissue gave higher backward SHG compared to respective control tissue, while hydration sample gave a lower backward SHG. The collagen signal decreased with increase in hydration of the dermal collagen. Hydration affected the packing of the collagen fibrils causing a change in the backward SHG signal. In this study, the use of multiphoton microscopy to study the effect of hydration on dermal structure was demonstrated in ex vivo tissue.

  8. Evaluation of calcium magnesium acetate and road salt for contact hypersensitivity potential and dermal irritancy in humans.

    PubMed

    Cushman, J R; Duff, V A; Buteau, G H; Aust, L B; Caldwell, N; Lazer, W

    1991-04-01

    Calcium magnesium acetate (CMA) and road salt are both de-icing agents to which workers may be dermally exposed. A commercial formulation of CMA (Chevron Ice-B-Gon Deicer) and road salt were tested in a human repeat insult patch test to evaluate the contact hypersensitivity potential of these materials and to evaluate irritation following single or multiple applications. 72 of the initial 82 panelists completed the study. CMA and road salt (each at 10% and 30% w/w in distilled water; 0.3 ml) were administered under occlusive patches on the forearm for 14 h 3 x per week for 3 weeks. The panelists were challenged 2 weeks later; 2 panelists who had mild reactions were subsequently rechallenged 6 weeks later. Neither CMA nor road salt produced contact hypersensitivity in any panelists. Following the first application, moderate acute irritation was observed only at 1 skin site exposed to 30% road salt. Repeated exposure to CMA or road salt produced mild to moderate irritation. The highest incidence of moderate irritation was observed with 30% road salt. Thus, neither material is expected to cause significant dermal effects in exposed workers. CMA is expected to cause dermal irritation equivalent to or less than that caused by road salt.

  9. [Effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells].

    PubMed

    Qiao, L; Yang, H Z; Li, X C; Huang, X Q; Yuan, B; Zhou, Z D

    2017-02-20

    Objective: To observe the effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells cultured in vitro. Methods: The dividing method and treatment of cells for the detection of all indexes in this study were as follows. Human dermal microvascular endothelial cells of the 4th passage were divided into 3 groups according to the random number table, with 12 wells in each group. Cells in control group (C) were cultured with complete culture solution containing 5 mmol/L D-glucose for 7 d. Cells in transient high glucose group (THG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 2 d and complete culture solution containing 5 mmol/L D-glucose for 5 d. Cells in prolonged high glucose group (PHG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 7 d. (1) The cell morphology in groups C and PHG on culture day 7 and that in group THG on culture day 2 and 7 was observed by inverted optical microscope. (2) On culture day 0, 2, 4, and 7, cell proliferation rate was determined by cell viability analyzing counter. (3) After culture day 2, the scratch experiment was performed, and the cells were further cultured. At post scratch hour (PSH) 0, 24, 48, 72, 96, and 120, the scratch area was measured, and the cell migration rates of the latter 5 time points were calculated. (4) On culture day 0, 2, 4, and 7, the cell apoptosis rate was determined by cell analyzer. (5) Cells were seeded into Matrigel to culture for 24 h after culture day 7. The formation of vessel-like structure was observed by inverted optical microscope. The length and number of branch point of vessel-like structure were calculated. (6) On culture day 2, 4, and 7, mRNA expression of vascularization-related gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with

  10. [Effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism].

    PubMed

    Zhao, B; Han, F; Zhang, W; Wang, X J; Zhang, J; Yang, F F; Shi, J H; Su, L L; Hu, D H

    2017-06-20

    Objective: To investigate the effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism. Methods: The third passage of healthy adult human dermal fibroblasts in logarithmic phase were cultured in DMEM medium containing 10% fetal bovine serum for the following five experiments. (1) In experiments 1, 2, and 3, cells were divided into normoxia group and hypoxia group according to the random number table, with 10 dishes in each group. Cells of normoxia group were cultured in incubator containing 21% oxygen, while those of hypoxia group with 1% oxygen. At post culture hour (PCH) 0 and 48, 5 dishes of cells were collected from each group, respectively. mRNA expressions of markers of myofibroblasts including alpha smooth muscle actin (α-SMA), type Ⅰ collagen, and type Ⅲ collagen of cells were determined with real time fluorescent quantitative reverse transcription polymerase chain reaction in experiment 1. Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting in experiment 2. The protein expression of nuclear factor-kappa B (NF-κB) of cells was determined with Western blotting in experiment 3. (2) In experiment 4, cells were divided into normoxia group, hypoxia group, and hypoxia+ pyrrolidine dithiocarbamate (PDTC) group according to the random number table, with 5 dishes in each group. Cells in the former two groups were treated the same as those in experiment 1. Cells in hypoxia+ PDTC group were treated the same as those in hypoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, the protein expression of NF-κB of cells was determined with Western blotting. (3) In experiment 5, cells were divided into normoxia group, hypoxia group, hypoxia+ PDTC group, and normoxia+ PDTC group according to the random number table, with 5 dishes in each group. Cells in the former three groups were treated the

  11. Early escharectomy and concurrent composite skin grafting over human acellular dermal matrix scaffold for covering deep facial burns.

    PubMed

    Tang, Bing; Zhu, Bin; Liang, Yue-Ying; Bi, Liang-Kuan; Chen, Bin; Hu, Zhi-Cheng; Zhang, Kai; Zhu, Jia-Yuan

    2011-04-01

    Although escharectomy and full-thickness skin autografting have been widely used to treat deep facial burns, the clinical outcomes remain unacceptable. Composite razor-thin skin grafting over acellular dermal matrix scaffold has been used successfully in repairing burns of the trunk and limbs, but its use in covering deep facial burns has rarely been reported. In this study, the authors investigated the clinical outcomes of early escharectomy and concurrent composite razor-thin skin autografting and acellular dermal matrix scaffold for treating deep facial burns. Patients with deep facial burns (n = 16) involving 8 to 30 percent of the total body surface area received early escharectomy by postburn day 3 and concurrent, one-stage, large, razor-thin skin autografting on top of human acellular dermal matrix scaffold. Wound dressings were changed on postoperative days 7, 9, and 12 to examine the survival of skin autografts. Patients were followed up for 12 months to evaluate their facial profiles. The take rate of composite skin autografts was 97.3 percent at postoperative day 12. At the follow-up visit, the skin autografts appeared normal in color, with soft texture and good elasticity. The skin junctures showed little scarring. The patients exhibited a chubby facial appearance and abundant expression, except for one patient with microstomia and two patients with ectropion who required further plastic surgical interventions. Early escharectomy and concurrent composite razor-thin skin autografting on top of acellular dermal matrix scaffold constitute an effective and favorable option for covering deep facial burns, especially for patients with limited donor sites.

  12. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    SciTech Connect

    De Abrew, K. Nadira; Thomas-Virnig, Christina L.; Rasmussen, Cathy A.; Bolterstein, Elyse A.; Schlosser, Sandy J.; Allen-Hoffmann, B. Lynn

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  13. 199 EFFECTS OF REPROGRAMMING-CONDITIONED MEDIUM ON ULTRAVIOLET RAY A-DAMAGED HUMAN DERMAL FIBROBLASTS.

    PubMed

    Kang, J; Lee, S G; Kang, J H; Park, S-M; Heo, S Y; Lee, S Y; Kim, S; Lo, E; Ahn, K S; Shim, H

    2016-01-01

    Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P<0.05) among the treatments in the order of Repro-, HDF-, and iPSC-CM. In the same context, apoptosis and senescence were significantly different (P<0.05) in the order of iPSC-, HDF-, and Repro-CM. Interestingly, iPSC-CM did not substantially ameliorate UVA-induced damage, suggesting that the conditions optimized to pluripotent stem cells may not be suitable for the recovery from damage in terminally differentiated cells, such as fibroblasts. The RNA-seq analysis was performed to assess the genome-wide transcriptional profile in the process of recovery. Repro- and HDF-CM were categorized more closely than iPSC-CM in hierarchical cluster analysis. In comparison with iPSC-CM, the up-regulated genes by Repro

  14. [Structural and biomechanical properties of accelular dermal matrix derived from human scar tissue].

    PubMed

    Xu, Jingjing; Cai, Jinglong; Wang, Qian; Li, Yi; Jiao, Hu; Zong, Xianlei

    2015-03-17

    To explore the structural and biomechanical properties of acellular dermal matrix (ADM) of human scar tissue. Randomly choose 8 human mature scar tissue, 8 human hypertrophic scar tissue, and 4 normal human tissue as experimental samples respectively. Then 0.5 mm split-thickness skin grafts were obtained by drum-type scalpel, further acellularized by 2.5 g/L trypsin-0.5% TritonX-100. Structural analyses were performed by macroscopic observation, hematoxylin and eosin histological staining and scanning electron microscope. Then human epithelial stem cells were inoculated, cultured on those materials and observe their adhesive properties. Finally the biomechanical properties of ADM were analyzed through the detection of stress-strain relation, stress relaxation, creep and ultimate stress strength to distinguish different origins. Prepared ADM was milky white in color despite their distinct origins, physiological and mature scar tissue derived ADM was soft and flexible in texture while ADM from hypertrophic scar showed a more tenacious character. Optical microscopic and electron microscopic analyses showed no sign of visible cellular structures. ADM from physiological group had relatively homogeneous and inerratic collagenous fibers, ADM from hypertrophic group presented with fibers of various diameters and arrayed in a compact and disordered manner. ADM from mature scar tissue possessed traits between the other two groups. Adhesion growth could be observed 2 weeks after inoculating human epithelial stem cells on ADM and cells grew in a cohesive fashion on ADM both from normal skin and mature scar tissue and in an adhesive fashion on hypertrophic scar tissue ADM. Stress-strain β value (3.024 ± 0.413, 2.595 ± 0.443, 2.590 ± 0.366), creep slope (0.018 ± 0.003, 0.019 ± 0.009, 0.023 ± 0.010) and ultimate stress strength (8.971 ± 2.434, 11.011 ± 1.492, 15.567 ± 2.931) of ADM showed no significant differences in hypertrophic scar, mature scar and normal skin

  15. Adhesion of Neisseria meningitidis to Dermal Vessels Leads to Local Vascular Damage and Purpura in a Humanized Mouse Model

    PubMed Central

    Melican, Keira; Michea Veloso, Paula; Martin, Tiffany; Bruneval, Patrick; Duménil, Guillaume

    2013-01-01

    Septic shock caused by Neisseria meningitidis is typically rapidly evolving and often fatal despite antibiotic therapy. Further understanding of the mechanisms underlying the disease is necessary to reduce fatality rates. Postmortem samples from the characteristic purpuric rashes of the infection show bacterial aggregates in close association with microvessel endothelium but the species specificity of N. meningitidis has previously hindered the development of an in vivo model to study the role of adhesion on disease progression. Here we introduced human dermal microvessels into SCID/Beige mice by xenografting human skin. Bacteria injected intravenously exclusively associated with the human vessel endothelium in the skin graft. Infection was accompanied by a potent inflammatory response with the secretion of human inflammatory cytokines and recruitment of inflammatory cells. Importantly, infection also led to local vascular damage with hemostasis, thrombosis, vascular leakage and finally purpura in the grafted skin, replicating the clinical presentation for the first time in an animal model. The adhesive properties of the type IV pili of N. meningitidis were found to be the main mediator of association with the dermal microvessels in vivo. Bacterial mutants with altered type IV pili function also did not trigger inflammation or lead to vascular damage. This work demonstrates that local type IV pili mediated adhesion of N. meningitidis to the vascular wall, as opposed to circulating bacteria, determines vascular dysfunction in meningococcemia. PMID:23359320

  16. Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

    PubMed Central

    Nuessle, Tiffany M.; Garneau, Nicole L.; Sloan, Meghan M.; Santorico, Stephanie A.

    2015-01-01

    The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller & Reedy’s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller & Reedy’s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller & Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies. PMID:26131644

  17. Salidroside protects against premature senescence induced by ultraviolet B irradiation in human dermal fibroblasts.

    PubMed

    Mao, G-X; Xing, W-M; Wen, X-L; Jia, B-B; Yang, Z-X; Wang, Y-Z; Jin, X-Q; Wang, G-F; Yan, J

    2015-06-01

    Salidroside, the predominant component of a Chinese herbal medicine, Rhodiola rosea L., becomes an attractive bio-agent due to its multifunction. Although it is well proposed that this herbal medicine may have photoprotective effect according to the folk hearsay, the direct supportive experimental evidences linking the drug with skin ageing have rarely been reported so far. The study was conducted to investigate the photoprotective role of salidrosdie and its related mechanisms in vitro. First, a premature senescence model induced by UVB irradiation (250 mJ cm(-2)) in human dermal fibroblasts (HDFs) was established, and senescent phenotypes were evaluated by cell morphology, cell proliferation, senescence-associated beta-galactosidase (SA-β-gal) activity and cell cycle distribution. Then the photoprotective effect of salidroside was investigated. Cells were pre-treated with various doses of salidroside (1, 5 and 10 μM) followed by the sublethal dosage of UVB exposure and then were harvested for various detections, including senescence-associated phenotypes and molecules, alteration of oxidative stress, matrix metalloproteinase-1 (MMP-1) secretion and inflammatory response. Pre-treatment of salidroside dose dependently reversed the senescent state of HDFs induced by UVB as evidenced by elevated cell viability, decreased SA-β-gal activity and relieving of G1/G0 cell cycle arrest. UVB-induced increased protein expression of cyclin-dependent kinase (CDK) inhibitors p21(WAF) (1) and p16(INK) (4) was also repressed by salidrosdie treatment in a dose-dependent manner. Meanwhile, the increment of malondialdehyde (MDA) level in UVB-irradiated HDFs was inhibited upon salidroside treatment. Additionally, salidroside significantly attenuated UVB-induced synthesis of MMP-1 as well as the production of IL-6 and TNF-α in HDFs. Our data provided the evidences for the protective role of salidroside against UVB-induced premature senescence in HDFs probably via its anti

  18. Ultraviolet A Enhances Cathepsin L Expression and Activity via JNK Pathway in Human Dermal Fibroblasts

    PubMed Central

    Xu, Qing-Fang; Zheng, Yue; Chen, Jian; Xu, Xin-Ya; Gong, Zi-Jian; Huang, Yun-Fen; Lu, Chun; Maibach, Howard I; Lai, Wei

    2016-01-01

    Background: Cathepsin L (CatL) is a cysteine protease with strong matrix degradation activity that contributes to photoaging. Mannose phosphate-independent sorting pathways mediate ultraviolet A (UVA)-induced alternate trafficking of CatL. Little is known about signaling pathways involved in the regulation of UVA-induced CatL expression and activity. This study aims to investigate whether a single UVA irradiation affects CatL expression and activity and whether mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway is involved in the regulation of UVA-induced CatL expression and activity in human dermal fibroblasts (HDFs). Methods: Primary HDFs were exposed to UVA. Cell proliferation was determined by a cell counting kit. UVA-induced CatL production and activity were studied with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric assay in cell lysates collected on three consecutive days after irradiation. Time courses of UVA-activated JNK and p38MAPK signaling were examined by Western blotting. Effects of MAPK inhibitors and knockdown of Jun and Fos on UVA-induced CatL expression and activity were investigated by RT-PCR, Western blotting, and fluorimetric assay. Data were analyzed by one-way analysis of variance. Results: UVA significantly increased CatL gene expression, protein abundance, and enzymatic activity for three consecutive days after irradiation (F = 83.11, 56.14, and 71.19, respectively; all P < 0.05). Further investigation demonstrated phosphorylation of JNK and p38MAPK activated by UVA. Importantly, inactivation of JNK pathway significantly decreased UVA-induced CatL expression and activity, which were not affected by p38MAPK inhibition. Moreover, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatL expression and activity. Conclusions: UVA enhances CatL production and activity in HDFs, probably by activating JNK and downstreaming AP-1. These

  19. In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute.

    PubMed

    Tinois, E; Tiollier, J; Gaucherand, M; Dumas, H; Tardy, M; Thivolet, J

    1991-04-01

    Using human type IV and type I + III collagens and a new, nontoxic cross-linking procedure, we have developed a cell-free bilayered human dermal substitute for organotypic culture and transplantation of human skin keratinocytes. We have studied the formation of the basement membrane, and the differentiation of keratinocytes grown on the type IV collagen layer of this dermal substitute, in vitro and after grafting onto nude mice. These studies demonstrated the formation of essential constituents of the basement membrane in culture: hemidesmosomes and deposition of extracellular matrix on the top of the type IV collagen were observed as early as 6 days after plating of human keratinocytes. Although the keratinocytes formed a well-organized multilayered epithelium, they exhibited limited differentiation when grown submerged in liquid medium. However, the multilayered sheet obtained after 14 days in submerged culture was composed of a regular basal cell layer, several nucleated suprabasal cell layers containing granular cells, and several dense, anucleated cell layers. The grafting experiments have shown a good biocompatibility of the dermal substitute. It is repopulated by fibroblasts, newly synthesized collagen, vessels, and a few mononuclear cells. At Day 14 after grafting, the type IV collagen layer was still present and very dense, and the basement membrane appeared as in culture, with numerous well-structured hemidesmosomes and deposition of extracellular matrix resembling lamina densa. At Day 55 after transplantation, even if the epidermal graft did not exhibit all the characteristics of the normal epidermis in vivo, it was very close to it. At this stage, the basement membrane was complete, with structures clearly indicative of anchoring fibrils. This new dermal substitute offers many advantages. It is stable and easy to handle. Its production is standardized. The oxidation induced by periodic acid led to a nontoxic cross-linked matrix. This dermal substitute

  20. Strategies to enhance epithelial-mesenchymal interactions for human hair follicle bioengineering.

    PubMed

    Ohyama, Manabu; Veraitch, Ophelia

    2013-05-01

    Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Accordingly, the enhancement of this crosstalk represents a promising approach to achieve successful bioengineering of human hair follicles. The present article summarizes the techniques, both currently available and potentially feasible, to promote epithelial-mesenchymal interactions (EMIs) necessary for human hair follicle regeneration. The strategies include the preparation of epithelial components with high receptivity to trichogenic dermal signals and/or mesenchymal cell populations with potent hair inductive capacity. In this regard, bulge epithelial stem cells, keratinocytes predisposed to hair follicle fate or keratinocyte precursor cells with plasticity may provide favorable epithelial cell populations. Dermal papilla cells sustaining intrinsic hair inductive capacity, putative dermal papilla precursor cells in the dermal sheath/neonatal dermis or trichogenic dermal cells derived from undifferentiated stem/progenitor cells are promising candidates as hair inductive dermal cells. The most established protocol for in vivo hair follicle reconstitution is co-grafting of epithelial and mesenchymal components into immunodeficient mice. In theory, combination of individually optimized cellular components of respective lineages should elicit most intensive EMIs to form hair follicles. Still, EMIs can be further ameliorated by the modulation of non-cell autonomous conditions, including cell compartmentalization to replicate the positional relationship in vivo and humanization of host environment by preparing human stromal bed. These approaches may not always synergistically intensify EMIs, however, step-by-step investigation probing optimal combinations should maximally enhance EMIs to achieve successful human hair follicle bioengineering.

  1. Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

    PubMed Central

    Jeremias, Talita da Silva; Machado, Rafaela Grecco; Visoni, Silvia Beatriz Coutinho; Pereima, Maurício José; Leonardi, Dilmar Francisco; Trentin, Andrea Gonçalves

    2014-01-01

    New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology. PMID:24586857

  2. The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

    SciTech Connect

    Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F. . E-mail: yves.poumay@fundp.ac.be

    2007-08-03

    Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.

  3. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    PubMed

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Isolation of fungi belonging to the genera Geotrichum and Trichosporum from human dermal lesions.

    PubMed

    Restrepo, A; De Uribe, L

    1976-08-30

    Isolates of Geotrichum and Trichosporum spp. obtained from patients with a variety of dermal lesions were studied. Among 2,202 cases examined, microorganisms of these genera were recovered from 100 (4,5%); there were 38 isolated of Geotrichum- and 62 of Trichosporum- spp. Most isolations were obtained from nails: 52 cases. The species most frequently found were T. beigelii (25 cases) and G. candidum (30 cases). In 50 of the patients, these fungi were isolated in pure culture, in an additional 40 Trichosporum spp. were found. Mixed cultures with C. albicans were observed in 28 patients, with other Candida spp. in 16 and with dermatophytes in 6. Among the patients whose isolations occurred in pure cultures, the number of colonies recovered was large in 20 cases, 1 with Geotrichum candidum - 19 with Trichosporum (16 T. beigelii, 3 T. capitatum). The relationship between the isolated yeast-like fungi and the dermal lesion was considered to be direct in these 20 patients.

  5. Effect of ultrasound irradiation on α-SMA and TGF-β1 expression in human dermal fibroblasts.

    PubMed

    Maeshige, Noriaki; Terashi, Hiroto; Aoyama, Michiko; Torii, Kazuhiro; Sugimoto, Masaharu; Usami, Makoto

    2011-05-11

    Ultrasound therapy is used to promote pressure ulcer healing as an adjunctive therapy. However, the efficacy and the scientific basis of this treatment are unclear. We investigated the effect of ultrasound irradiation on alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta1 (TGF-β1) expression in human dermal fibroblasts. These are important factors for acceleration of wound closure. We used pulsed ultrasound of 0, 0.1, 0.5, and 1.0 W/cm2. TGF-β1 and α-SMA mRNA was measured by quantitative real-time polymerase chain reaction, α-SMA protein was examined by western blot, and localization of α-SMA was evaluated by immunofluorescence staining. Expression of α-SMA and TGF-β1 mRNA was increased at 24 h but not at 48 h after ultrasound irradiation. There were significant differences between controls of 0 W/cm² and 0.1 W/cm² with a 1.34 ± 0.26 fold increase in α-SMA (P < 0.05) and a 1.78 ± 0.57 fold increase in TGF-β1 (P < 0.05). Protein levels of α-SMA were also increased and detected in ultrasound irradiated fibroblasts at 24 h. Ultrasound irradiation promotes α-SMA expression in human dermal fibroblasts and this suggests the biological mechanism of ultrasound efficacy on chronic wound treatment.

  6. Dermal exposure to methamphetamine hydrochloride contaminated residential surfaces: surface pH values, volatility, and in vitro human skin.

    PubMed

    Salocks, Charles B; Hui, Xiaoying; Lamel, Sonia; Qiao, Peter; Sanborn, James R; Maibach, Howard I

    2012-12-01

    This study evaluated pH effects on [(14)C] d-methamphetamine hydrochloride ([(14)C]-meth HCl) percutaneous penetration in vitro and volatility and stability in aqueous solution, on solid surface, or human skin using the finite dose technique and flow through diffusion cells. Results show that when the pH level exceeds 4 or 5, the nonvolatile [(14)C]-meth HCl salt becomes unstable, likely converting to its volatile freebase form. Additionally, contaminated smooth, dense surfaces retain and transfer more [(14)C]-meth HCl than those with rough, loose surfaces, especially under acidic conditions. Skin surface pH is a critical factor affecting the rate and magnitude of dermal absorption. [(14)C]-Meth HCl penetrates into and through the human cadaver skin quickly following exposure. [(14)C]-Meth HCl retained in the skin layer is released into the receptor fluid even if the contact material has been removed. Future exploration of decontaminant and removal procedure efficacies and their effect on dermal penetration of [(14)C]-meth HCl is recommended. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway

    PubMed Central

    Tandon, Nina; Cimetta, Elisa; Villasante, Aranzazu; Kupferstein, Nicolette; Southall, Michael D.; Fassih, Ali; Xie, Junxia; Sun, Ying; Vunjak-Novakovic, Gordana

    2015-01-01

    Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing. PMID:24113575

  8. Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway.

    PubMed

    Tandon, Nina; Cimetta, Elisa; Villasante, Aranzazu; Kupferstein, Nicolette; Southall, Michael D; Fassih, Ali; Xie, Junxia; Sun, Ying; Vunjak-Novakovic, Gordana

    2014-01-01

    Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing. © 2013 Elsevier Inc. All rights reserved.

  9. In vitro human epidermal permeation of nicotine from electronic cigarette refill liquids and implications for dermal exposure assessment.

    PubMed

    Frasch, H Frederick; Barbero, Ana M

    2016-12-07

    Nicotine plus flavorings in a propylene glycol (PG) vehicle are the components of electronic cigarette liquids (e-liquids), which are vaporized and inhaled by the user. Dermal exposure to nicotine and e-liquids may occur among workers in mixing and filling of e-cigarettes in the manufacturing process. Inadvertent skin contact among consumers is also a concern. In vitro nicotine permeation studies using heat-separated human epidermis were performed with surrogate and two commercial e-liquids, neat and aqueous nicotine donor formulations. Steady-state fluxes (Jss), and lag times (tlag) were measured for each formulation. In addition, transient (4 h) exposure and finite dose (1-10 μl/cm(2)) experiments were undertaken using one commercial e-liquid. Average Jss (μg/cm(2)/h) from formulations were: nicotine in PG (24 mg/ml): 3.97; commercial e-liquid containing menthol (25 mg/ml nicotine): 10.2; commercial e-liquid containing limonene (25 mg/ml nicotine): 23.7; neat nicotine: 175. E-liquid lag times ranged from 5 to 10 h. Absorbed fraction of nicotine from finite doses was ≈0.3 at 48 h. The data were applied to transient exposure and finite dose dermal exposure assessment models and to a simple pharmacokinetic model. Three illustrative exposure scenarios demonstrate use of the data to predict systemic uptake and plasma concentrations from dermal exposure. The data demonstrate the potential for significant nicotine absorption through skin contact with e-cigarette refill solutions and the neat nicotine used to mix them.Journal of Exposure Science and Environmental Epidemiology advance online publication, 7 December 2016; doi:10.1038/jes.2016.68.

  10. Comparison of interleukin 10 homologs on dermal wound healing using a novel human skin ex vivo organ culture model

    PubMed Central

    Balaji, Swathi; Moles, Chad M.; Bhattacharya, Sukanta S.; LeSaint, Maria; Dhamija, Yashu; Le, Louis D.; King, Alice; Kidd, Mykia; Bouso, Muhammad F.; Shaaban, Aimen; Crombleholme, Timothy M.; Bollyky, Paul; Keswani, Sundeep G.

    2015-01-01

    Background Anti-inflammatory cytokine interleukin (IL)-10 has been shown to induce regenerative healing in postnatal wounds. A viral homolog of IL-10 produced by human cytomegalovirus (CMV IL-10) similarly generates potent immunoregulatory effects, but its effects on wound healing have not been investigated. Currently, there are limited cost-effective methods of screening vulnerary therapeutics. Taken together, we aim to develop and validate a novel human ex vivo dermal wound model and hypothesize that CMV IL-10 will enhance dermal wound healing. Methods Full-thickness circular (6-mm) explants were taken from surgical skin samples and 3-mm full-thickness wounds were created. Explants were embedded in collagen I matrix and maintained in specially formulated media with the epidermis at air–liquid interface, and treated with human IL-10 or CMV IL-10 (200 ng/mL). The viability of cultured explants was validated by histology and lactate dehydrogenase (LDH) activity. Epithelial gap, epithelial height, basal keratinocyte migration, vascular endothelial growth factor levels, and neovascularization were measured at days 3 and 7 to determine IL-10 effects on wound healing. Results Culture explants at day 7 appeared similar to fresh skin in morphology, cell, and vessel density. By day 14, the epidermis separated from the dermis and the cell density diminished. Day 7 wounds appeared viable with advancing epithelial and basal keratinocyte migration with no evidence of necrosis. Cytotoxicity analysis via the quantification of LDH revealed no differences between controls and treated groups. There was a slight increase in the quantity of LDH in media at day 3; however, this decreased at day 5 and continued to decline up to day 21. CMV IL-10 treatment resulted in a significant decrease in the epithelial gap and an increase in epithelial height. There were no differences in the rates of basal keratinocyte migration at day 7 between treated and control groups. Interestingly, human

  11. Dermal uptake and percutaneous penetration of ten flame retardants in a human skin ex vivo model.

    PubMed

    Frederiksen, Marie; Vorkamp, Katrin; Jensen, Niels Martin; Sørensen, Jens Ahm; Knudsen, Lisbeth E; Sørensen, Lars S; Webster, Thomas F; Nielsen, Jesper B

    2016-11-01

    The dermal uptake and percutaneous penetration of ten organic flame retardants was measured using an ex vivo human skin model. The studied compounds were DBDPE, BTBPE, TBP-DBPE, EH-TBB, BEH-TEBP, α, β and γ-HBCDD as well as syn- and anti-DDC-CO. Little or none of the applied flame retardants was recovered in either type of the receptor fluids used (physiological and worst-case). However, significant fractions were recovered in the skin depot, particularly in the upper skin layers. The primary effect of the worst-case receptor fluid was deeper penetration into the skin. The recovered mass was used to calculate lower- and upper-bound permeability coefficients kp. Despite large structural variation between the studied compounds, a clear, significant decreasing trend of kp was observed with increasing log Kow. The results indicate that the dermis may provide a significant barrier for these highly lipophilic compounds. However, based on our results, dermal uptake should be considered in exposure assessments, though it may proceed in a time-lagged manner compared to less hydrophobic compounds.

  12. Phytosphingosine-1-phosphate and epidermal growth factor synergistically restore extracellular matrix in human dermal fibroblasts in vitro and in vivo.

    PubMed

    Kwon, Seung Bin; An, Sungkwan; Kim, Min Jung; Kim, Ka Ram; Choi, Young Min; Ahn, Kyu Joong; An, In-Sook; Cha, Hwa Jun

    2017-03-01

    Phytosphingosine-1-phosphate (PhS1P), which is found in plants and fungi, is generated by the phosphorylation of phytosphingosine and is structurally similar to molecules that promote cellular growth and proliferation. The aim of this study was to ascertain whether PhS1P displays synergistic effects together with epidermal growth factor (EGF), which is also critical for activating proliferation, migration and survival pathways. We utilized cultured human dermal fibroblasts (HDFs) and a number of assays, including western blotting, cell migration assays, quantitative (real-time) PCR, and viability assays. We found that PhS1P promoted the activity of EGF in vitro. We then conducted a clinical trial in females over 35 years of age, with visible signs of skin aging. By evaluating skin hydration, dermal density and thickness, length of fine wrinkles, and skin elasticity, we verified the clinical efficacy of a combined treatment of PhS1P and EGF in vivo. On the whole, our data suggest that PhS1P displays a synergistic anti-aging effect together with EGF, both in vitro and in vivo.

  13. Phytosphingosine-1-phosphate and epidermal growth factor synergistically restore extracellular matrix in human dermal fibroblasts in vitro and in vivo.

    PubMed

    Kwon, Seung Bin; An, Sungkwan; Kim, Min Jung; Kim, Ka Ram; Choi, Young Min; Ahn, Kyu Joong; An, In-Sook; Cha, Hwa Jun

    2017-01-23

    Phytosphingosine-1-phosphate (PhS1P), which is found in plants and fungi, is generated by the phosphorylation of phytosphingosine and is structurally similar to molecules that promote cellular growth and proliferation. The aim of this study was to ascertain whether PhS1P displays synergistic effects together with epidermal growth factor (EGF), which is also critical for activating proliferation, migration and survival pathways. We utilized cultured human dermal fibroblasts (HDFs) and a number of assays, including western blotting, cell migration assays, quantitative (real-time) PCR, and viability assays. We found that PhS1P promoted the activity of EGF in vitro. We then conducted a clinical trial in females over 35 years of age, with visible signs of skin aging. By evaluating skin hydration, dermal density and thickness, length of fine wrinkles, and skin elasticity, we verified the clinical efficacy of a combined treatment of PhS1P and EGF in vivo. On the whole, our data suggest that PhS1P displays a synergistic anti-aging effect together with EGF, both in vitro and in vivo.

  14. Plasma Rich in Growth Factors Inhibits Ultraviolet B Induced Photoageing of the Skin in Human Dermal Fibroblast Culture.

    PubMed

    Anitua, Eduardo; Pino, Ander; Orive, Gorka

    Ultraviolet irradiation is able to deeply penetrate into the dermis and alter fibroblast structure and function, leading to a degradation of the dermal extracellular matrix. The regenerative effect of plasma rich in growth factors (PRGF) on skin ageing was investigated using UVB photo-stressed human dermal fibroblasts as an in vitro culture model. PRGF was assessed over the main indicative features of ultraviolet B irradiation, including ROS formation, cell viability and death detection, apoptosis/ necrosis analysis and biosynthetic activity measurement. Four different UV irradiation protocols were tested in order to analyze the beneficial effects of PRGF. Ultraviolet irradiation exhibited a dose dependent cytotoxicity and dose of 400mJ/cm2 was selected for subsequent experiments. PRGF increased the cell viability and decreased the cell death comparing to the non-treated group. The apoptosis and necrosis were significantly lower in PRGF treated fibroblasts. ROS production after UV irradiation was significantly reduced in the presence of PRGF. Procollagen type I, hyaluronic acid and TIMP-1 levels were higher in the when treated with PRGF. This preliminary in vitro study suggests that PRGF is able to prevent UVB derived photooxidative stress and to diminish the cell damage caused by ultraviolet irradiation.

  15. Assessment of dermal absorption of DEET-containing insect repellent and oxybenzone-containing sunscreen using human urinary metabolites.

    PubMed

    Yiin, Lih-Ming; Tian, Jia-Ni; Hung, Chien-Che

    2015-05-01

    Mutual enhancement of dermal absorption of N,N-diethyl-m-toluamide (DEET) and oxybenzone (OBZ) has been reported recently with DEET and OBZ being active ingredients of insect repellent and sunscreen, respectively. To assess the reported enhancing effect directly, we used human urinary metabolites as biomarkers; besides, we also sought to determine the best way for concurrent use of these two products without extra absorption of either. Four dermal application methods were used: DEET only (S1), OBZ only (S2), DEET on top of OBZ (S3), and OBZ on top of DEET (S4). Among the study methods, there was a significant difference (p = 0.013), which was attributed to the difference between S1 and S4, suggesting that applying OBZ over DEET on the skin lead to significantly higher absorption of DEET. Using both products in reverse order, (S3) did not result in extra DEET absorption significantly. As for OBZ permeation, no significant difference was observed among the methods. In summary, the enhancement of DEET absorption is confirmed for OBZ being applied over DEET on the skin; should concurrent use of both be necessary, applying sunscreen (OBZ) first and then insect repellent (DEET) with a 15-min interval is recommended.

  16. Mitochondrial tolerance to single and repeat exposure to simulated sunlight in human epidermal and dermal skin cells.

    PubMed

    Kelly, J; Murphy, J E J

    2016-12-01

    Sunlight represents the primary threat to mitochondrial integrity in skin given the unique nature of the mitochondrial genome and its proximity to the electron transport chain. The accumulation of mitochondrial DNA (mtDNA) mutations is a key factor in many human pathologies and this is linked to key roles of mitochondrial function in terms of energy production and cell regulation. The main objective of this study was to evaluate solar radiation induced changes in mitochondrial integrity, function and dynamics in human skin cells using a Q-Sun solar simulator to deliver a close match to the intensity of summer sunlight. Spontaneously immortalised human skin epidermal keratinocytes (HaCaT) and Human Dermal Fibroblasts (HDFn) were divided into two groups. Group A were irradiated once and Group B twice 7days apart and evaluated using cell survival, viability and mitochondrial membrane potential (MMP) and mass at 1, 4 and 7days post one exposure for Group A and 1, 4, 7 and 14days post second exposure for Group B. Viability and survival of HaCaT and HDFn cells decreased after repeat exposure to Simulated Sunlight Irradiation (SSI) with no recovery. HDFn cells showed no loss in MMP after one or two exposures to SSI compared to HaCaT cells which showed a periodic loss of MMP after one exposure with a repeat exposure causing a dramatic decrease from which cells did not recover. Mitochondrial Mass in exposed HDFn cells was consistent with control after one or two exposures to SSI; however mitochondrial mass was significantly decreased in HaCaT cells. Data presented here suggests that mitochondria in epidermal cells are more sensitive to sunlight damage compared to mitochondria in dermal cells, despite their origin, confirming a skin layer specific sensitivity to sunlight, but not as expected. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. The Effects of Chronological Age on the Cellular Mechanics of Human Dermal Fibroblasts

    NASA Astrophysics Data System (ADS)

    Pan, Z.; Hung, V.; Kambhampati, S.; Ge, S. R.; Rafailovich, M.; Ghosh, K.; Clark, R.; Liu, Y. J.; Nakamura, T.; Shu, X. Z.; Prestwich, G.

    2006-03-01

    It is often observed that older people display diminished wound healing abilities. Understanding of this phenomenon is important for many in vivo applications of tissue engineering. In this study, the cell mechanics of dermal fibroblasts from 25, 40 and 84 years old female subjects were compared. These cells were cultured on functionalized hyaluronic acid hydrogel substrates which emulated physiological conditions in dermal tissue. The deformation of the substrate caused by cellular traction forces was detected by tracing the displacement of fluorescent beads embedded in the substrate using Digital Image Speckle Correlation. Then cellular traction forces were quantitatively determined by Finite Element Method in a linear elastic model with a high spatial resolution. These results were correlated with auxiliary measurements of substrate modulus, cell modulus and migration. We found that with increasing age, the magnitude of the cellular traction forces diminished. Similarly, the ability of the cells to adapt to changes in the mechanical properties of their environment and migrate was also impaired. The interrelationship between these factors and wound healing will be discussed. This work is supported by NSF- MRSEC program.

  18. Expression of catalytically active Matrix Metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

    PubMed Central

    Xia, Wei; Hammerberg, Craig; Li, Yong; He, Tianyuan; Quan, Taihao; Voorhees, John J; Fisher, Gary J

    2013-01-01

    Summary Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-β pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts, and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function. PMID:23601157

  19. Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

    PubMed

    Hara, Tomoya; Kakudo, Natsuko; Morimoto, Naoki; Ogawa, Takeshi; Lai, Fangyuan; Kusumoto, Kenji

    2016-12-01

    Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.

  20. Aligned nanofibers direct human dermal fibroblasts to tenogenic phenotype in vitro and enhance tendon regeneration in vivo.

    PubMed

    Wang, Wenbo; He, Jing; Feng, Bei; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Fu, Wei; Liu, Wei

    2016-05-01

    To explore the effect of aligned nanofibers on inducing tenogenic phenotype of human dermal fibroblasts (hDFs) in vitro and on inducing de novo tendon regeneration in vivo. Random and aligned nanofibers were electrospun, seeded with hDFs and cultured in vitro, and in vivo implanted without cell seeding to bridge segmental defect of rat Achilles tendon. In vitro, the well-aligned nanofibers could elongate hDFs, induce a tenogenic phenotype and form better organized neotendon respectively compared with random nanofibers. In vivo, the bridged nanofibers of aligned group could better recruit host cells and regenerate Achilles tendon de novo with enhanced tenogenic gene expression. Aligned nanofibers could induce tenogenic phenotype in vitro and regenerate tendon in vivo.

  1. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures

    PubMed Central

    Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

    2016-01-01

    Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients. PMID:27621603

  2. Large-Scaled Metabolic Profiling of Human Dermal Fibroblasts Derived from Pseudoxanthoma Elasticum Patients and Healthy Controls

    PubMed Central

    Kuzaj, Patricia; Kuhn, Joachim; Michalek, Ryan D.; Karoly, Edward D.; Faust, Isabel; Dabisch-Ruthe, Mareike; Knabbe, Cornelius; Hendig, Doris

    2014-01-01

    Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization. PMID:25265166

  3. Chum salmon egg extracts induce upregulation of collagen type I and exert antioxidative effects on human dermal fibroblast cultures.

    PubMed

    Yoshino, Atsushi; Polouliakh, Natalia; Meguro, Akira; Takeuchi, Masaki; Kawagoe, Tatsukata; Mizuki, Nobuhisa

    2016-01-01

    Components of fish roe possess antioxidant and antiaging activities, making them potentially very beneficial natural resources. Here, we investigated chum salmon eggs (CSEs) as a source of active ingredients, including vitamins, unsaturated fatty acids, and proteins. We incubated human dermal fibroblast cultures for 48 hours with high and low concentrations of CSE extracts and analyzed changes in gene expression. Cells treated with CSE extract showed concentration-dependent upregulation of collagen type I genes and of multiple antioxidative genes, including OXR1, TXNRD1, and PRDX family genes. We further conducted in silico phylogenetic footprinting analysis of promoter regions. These results suggested that transcription factors such as acute myeloid leukemia-1a and cyclic adenosine monophosphate response element-binding protein may be involved in the observed upregulation of antioxidative genes. Our results support the idea that CSEs are strong candidate sources of antioxidant materials and cosmeceutically effective ingredients.

  4. UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

    PubMed Central

    Lamore, Sarah D.; Wondrak, Georg T.

    2013-01-01

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

  5. UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts.

    PubMed

    Lamore, Sarah D; Wondrak, Georg T

    2013-06-05

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display 'UVA-mimetic' effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts.

  6. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    SciTech Connect

    Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  7. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling. Copyright © 2016 the American Physiological Society.

  8. Assessment of the in vitro dermal irritation potential of cerium, silver, and titanium nanoparticles in a human skin equivalent model.

    PubMed

    Miyani, Vivek A; Hughes, Michael F

    2017-06-01

    Metal nanoparticles can potentially contact human skin during their manufacture and use in commercial products. This study examined the potential of metal nanoparticles to elicit irritant contact dermatitis in a human skin equivalent model (HSEM) derived from keratinocytes. Ag (10-100 nm), TiO2 (22-214 nm), and CeO2 (15-40 nm) nanoparticles were studied. The Ag particles were either coated/shelled with silica or capped with citrate or polyvinylpyrrolidone and were in water. The TiO2 and CeO2 particles were suspended in media containing 10% fetal bovine serum. The particles (1 mg/ml) were applied to the epidermal surface of the HSEM. Positive (5% sodium dodecyl sulfate (SDS)) and negative controls (saline or media) were included. After 1-h exposure at 37 °C, the HSEM was washed with saline to remove the nanoparticles. Following a 42-h incubation (37 °C), HSEM viability was assessed using the MTT assay. A test substance is considered a dermal irritant if the HSEM viability is < 50%. The mean viability for the SDS-treated HSEM was 7.8%. The viabilities of the nanoparticle-treated HSEM were 91% or greater. The Ag, TiO2, and CeO2 nanoparticles examined were not dermal irritants under the conditions used in this study. The stratum corneum of the HSEM may limit penetration of metal nanoparticles to induce toxicity.

  9. Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells.

    PubMed

    Milan, P Brouki; Lotfibakhshaiesh, N; Joghataie, M T; Ai, J; Pazouki, A; Kaplan, D L; Kargozar, S; Amini, N; Hamblin, M R; Mozafari, M; Samadikuchaksaraei, A

    2016-11-01

    There is an unmet clinical need for novel wound healing strategies to treat full thickness skin defects, especially in diabetic patients. We hypothesized that a scaffold could perform dual roles of a biomechanical support and a favorable biochemical environment for stem cells. Human umbilical cord perivascular cells (HUCPVCs) have been recently reported as a type of mesenchymal stem cell that can accelerate early wound healing in skin defects. However, there are only a limited number of studies that have incorporated these cells into natural scaffolds for dermal tissue engineering. The aim of the present study was to promote angiogenesis and accelerate wound healing by using HUCPVCs and decellularized dermal matrix (DDM) in a rat model of diabetic wounds. The DDM scaffolds were prepared from harvested human skin samples and histological, ultrastructural, molecular and mechanical assessments were carried out. In comparison with the control (without any treatment) and DDM alone group, full thickness excisional wounds treated with HUCPVCs-loaded DDM scaffolds demonstrated an accelerated wound closure rate, faster re-epithelization, more granulation tissue formation and decreased collagen deposition. Furthermore, immunofluorescence analysis showed that the VEGFR-2 expression and vascular density in the HUCPVCs-loaded DDM scaffold treated group were also significantly higher than the other groups at 7days post implantation. Since the rates of angiogenesis, re-epithelization and formation of granulation tissue are directly correlated with full thickness wound healing in patients, the proposed HUCPVCs-loaded DDM scaffolds may fulfil a role neglected by current treatment strategies. This pre-clinical proof-of-concept study warrants further clinical evaluation.

  10. Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts from prolonged stress and contribute to survival and rejuvenation of human skin equivalents.

    PubMed

    Khan, Tapan K; Wender, Paul A; Alkon, Daniel L

    2017-06-07

    Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine. © 2017 Wiley Periodicals, Inc.

  11. Sensing in the mouth: A model for filiform papillae as strain amplifiers

    NASA Astrophysics Data System (ADS)

    Lauga, Eric; Pipe, Christopher; Le Révérend, Benjamin

    2016-08-01

    Texture perception of foods is a common yet remarkably unstudied biophysical problem. Motivated by recent experiments reporting the presence of corpuscular endings in tongue filiform papillae, we develop in this work a mechanical model of the human tongue covered with filiform papillae in the form of elastic beams. Considering the typical flows that occur in the mouth during oral evaluation of Newtonian liquids, we suggest that filiform papillae may act either as direct strain sensors and/or as indirect strain amplifiers for the underlying mucosal tissue. Application of this model may also be valid for other biological appendages, such as primary cilliae and superficial neuromasts.

  12. A COMPARATIVE INVESTIGATION OF THE INFLUENCE OF DERMAL APPENDAGES (HAIR FOLLICLES) ON THE PERCUTANEOUS ABSORPTION OF ORGANOPHOSPHORUS (OP) INSECTICIDES USING QSAR AND PBPK/PD MODELS FOR HUMAN RISK ASSESSMENT

    EPA Science Inventory

    The successful use of the Exposure Related Dose Estimating Model (ERDEM) for assessment of dermal exposure of humans to OP pesticides requires the input of representative and comparable input parameters. In the specific case of dermal exposure, regional anatomical variation in...

  13. A COMPARATIVE INVESTIGATION OF THE INFLUENCE OF DERMAL APPENDAGES (HAIR FOLLICLES) ON THE PERCUTANEOUS ABSORPTION OF ORGANOPHOSPHORUS (OP) INSECTICIDES USING QSAR AND PBPK/PD MODELS FOR HUMAN RISK ASSESSMENT

    EPA Science Inventory

    The successful use of the Exposure Related Dose Estimating Model (ERDEM) for assessment of dermal exposure of humans to OP pesticides requires the input of representative and comparable input parameters. In the specific case of dermal exposure, regional anatomical variation in...

  14. Stereo architecture of the connective tissue cores of the lingual papillae in the treeshrew (Tupaia glis).

    PubMed

    Kobayashi, K; Wanichanon, C

    1992-12-01

    The stereo architecture of the lingual connective tissue cores (CTC) in the treeshrew (Tupaia glis) (which has the primitive characteristics of primates) was observed by scanning electron microscopy, and compared to that of other animal orders. The tongue of the treeshrew has three vallate papillae which are situated in the posterior part of the tongue, while some macaques have several vallate papillae. Among numerous filiform papillae, fungiform papillae are sporadically distributed. A filiform papilla consists of a bundle of several slender spine-like processes arranged in a circle at the basal margin. After removal of the epithelium, the CTC of the filiform papilla looks like a human hand raised with the palm facing towards the tongue tip. The fungiform CTC in the threeshrew is columnar in shape (rather similar to that of Insectivora and Rodentia) and at the top there are several round depressions for taste buds. In the treeshrew several large rod-shaped processes are derived from the postero-lateral margin of the tongue, as in Carnivora (dogs and cats), where foliate papillae are located in many other animal species. The treeshrew has numerous characteristics similar to those of the crab-eating macaque (Primates), but at the same time it has some characteristics similar to those of Insectivora, Rodentia, Carnivora and Artiodactyla.

  15. Steady state temperature distribution in dermal regions of an irregular tapered shaped human limb with variable eccentricity.

    PubMed

    Agrawal, M; Pardasani, K R; Adlakha, N

    2014-08-01

    The investigators in the past have developed some models of temperature distribution in the human limb assuming it as a regular circular or elliptical tapered cylinder. But in reality the limb is not of regular tapered cylindrical shape. The radius and eccentricity are not same throughout the limb. In view of above a model of temperature distribution in the irregular tapered elliptical shaped human limb is proposed for a three dimensional steady state case in this paper. The limb is assumed to be composed of multiple cylindrical substructures with variable radius and eccentricity. The mathematical model incorporates the effect of blood mass flow rate, metabolic activity and thermal conductivity. The outer surface is exposed to the environment and appropriate boundary conditions have been framed. The finite element method has been employed to obtain the solution. The temperature profiles have been computed in the dermal layers of a human limb and used to study the effect of shape, microstructure and biophysical parameters on temperature distribution in human limbs. The proposed model is one of the most realistic model as compared to conventional models as this can be effectively employed to every regular and nonregular structures of the body with variable radius and eccentricity to study the thermal behaviour.

  16. Dermal penetration of propylene glycols: measured absorption across human abdominal skin in vitro and comparison with a QSAR model.

    PubMed

    Fasano, William J; ten Berge, Wil F; Banton, Marcy I; Heneweer, Marjoke; Moore, Nigel P

    2011-12-01

    The dermal penetration of undiluted monopropylene glycol (MPG) and dipropylene glycol (DPG) has been measured in vitro using human abdominal skin under conditions of infinite dose application, and the results compared with predictions from the SKINPERM QSAR model (ten Berge, 2009). The measured steady-state penetration rates (Jss) for MPG and DPG were 97.6 and 39.3 μg/cm2/h, respectively, and the permeability coefficients (Kp) were 9.48×10(-5) cm/h for MPG and 3.85×10(-5) cm/h for DPG. In comparison, the SKINPERM model slightly over-predicted Jss and Kp for MPG and DPG by between 2.6- and 5.1-fold, respectively. The model predictions of 254 μg/cm2/h and 24.6×10(-5) cm/h for MPG, and 202 μg/cm2/h and 19.8×10(-5) cm/h for DPG were in fairly good agreement with the measured values. Further, the model predicted a Jss of 101 μg/cm2/h and a Kp of 9.9×10(-5) cm/h for the homologue tripropylene glycol. Assuming that the measured Jss was the same under conditions of finite dose application (taken to be 10 μL/ cm2) and was maintained over a 24-h period (both conservative assumptions), the relative dermal absorption of the applied dose was estimated to be 23% (0.96%/h) for MPG and 9% (0.39%/h) for DPG. However, the extrapolation for MPG may be further overestimated due to possible residence in the stratum corneum under infinite conditions of exposure that would not be applicable to a finite loading dose. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Role of NF-κB-p53 crosstalk in ultraviolet A-induced cell death and G1 arrest in human dermal fibroblasts.

    PubMed

    Lee, Yun Kyung; Cha, Hwa Jun; Hong, Misun; Yoon, Yeongmin; Lee, Hyunjin; An, Sungkwan

    2012-01-01

    Photoaging is the premature aging of the skin caused by repeated exposure to sunlight and is characterized by a depletion of the dermal extracellular matrix. This depletion is due to the loss of fibroblast cells and their multiple functions. UVA was revealed as a major inducer of photoaging in various clinical studies. As UVA photons have long wavelength spectra, UVA penetrates deeper into the dermis than UVB and UVC, leading to the induction of cell death, the destruction of the dermal extracellular matrix through the induction of matrix metalloproteinase expression, and the repression of collagen expression. However, the exact effects of UVA on the skin remain a matter of debate. Here, we assess cell cycle stage to demonstrate that NF-κB-p53 crosstalk induces apoptosis and growth arrest in UVA-irradiated human dermal fibroblasts. In addition, UVA irradiation led to an increase of NF-κB-HDAC1 complexes, which in turn repressed cyclin D1 expression in UVA-irradiated human dermal fibroblasts. We provide direct evidence that UVA irradiation induces changes in the p53-dependent NF-κB complex that lead to growth arrest and apoptosis through the repression of cyclin D1. These studies uncovered that NF-κB-p53 crosstalk is a key regulator of UVA-dependent growth arrest and apoptosis.

  18. Morphology of the lingual papillae in the jaguar.

    PubMed

    Emura, Shoichi; Okumura, Toshihiko; Chen, Huayue

    2013-01-01

    We examined the dorsal lingual surfaces of an adult jaguar (Panthera onca) by scanning electron microscopy. The tongue of the jaguar was about 17 cm long, and the center of the lingual apex became hollow. There were 7 vallate papillae in total. The filiform papilla on the lingual apex consisted of a larger main papilla and some secondary papillae. The connective tissue core of the filiform papilla was mountain-like in shape. The connective tissue core of the fungiform papilla was mushroom-like in shape. The filiform papilla on the anterior part of the lingual body was large and cylinder-like in shape. The connective tissue core of the filiform papilla consisted of a big conical papilla and many rod-like papillae. The filiform papilla on the central part of the lingual body was a big conical papilla. The connective tissue core of the filiform papilla consisted of a rod process and bowl-like structure. The vallate papillae were located on both sides of the posterolateral aspects. The vallate papillae were flattened-oval in shape and the papillae were surrounded by a groove and pad. The top of the connective tissue core of the vallate papilla had a rough surface with no spines.

  19. Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla

    PubMed Central

    Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

    2014-01-01

    Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs. PMID:24864235

  20. Profibrillin-1 Maturation by Human Dermal Fibroblasts: Proteolytic Processing and Molecular Chaperones

    PubMed Central

    Wallis, Debra D.; Putnam, Elizabeth A.; Cretoiu, Jill S.; Carmical, Sonya G.; Cao, Shi-Nian; Thomas, Gary; Milewicz, Dianna M.

    2006-01-01

    Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A1 and incubation of dermal fibroblasts at 22°C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, α-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway. PMID:14523997

  1. FGF2 Overrides TGFβ1-Driven Integrin ITGA11 Expression in Human Dermal Fibroblasts.

    PubMed

    Grella, Alexandra; Kole, Denis; Holmes, William; Dominko, Tanja

    2016-04-01

    Deposition of collagen-based extracellular matrix by fibroblasts during wound healing leads to scar formation--a typical outcome of the healing process in soft tissue wounds. The process can, however, be skewed in favor of tissue regeneration by manipulation of wound environment. Low oxygen conditions and supplementation with FGF2 provide extracellular cues that drive wound fibroblasts towards a pro-regenerative phenotype. Under these conditions, fibroblasts dramatically alter expression of many genes among which the most significantly deregulated are extracellular matrix and adhesion molecules. Here we investigate the mechanism of a collagen I binding integrin α11 (ITGA11) deregulation in response to low oxygen-mediated FGF2 effects in dermal fibroblasts. Using RT-PCR, qRT-PCR, Western blotting, and immunocytochemistry, we describe significant down-regulation of ITGA11. Decrease in ITGA11 is associated with its loss from focal adhesions. We show that loss of ITGA11 requires FGF2 induced ERK1/2 activity and in the presence of FGF2, ITGA11 expression cannot be rescued by TGFβ1, a potent activator of ITGA11. Our results indicate that FGF2 may be redirecting fibroblasts towards an anti-fibrotic phenotype by overriding TGFβ1 mediated ITGA11 expression. © 2015 Wiley Periodicals, Inc.

  2. Assessment of relevant factors and relationships concerning human dermal exposure to pesticides in greenhouse applications.

    PubMed

    Martínez Vidal, Jose L; Egea González, Francisco J; Garrido Frenich, Antonia; Martínez Galera, María; Aguilera, Pedro A; López Carrique, Enrique

    2002-08-01

    Principal component analysis (PCA) was applied to the gas chromatographic data obtained from 23 different greenhouse trials. This was used to establish which factors, including application technique (very small, small, medium and large drop-size), crop characteristics (short/tall, thin/dense) and pattern application of the operator (walking towards or away from the treated area) are relevant to the dermal exposure levels of greenhouse applicators. The results showed that the highest exposure by pesticides during field applications in greenhouses, in the climatic conditions and in the crop conditions typical of a southern European country, occurs on the lower legs and front thighs of the applicators. Similar results were obtained by hierarchical cluster analysis (HCA). Drop-size seems to be very important in determining total exposure, while height and density of crops have little influence on total exposure under the conditions of the present study. No pesticide type is a major factor in total exposure. The application of multiple regression analysis (MRA) allowed assessment of the relationships between the pesticide exposure of the less affected parts of the body with the most affected parts.

  3. Human skin fibroblasts in culture: procollagen synthesis in the presence of sera from normal human subjects and from patients with dermal fibroses.

    PubMed

    Tan, E M; Uitto, J; Bauer, E A; Eisen, A Z

    1981-06-01

    Various dermal fibrotic conditions, such as progressive systemic sclerosis, localized morphea and familial cutaneous collagenoma, are characterized by excessive deposition of collagen in the skin. In the present study, we examined the possibility that a circulating serum factor(s) is responsible for increased collagen production in these diseases. The effects of human serum on the synthesis of procollagen were examined by incubating normal human dermal fibroblasts with [3H]proline and varying concentrations of dialyzed heat-inactivated serum. The synthesis of procollagen was measured as formation of nondialyzable [3H]hydroxyproline and collagenase-digestible 3H]polypeptides. In the absence of serum little procollagen was formed but the synthesis was markedly stimulated by the addition of normal serum in a concentration-dependent manner. THe ratio of genetically distinct 3H-procollagens of type I and type III, assayed by DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis after limited pepsin proteolysis, was unaffected by the addition of serum. Thus, normal human serum contains a nondialyzable factor(s) which stimulates the synthesis of procollagens type I and type III equally. Sera from 5 patients with progressive systemic sclerosis, 3 with localized scleroderma, and 2 with familial cutaneous collagenoma were also tested. Sera from these patients failed to stimulate 3H-procollagen production more than sera from healthy age-matched controls. Therefore, no increased quantities or qualitatively aberrant factors were shown to be present in the sera of these patients.

  4. Assessment of in vitro human dermal absorption studies on pesticides to determine default values, opportunities for read-across and influence of dilution on absorption.

    PubMed

    Aggarwal, M; Battalora, M; Fisher, P; Hüser, A; Parr-Dobrzanski, R; Soufi, M; Mostert, V; Strupp, C; Whalley, P; Wiemann, C; Billington, R

    2014-04-01

    Dermal absorption is an integral part of non-dietary human safety risk assessments for agrochemicals. Typically, dermal absorption data for agrochemical active substances are generated from the undiluted formulation concentrate and its spray dilutions. European Food Safety Authority (EFSA) guidance, which combines highly conservative default values, very limited opportunities for read-across from existing data and other overly conservative conclusions, was the driver for this assessment. To investigate the reliability of the EFSA guidance, a homogeneous data-set of 190 GLP and OECD guideline compliant in vitro human skin studies, chosen to match the test method preferred by EU data requirements, was evaluated. These studies represented a wide range of active substances, formulation types, and concentrations. In alignment with EFSA guidance on human exposure assessment, a conservative estimate of absorption (95th percentile) was chosen to define defaults, which were also based on the EFSA worst-case assumption that all material in skin, excluding the first two tape strips, is absorbed. The analysis supports dermal absorption defaults of 6% for liquid concentrates, 2% for solid concentrates, and 30% for all spray dilutions, irrespective of the active substance concentration. Relatively high dermal absorption values for organic solvent-based formulations, compared to water-based or solid concentrates, support their use as worst-case surrogate data for read-across to other formulation types. The current review also shows that dermal absorption of sprays does not increase linearly with increasing dilution, and provides a novel, science-based option for extrapolation from existing data.

  5. Clinical performance of a dermal filler containing natural glycolic Acid and a polylactic Acid polymer: results of a clinical trial in human immunodeficiency virus subjects with facial lipoatrophy.

    PubMed

    Tagle, Jorge M; Macchetto, Pedro Cervantes; Durán Páramo, Rosa Margarita

    2010-02-01

    Lipoatrophy is a condition that affects certain individuals, most commonly those who are infected with the human immunodeficiency virus.(1-3) Injectable fillers are used for the treatment of these dermal contour deformities to smooth dermal depressions formed by the loss of volume. These dermal fillers (also known as soft tissue augmentation devices) can correct contour deformities caused by lipoatrophy in patients who are human immunodeficiency virus positive or negative. The product used in this study is a patented, second-generation, injectable, dermal collagen stimulator that combines glycolic acid and polylactic acid. The glycolic acid used is not a polymer, but rather an acid derived from sugar cane. Its chemical structure corresponds to that of an alpha-hydroxy acid. Glycolic acid is a well-characterized agent that is present in a number of cosmetic products. Polylactic acid is a synthetic, biocompatible, biodegradable, inert, synthetic polymer from the poly a-hydroxy-acid family that is believed to stimulate fibroblasts to produce more collagen, thus increasing facial volume. Together, polylactic acid and glycolic acid act in concert to 1) stimulate collagen production and 2) hydrate the outer layers of the skin. A multicenter, clinical investigation authorized by the Mexican Secretariat of Health was conducted between September 20, 2002, and September 19, 2004. This clinical study was conducted in male patients between 32 and 60 years of age with lipoatrophy as a result of highly active antiretroviral therapy for human immunodeficiency virus infection. The study objective was to measure the improvement of contour deformities after the injection of a dermal collagen stimulator containing glycolic acid and polylactic acid. In addition to safety, this dermal filler was assessed when used to correct volume deformities caused by lipoatrophy in subjects who are human immunodeficiency virus positive. Thirty male subjects participated and were treated as follows

  6. Efficacy of low-power laser irradiation in the prevention of D-galactose-induced senescence in human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Meng, Chengbo; Wu, Shengnan; Xing, Da

    2011-03-01

    Low-power laser (He-Ne) irradiation (LPLI) has been found to modulate various biological effects, especially those involved in promoting cell proliferation and metabolic regulation. However, the underlying mechanisms that LPLI prevents human cell senescence remain undefined. Herein, we devised a model enabling cell senescence using D-galactose for two days then treat with or without LPLI(< 15J/cm2), and investigated whether LPLI delays cell senescent in human dermal fibroblasts cells (HDF-a). First in this study, using SA-β-gal staining, compared with control cell we detected a lower frequency of SA-β-gal staining under the treatment of LPLI. Moreover, we found the growth rates of cell with LPLI was higher using CCK-8 analysis. Additionally, we also found LPLI induced HDF-a entered the irreversible G1 arrest measured by flow cytometry system. Therefore, LPLI may promote cell proliferation by stimulating cell-cycle progression and delay human cell senescence. Taken together, Low-power laser irradiation delay HDF-a cells senescence provides new information for the mechanisms of biological effects of LPLI.

  7. Molecular modifications of dermal and epidermal biomarkers following UVA exposures on reconstructed full-thickness human skin.

    PubMed

    Meloni, Marisa; Farina, Anne; de Servi, Barbara

    2010-04-01

    Ultraviolet A (UVA) radiation adversely affects skin health and appearance via multiple molecular pathways. Biologically relevant UVA damage are classified as short-term effects (e.g. formation of reactive oxygen species [ROS], inflammation, photo-oxidation, DNA damage, immunosuppression, photoallergy and cell-mediated contact hypersensitivity) or long-term effects (elastosis, photoageing and photocarcinogenesis). Single and chronic experimental exposure to UVA are limited in humans by ethical concerns, and furthermore it is impossible to quantify long-term endpoints such as photoageing over the life-span of a human volunteer. The aim of the present study was to investigate the biological relevance of the Phenion FT skin model for use in photobiological studies. Biological responses to acute and repeated UVA exposures were investigated by monitoring the kinetics of gene expression during the post-irradiation period. By using a dynamic approach, we were able to define early and stable markers of UVA-induced effects that could be predictive of UVA damage in vivo. The transcriptomic approach applied to 3D human tissues appears to be an encouraging method for gaining a deeper understanding of the UVA effects on skin and for studying the dermal response with non-invasive techniques.

  8. Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts.

    PubMed

    Belvedere, Raffaella; Bizzarro, Valentina; Parente, Luca; Petrella, Francesco; Petrella, Antonello

    2017-08-10

    Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

  9. Defining dermal adipose tissue.

    PubMed

    Driskell, Ryan R; Jahoda, Colin A B; Chuong, Cheng-Ming; Watt, Fiona M; Horsley, Valerie

    2014-09-01

    Here, we explore the evolution and development of skin-associated adipose tissue with the goal of establishing nomenclature for this tissue. Underlying the reticular dermis, a thick layer of adipocytes exists that encases mature hair follicles in rodents and humans. The association of lipid-filled cells with the skin is found in many invertebrate and vertebrate species. Historically, this layer of adipocytes has been termed subcutaneous adipose, hypodermis and subcutis. Recent data have revealed a common precursor for dermal fibroblasts and intradermal adipocytes during development. Furthermore, the development of adipocytes in the skin is independent from that of subcutaneous adipose tissue development. Finally, the role of adipocytes has been shown to be relevant for epidermal homoeostasis during hair follicle regeneration and wound healing. Thus, we propose a refined nomenclature for the cells and adipose tissue underlying the reticular dermis as intradermal adipocytes and dermal white adipose tissue, respectively.

  10. Morphology of the lingual papillae of the eland (Taurotragus oryx).

    PubMed

    Emura, Shoichi

    2016-01-01

    We examined the dorsal lingual surfaces of an adult eland (Taurotragus oryx) by scanning electron microscopy. Filiform, fungiform and vallate papillae were observed. The filiform papillae of the lingual apex consisted of a larger main papilla and smaller secondary papillae. The connective tissue core of the filiform papilla was U-shaped. The fungiform papillae were round in shape. The connective tissue core of the fungiform papilla was flower-bud shaped. The filiform papillae of the lingual body consisted of a main papilla and were big as compared to that of the lingual apex. The connective tissue core of the filiform papilla resembled that of the lingual apes. The lenticular papillae of large size were limited on the lingual prominence. The connective tissue core of the lenticular papilla consisted of numerous small spines. The vallate papillae were located on both sides of the posterolateral aspects. The vallate papillae were flattened-oval shaped and the papillae were surrounded by a semicircular trench. The connective tissue core of the vallate papilla was covered with numerous small spines. The lingual surface of the eland closely resembled that of the family Bovidae.

  11. Hypoxia drives the transition of human dermal fibroblasts to a myofibroblast-like phenotype via the TGF-β1/Smad3 pathway

    PubMed Central

    Zhao, Bin; Guan, Hao; Liu, Jia-Qi; Zheng, Zhao; Zhou, Qin; Zhang, Jian; Su, Lin-Lin; Hu, Da-Hai

    2017-01-01

    Keloids, partially considered as benign tumors, are characterized by the overgrowth of fibrosis beyond the boundaries of the wound and are regulated mainly by transforming growth factor (TGF)-β1, which induces the transition of fibroblasts to myofibroblasts. Hypoxia is an important driving force in the development of lung and liver fibrosis by activating hypoxia inducible factor-1α and stimulating epithelial-mesenchymal transition. However, it is unknown whether and hypoxia can influence human dermal scarring. The aim of this study was to investigate whether hypoxia drives the transition of dermal fibroblasts to myofibroblasts and to clarify the potential transduction mechanisms involved. First, we observed that keloids are a relatively hypoxic tissue. Second, we found that hypoxia drives the transition of normal dermal fibroblasts to a myofibroblast-like phenotype [high expression of α-smooth muscle actin (α-SMA) and collagen I and III]. Finally, hypoxia effectively facilitated the nuclear import of the Smad2 and Smad3 complex, while blockade with the Smad3 inhibitor, SIS3, significantly impaired the expression of hypoxia-induced fibrosis-related molecules. Taken together, to the best of our knowledge, this study demonstrates for the first time that hypoxia facilitates the transition of dermal fibroblasts to myofibroblasts through the activation of the TGF-β1/Smad3 signaling pathway and our findings may provide a potential target for the treatment of keloids. PMID:27909731

  12. Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

    PubMed

    Kim, Ha Won; Chan, Qilin; Afton, Scott E; Caruso, Joseph A; Lai, Barry; Weintraub, Neal L; Qin, Zhenyu

    2012-02-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

  13. Human Macrophage ATP7A is Localized in the trans-Golgi Apparatus, Controls Intracellular Copper Levels, and Mediates Macrophage Responses to Dermal Wounds

    PubMed Central

    Kim, Ha Won; Chan, Qilin; Afton, Scott E.; Caruso, Joseph A.; Lai, Barry; Weintraub, Neal L.; Qin, Zhenyu

    2013-01-01

    The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound

  14. In vivo quantification of epidermis pigmentation and dermis papilla density with reflectance confocal microscopy: variations with age and skin phototype.

    PubMed

    Lagarrigue, Sophie Garrido; George, Jerome; Questel, Emmanuel; Lauze, Christophe; Meyer, Nicolas; Lagarde, Jean-Michel; Simon, Michel; Schmitt, Anne-Marie; Serre, Guy; Paul, Carle

    2012-04-01

    Reflectance confocal microscopy (RCM) may help to quantify variations of skin pigmentation induced by different stimuli such as UV radiation or therapeutic intervention. The objective of our work was to identify RCM parameters able to quantify in vivo dermis papilla density and epidermis pigmentation potentially applicable in clinical studies. The study included 111 healthy female volunteers with phototypes I-VI. Photo-exposed and photo-protected anatomical sites were imaged. The effect of age was also assessed. Four epidermis components were specifically investigated: stratum corneum, stratum spinosum, basal epidermal layer and dermo-epidermal junction. Laser power, diameter of corneocytes and upper spinous keratinocytes, brightness of upper spinous and interpapillary spinous keratinocytes, number of dermal papillae and papillary contrast were systematically assessed. Papillary contrast measured at the dermo-epidermal junction appeared to be a reliable marker of epidermis pigmentation and showed a strong correlation with skin pigmentation assessed clinically using the Fitzpatrick's classification. Brightness of upper spinous and interpapillary spinous keratinocytes was not influenced by the skin phototype. The number of dermal papillae was significantly lower in subjects with phototypes I-II as compared with darker skin subjects. A dramatic reduction in the number of dermal papillae was noticed with age, particularly in subjects with fair skin. The method presented here provides a new in vivo investigation tool for quantification of dermis papilla density and epidermal pigmentation. Papillary contrast measured at the dermo-epidermal junction may be selected as a marker of skin pigmentation for evaluation in clinical studies. © 2012 John Wiley & Sons A/S.

  15. Raman and infrared spectroscopy differentiate senescent from proliferating cells in a human dermal fibroblast 3D skin model.

    PubMed

    Eberhardt, Katharina; Matthäus, Christian; Winter, Doreen; Wiegand, Cornelia; Hipler, Uta-Christina; Diekmann, Stephan; Popp, Jürgen

    2017-08-15

    Senescent cells contribute to tissue aging and dysfunction. Therefore, detecting senescent cells in skin is of interest for skin tumor diagnostics and therapy. Here, we studied the transition into senescence of human dermal fibroblasts (HDFs) in a three-dimensional (3D) human fibroblast-derived matrix (FDM). Senescent and proliferating cells were imaged by Raman spectroscopy (RS) and Fourier transform infrared (FTIR) spectroscopy. The obtained averaged spectra were analyzed using PLS-LDA. For these 3D cultured cells, RS and FTIR could clearly distinguish senescent from proliferating cells. For both techniques, we detected senescence-associated alterations in almost all cellular macromolecules. Furthermore, we identified different biochemical properties of 3D compared to two-dimensional (2D) cultured cells, indicating that cells in their natural, skin-like 3D environment act differently than in (2D) cell cultivations in vitro. Compared to 2D cultured cells, cells grown in 3D models displayed a sharper contrast between the proliferating and senescent state, also affecting the abundance of biomolecules including nucleic acids. The training accuracies of both vibrational spectroscopic techniques were >96%, demonstrating the suitability of these label-free measurements for detecting these cellular states in 3D skin models.

  16. Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

    PubMed

    Song, Eunju; Chung, Haeyon; Shim, Eugene; Jeong, Jung-Ky; Han, Bok-Kyung; Choi, Hyuk-Joon; Hwang, Jinah

    2016-11-01

    Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

  17. Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques.

    PubMed

    Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Ghasemi-Dehkordi, Payam; Sadeghiani, Marzieh; Saffari-Chaleshtori, Javad; Hashemzadeh-Chaleshtori, Morteza; Khosravi-Farsani, Somayeh

    2014-10-01

    Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other.

  18. Protective effects of fermented Citrus unshiu peel extract against ultraviolet-A-induced photoageing in human dermal fibrobolasts.

    PubMed

    Bae, Jun Tae; Ko, Hyun Ju; Kim, Gyoung Bum; Pyo, Hyeong Bae; Lee, Geun Soo

    2012-12-01

    The aqueous extracts of Citrus unshiu peel containing flavonoid glycosides was used as co-substrate with Schizophyllum commune mycelia producing β-glucosidase and its biological activities were studied. β-glucosidase-produced S. commune mycelia converted the glycosides (narirutin and hesperidin) into aglycones (naringenin and hesperetin). The photoprotective potential of fermented C. unshiu peel extract with S. commune (S-CPE) was tested in human dermal fibroblasts (HDFs) exposed to UVA. It was revealed that S-CPE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with S-CPE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. The UVA irradiation raised the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells in comparison with the normal control group. The treatment of UVA-irradiated HDFs with S-CPE was shown to decrease the level of SA-β-gal (by approximately 45% at an S-CPE concentration 0.1%, w/v) compared with the UVA-irradiated HDFs. It was found that S-CPE containing hesperetin has notable collagen biosynthetic activity for fibroblasts, indicating that S-CPE can be promising cosmetic ingredients. Copyright © 2012 John Wiley & Sons, Ltd.

  19. In vitro dermal absorption of di(2-ethylhexyl) adipate (DEHA) in a roll-on deodorant using human skin.

    PubMed

    Zhou, Simon Ningsun; Moody, Richard P; Aikawa, Bio; Yip, Anna; Wang, Bing; Zhu, Jiping

    2013-01-01

    In vitro dermal absorption experiments were conducted using a roll-on deodorant that contains 1.56% di(2-ethylhexyl) adipate (DEHA), a plasticizer widely used in consumer products. Human skin specimens were fitted in Bronaugh flow-through Teflon diffusion cells. The diffusion cells were maintained at 32 °C to reflect the skin temperature. Two amounts (low dose: 5 mg of the product; high dose: 100 mg) were applied, in triplicate, each on four different human skins. DEHA was determined in the receiver solution at 6-h intervals, using headspace solid-phase microextraction gas chromatography-mass spectrometry (GC-MS). After 24 h, the experiment was terminated and masses of DEHA in the skin depot, skin wash, and upper and lower chambers of the diffusion cell were determined. A significant portion of applied DEHA, 28% in the low amount application and 34% in the high one, was found in the skin depot. In comparison, only 0.04% and 0.002% of applied DEHA were found in the receiver solutions for the low and high doses, respectively. Under our experimental conditions, an apparent steady-state flux of low DEHA mass penetrating from skin into the receiver solution was observed with a penetration rate of 2.2 ng/cm(2)/h for both the low and high doses. The average mass recovery was 81% for the low dose application and 56% for the high dose.

  20. In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

    PubMed

    Seker, Sükran; Elçin, A Eser; Yumak, Tuğrul; Sınağ, Ali; Elçin, Y Murat

    2014-12-01

    The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

  1. Evaluation of X-Inactivation Status and Cytogenetic Stability of Human Dermal Fibroblasts after Long-Term Culture

    PubMed Central

    Chen, Juan; Shi, Zhan-Ping; Dong, Juan; Liao, Ting-Ting; Wang, Yan-Peng; Sun, Xue-Ping; Yan, Zheng-Jie; Qian, Xiao-Qiao; Cui, Yu-Gui; Xue, Zhi-Gang; Fan, Guoping; Liu, Jia-Yin

    2010-01-01

    Human primary fibroblasts are a popular type of somatic cells for the production of induced pluripotent stem (iPS) cells. Here we characterized biological properties of primary fibroblasts in terms of cell-growth rate, cytogenetic stability, and the number of inactive X chromosomes during long-term passaging. We produced eight lines of female human dermal fibroblasts (HDFs) and found normal karyotype and expected pattern of X chromosome inactivation (XCI) at low passages (Passage P1-5). However, four out of the eight HDF lines at high passage numbers (≥ P10) exhibited duplicated hallmarks of inactive X chromosome including two punctuate signals of histone H3 lysine 27 trimethylation (H3K27me3) and X inactive-specific transcript (XIST) RNA signals in approximately 8.5–18.5% of the cells. Our data suggest that the copy number of inactive X chromosomes in a subset of female HDF is increased by a two-fold. Consistently, DNA fluorescent in situ hybridization (FISH) identified 3-4 copies of X chromosomes in one nucleus in this subset of cells with two inactive Xs. We conclude that female HDF cultures exhibit a higher risk of genetic anomalies such as carrying an increased number of X chromosomes including both active and inactive X chromosomes at a high passage (≥ P10). PMID:21234375

  2. Morphology of the lingual papillae in the tiger.

    PubMed

    Emura, Shoichi; Hayakawa, Daisuke; Chen, Huayue; Shoumura, Shizuko

    2004-08-01

    The dorsal lingual surfaces of an adult tiger (Panthera tigris altaica) was examined by macroscopical and scanning electron microscopical observations. Filiform, fungiform and vallate papillae were observed. The filiform papillae were distributed over the entire dosal surface of the tongue. The fungiform papillae were present rounded bodies, and more densely distributed on the lingual apex. There were 4 vallate papillae in total on borderline between the lingual body and lingual radix. Each papilla was surrounded by a groove. No foliate papillae were seen on the dorsal surface. Openings of the glandular ducts on the regions of the vallate papillae were found.

  3. Morphology of the lingual papillae in the eastern grey kangaroo.

    PubMed

    Emura, Shoichi

    2016-01-01

    We examined the dorsal lingual surface of an adult eastern grey kangaroo (Macropus gigantues) by scanning electron microscopy. The filiform papillae on the lingual apex and anterior body consisted of a main papilla and secondary papillae. The connective tissue core of the filiform papillae on the lingual apex had several processes. The filiform papillae on the lingual posterior body were thread-like in shape. The connective tissue core of the filiform papillae on the lingual posterior body consisted of many slender processes. The fungiform papillae were round in shape. Three vallate papillae with the apex of the triangle directed posteriorly consisted of a groove and pad. Several conical papillae derived from the posterolateral margin of the tongue where foliate papillae have been shown to be distributed in many other animal species. The surface structure of the tongue in the eastern grey kangaroo resembles that of the red kangaroo.

  4. In vitro biological responses to nanofibrillated cellulose by human dermal, lung and immune cells: surface chemistry aspect.

    PubMed

    Lopes, Viviana R; Sanchez-Martinez, Carla; Strømme, Maria; Ferraz, Natalia

    2017-01-10

    Nanocellulose, and particularly nanofibrillated cellulose (NFC), has been proposed for a diversity of applications in industry and in the biomedical field. Its unique physicochemical and structural features distinguish nanocellulose from traditional materials and enable its use as an advance nanomaterial. However, its nanoscale features may induce unknown biological responses. Limited studies with NFC are available and the biological impacts of its use have not been thoroughly explored. This study assesses the in vitro biological responses elicited by wood-derived NFC gels, when human dermal fibroblasts, lung MRC-5 cells and THP-1 macrophage cells are exposed to the nanomaterial. Furthermore, whether the presence of surface charged groups (i.e. carboxymethyl and hydroxypropyltrimethylammonium groups) on NFC can induce distinct biological responses is investigated. The introduction of surface charged groups resulted in individual nanofibrils, while fibril aggregates predominated in the unmodified NFC gel suspensions as observed by transmission electron microscopy. In the presence of proteins, the surface modified NFCs formed compact agglomerates while the agglomeration pattern of the unmodified NFC was similar in the presence of proteins and in physiological buffer. Unmodified and modified NFC gels did not induce cytotoxicity in human dermal fibroblasts, lung and macrophage cells. No significant ROS production by THP-1 macrophages was found and no cellular uptake was observed. However, an inflammatory response was detected when THP-1 macrophages were treated with unmodified NFC as assessed by an increase in TNF-α and IL1-β levels, an effect that was absent when surface charged groups were introduced into NFC. Taken together, the data presented here show the absence of cytotoxic effects associated with the exposure to unmodified, carboxymethylated and hydroxypropyltrimethylammonium-modified NFCs. Unmodified NFC presented a pro-inflammatory effect which can be

  5. Osmoprotective proteome adjustments in mouse kidney papilla.

    PubMed

    Gabert, B J; Kültz, D

    2011-03-01

    The papilla of the mammalian kidney must tolerate greatly varying degrees of hyperosmotic stress during urine concentration and depending on whole organism hydration state. To identify proteome adaptations supporting cell function and survival in such a harsh environment we compared the proteome of a) the hyperosmotic renal papilla with that of adjacent iso-osmotic cortex tissue and b) the renal papilla of diuretic versus that of anti-diuretic mice. Though functionally distinct the papilla is in close physical proximity to the renal cortex, an iso-osmotic region. Proteomic differences between the papilla and cortex of C57BL6 mice were identified using two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrometry. We found 37 different proteins characteristic of the cortex and 16 proteins over-represented in the papilla. Regional specificity was confirmed by Western blot and further substantiated by immunohistochemistry for selected proteins. Proteins that are characteristic of the renal papilla include αB crystallin, Hsp beta-1, Hsp90, 14-3-3 protein, glutathione S-transferase, aldose reductase, actin and tropomyosin. Gene ontology analysis confirmed a significant increase in molecular functions associated with protein chaperoning and cell stabilization. Proteins over-represented in the cortex were largely related to routine metabolism. During antidiuresis 15 different proteins changed significantly while 18 different proteins changed significantly during diuresis relative to normally hydrated controls. Changes were confirmed by Western blot for selected proteins. Proteins that are significantly altered by diuretic state are associated with cell structure (actin, tubulin), signaling (Rho GDP dissociation inhibitor, abhydrolase domain-containing protein 14B), chaperone functioning (Hsp beta-1, αB crystallin, T complex protein-1) and anti-oxidant functions (α-enolase, GAPDH and LDH). Taken together our study reveals that specific proteins involved in

  6. Probe depth matters in dermal microdialysis sampling of benzoic acid after topical application: an ex vivo study in human skin.

    PubMed

    Holmgaard, R; Benfeldt, E; Bangsgaard, N; Sorensen, J A; Brosen, K; Nielsen, F; Nielsen, J B

    2012-01-01

    Microdialysis (MD) in the skin - dermal microdialysis (DMD) - is a unique technique for sampling of topically as well as systemically administered drugs at the site of action, e.g. sampling of dermatological drug concentrations in the dermis. Debate has concerned the existence of a correlation between the depth of the sampling device - the probe - in the dermis and the amount of drug sampled following topical drug administration. This study evaluates the relation between probe depth and drug sampling using dermal DMD sampling ex vivo in human skin. We used superficial (<1 mm), intermediate (1-2 mm) and deep (>2 mm) positioning of the linear MD probe in the dermis of human abdominal skin, followed by topical application of 4 mg/ml of benzoic acid (BA) in skin chambers overlying the probes. Dialysate was sampled every hour for 12 h and analysed for BA content by high-performance liquid chromatography. Probe depth was measured by 20-MHz ultrasound scanning. The area under the time-versus-concentration curve (AUC) describes the drug exposure in the tissue during the experiment and is a relevant parameter to compare for the 3 dermal probe depths investigated. The AUC(0-12) were: superficial probes: 3,335 ± 1,094 μg·h/ml (mean ± SD); intermediate probes: 2,178 ± 1,068 μg·h/ml, and deep probes: 1,159 ± 306 μg·h/ml. AUC(0-12) sampled by the superficial probes was significantly higher than that of samples from the intermediate and deeply positioned probes (p value <0.05). There was a significant inverse correlation between probe depth and AUC(0-12) sampled by the same probe (p value <0.001, r(2) value = 0.5). The mean extrapolated lag-times (±SD) for the superficial probes were 0.8 ± 0.1 h, for the intermediate probes 1.7 ± 0.5 h, and for the deep probes 2.7 ± 0.5 h, which were all significantly different from each other (p value <0.05). In conclusion, this paper demonstrates that there is an inverse relationship between the depth of the probe in the dermis

  7. Development of a Human Physiologically Based Pharmacokinetics (PBPK) Model For Dermal Permeability for Lindane

    EPA Science Inventory

    Lindane is a neurotoxicant used for the treatment of lice and scabies present on human skin. Due to its pharmaceutical application, an extensive pharmacokinetic database exists in humans. Mathematical diffusion models allow for calculation of lindane skin permeability coefficient...

  8. Development of a Human Physiologically Based Pharmacokinetics (PBPK) Model For Dermal Permeability for Lindane

    EPA Science Inventory

    Lindane is a neurotoxicant used for the treatment of lice and scabies present on human skin. Due to its pharmaceutical application, an extensive pharmacokinetic database exists in humans. Mathematical diffusion models allow for calculation of lindane skin permeability coefficient...

  9. In vitro characterization of human hair follicle dermal sheath mesenchymal stromal cells and their potential in enhancing diabetic wound healing.

    PubMed

    Ma, Dongrui; Kua, Jonah Ee Hsiang; Lim, Wee Keng; Lee, Seng Teik; Chua, Alvin Wen Choong

    2015-08-01

    Little is published on the characterization and therapeutic potential of human mesenchymal cells derived from hair follicle (HF) dermal sheath (DS). In this study, we isolated and characterized HF DS-mesenchymal stromal cells (DS-MSCs) with respect to the bone marrow mesenchymal stromal cells (BM-MSCs). We further tested if DS-MSC-conditioned medium (CM), like what was previously reported for BM-MSC CM, has superior wound-healing properties, in both in vitro and in vivo wound models compared with skin fibroblast CM. DS-MSCs were isolated from HF and cultured in vitro to assess long-term growth potential, colony-forming efficiency (CFE), expression of CD surface markers and differentiation potential. The cytokine expression of DS-MSC CM was determined through an antibody-based protein array analysis. The wound-healing effects of the CM were tested in vitro with the use of human cell cultures and in vivo with the use of a diabetic mouse wound model. In vitro results revealed that DS-MSCs have high growth capacity and CFE while displaying some phenotypes similar to BM-MSCs. DS-MSCs strongly expressed many surface markers expressed in BM-MSCs and could also differentiate into osteoblasts, chondrocytes and adipocytes. DS-MSCs secreted significantly higher proportions of paracrine factors such as interleukin-6 (IL-6), IL-8 and growth-related oncogene. DS-MSC-CM demonstrated enhanced wound-healing effects on human skin keratinocytes, fibroblasts and endothelial cells in vitro, and the wound-healing time in diabetic mice was found to be shorter, compared with vehicle controls. Human HF DS stromal cells demonstrated MSC-like properties and might be an alternative source for therapeutic use in wound healing. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  10. Monophasic Pulsed 200-μA Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin α2β1 in Human Dermal Fibroblasts

    PubMed Central

    Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto

    2016-01-01

    Objective: The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting galvanotaxis and the effects of electrical stimulation on integrin α2β1 and actin filaments in human dermal fibroblasts. Methods: Human dermal fibroblasts were treated with the monophasic pulsed microcurrent of 0, 100, 200, or 300 μA for 8 hours, and cell migration and cell viability were measured 24 hours after starting monophasic pulsed microcurrent stimulation. Polarization of integrin α2β1 and lamellipodia formation were detected by immunofluorescent staining 10 minutes after starting monophasic pulsed microcurrent stimulation. Results: The migration toward the cathode was significantly higher in the cells treated with the 200-μA monophasic pulsed microcurrent than in the controls (P < .01) without any change in cell viability; treatment with 300-μA monophasic pulsed microcurrent did not alter the migration ratio. The electrostimulus of 200 μA also promoted integrin α2β1 polarization and lamellipodia formation at the cathode edge (P < .05). Conclusion: The results show that 200 μA is an effective monophasic pulsed microcurrent intensity to promote migration toward the cathode, and this intensity could regulate polarization of migration-related intracellular factors in human dermal fibroblasts. PMID:26819649

  11. Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro

    PubMed Central

    Wermuth, Peter J.; Addya, Sankar; Jimenez, Sergio A.

    2011-01-01

    Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel

  12. Human volunteer study on the inhalational and dermal absorption of N-methyl-2-pyrrolidone (NMP) from the vapour phase.

    PubMed

    Bader, Michael; Wrbitzky, Renate; Blaszkewicz, Meinolf; Schäper, Michael; van Thriel, Christoph

    2008-01-01

    N-Methyl-2-pyrrolidone (NMP) is a versatile organic solvent frequently used for surface cleaning such as paint stripping or graffiti removal. Liquid NMP is rapidly absorbed through the skin but dermal vapour phase absorption might also play an important role for the uptake of the solvent. This particular aspect was investigated in an experimental study with 16 volunteers exposed to 80 mg/m(3) NMP for 8 h under either whole-body, i.e. inhalational plus dermal, or dermal-only conditions. Additionally, the influence of moderate physical workload on the uptake of NMP was studied. The urinary concentrations of NMP and its metabolites 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and 2-hydroxy-N-methylsuccinimide (2-HMSI) were followed for 48 h and analysed by gas chromatography-mass spectrometry (GC-MS). Percutaneous uptake delayed the elimination peak times and the apparent biological half-lives of NMP and 5-HNMP. Under resting conditions, dermal-only exposure resulted in the elimination of 71 +/- 8 mg NMP equivalents as compared to 169 +/- 15 mg for whole-body exposure. Moderate workload yielded 79 +/- 8 mg NMP (dermal-only) and 238 +/- 18 mg (whole-body). Thus, dermal absorption from the vapour phase may contribute significantly to the total uptake of NMP, e.g. from workplace atmospheres. As the concentration of airborne NMP does not reflect the body dose, biomonitoring should be carried out for surveillance purposes.

  13. Comparison of histological structure and biocompatibility between human acellular dermal matrix (ADM) and porcine ADM.

    PubMed

    Ge, Liangpeng; Zheng, Shuquan; Wei, Hong

    2009-02-01

    The present study was to compare the difference of histological structure and biocompatibility between human ADM and porcine ADM. The scaffold structure, collagen arrangement and collagen structure of human ADM and those of porcine ADM were very similar except for a slight difference in their black and white bands assessed by both light microscopy and electron microscopy. The positive immunohistochemical staining results of porcine ADM using human antibodies of collagen I, collagen III, collagen IV, fibronectin, laminin and vimentin and the result of SDS-PAGE implied a strong homology between the main proteins of human ADM and porcine ADM. In addition, statistical analysis indicated that there was no significant difference (P<0.05) between the biocompatibility of the two ADMs. Based on these results, we conclude that porcine ADM bears a strong similarity to human ADM, and might be a substitute for human ADM in the future.

  14. Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in-house and in vitro cultured human fibroblasts.

    PubMed

    Łabuś, Wojciech; Glik, Justyna; Klama-Baryła, Agnieszka; Kitala, Diana; Kraut, Małgorzata; Maj, Mariusz; Nowak, Mariusz; Misiuga, Marcelina; Marcinkowski, Andrzej; Trzebicka, Barbara; Poloczek, Ryszard; Kawecki, Marek

    2017-03-21

    The most efficient method in III° burn treatment is the use of the autologous split thickness skin grafts that were donated from undamaged body area. The main limitation of this method is lack of suitable donor sites. Tissue engineering is a useful tool to solve this problem. The goal of this study was to find the most efficient way of producing biovital skin substitute based on in house produced acellular dermal matrix ADM and in vitro cultured fibroblasts. Sixty samples of sterilized human allogeneic skin (that came from 10 different donors) were used to examine the influence of decellularizing substances on extracellular matrix and clinical usefulness of the test samples of allogeneic human dermis. Six groups of acellular dermal matrix were studied: ADM-1 control group, ADM-2 research group (24 h incubation in 0.05% trypsin/EDTA solution), ADM-3 research group (24 h incubation in 0.025% trypsin/EDTA solution), ADM-4 research group (24 h incubation in 0.05% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), ADM-5 research group (24 h incubation in 0.025% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), and ADM-6 research group (24 h incubation in 0,1% SDS). Obtained ADMs were examined histochemically and by atomic force microscopy (AFM). ADMs were settled by human fibroblasts. The number of cultured cells and their vitality were measured. The obtained results indicated that the optimal method for production of living skin substitutes is colonization of autologous fibroblasts on the scaffold prepared by the incubation of human allogeneic dermis in 0.05% trypsin/EDTA. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  15. A comparative study on the possible cytotoxic effects of different nanostructured lipid carrier (NLC) compositions in human dermal fibroblasts.

    PubMed

    Brugè, Francesca; Damiani, Elisabetta; Marcheggiani, Fabio; Offerta, Alessia; Puglia, Carmelo; Tiano, Luca

    2015-11-30

    Nanostructured lipid carriers (NLC) are widely used for topical delivery of active ingredients into the skin for both local and systemic treatment. But concerns have been raised regarding their potential nanotoxicity. To understand the role of NLC composition in terms of cytotoxicity and pro-oxidant effects, we investigated cell viability and intracellular levels of ROS (reactive oxygen species) production in human dermal fibroblasts (HDF) incubated with five NLC formulations differing in their solid lipid composition. HDF and NLC were also exposed to UVA irradiation in order to evaluate the behavior of NLC under realistic environmental conditions which might promote their instability. Using the Guava via-count assay, all nanoparticles, except for those formulated with Compritol 888 ATO, showed a significant decrease in live cells and a parallel increase in apoptotic or dead cells compared to the control, either before and/or after UVA irradiation (18 J/cm(2)). NLC formulated with Geleol™ Mono Diglycerides resulted the most cytotoxic. A similar trend was also observed when intracellular ROS levels were measured in HDF incubated with NLC: there was increased ROS content compared to the control, further exacerbated following UVA. NLC formulated with Dynasan 118 were particularly susceptible to UVA exposure. The results indicate which could be the most suitable candidates for formulating NLC that are biocompatible and non-cytotoxic even when exposed to UVA and hence help direct future choices during the formulation strategies of these delivery systems. Of those tested, Compritol 888 ATO appears to be the best choice.

  16. A Pilot Study of the Photoprotective Effects of Strawberry-Based Cosmetic Formulations on Human Dermal Fibroblasts

    PubMed Central

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Yuliett; Afrin, Sadia; Alvarez-Suarez, José Miguel; Gonzàlez-Paramàs, Ana Maria; Santos-Buelga, Celestino; Bompadre, Stefano; Quiles, José Luis; Mezzetti, Bruno; Giampieri, Francesca

    2015-01-01

    Strawberry polyphenols have been extensively studied over the last two decades for their beneficial properties. Recently, their possible use in ameliorating skin conditions has also been proposed; however, their role in preventing UVA-induced damage in cosmetic formulation has not yet been investigated. Skin is constantly exposed to several environmental stressors, such as UVA radiation, that induce oxidative stress, inflammation and cell death via the production of reactive oxygen species (ROS). In the present study, we assessed the potential photoprotective capacity of different strawberry-based formulations, enriched with nanoparticles of Coenzyme Q10 and with sun protection factor 10 (SPF10), in human dermal fibroblasts (HuDe) exposed to UVA radiation. We confirmed that strawberries are a very rich source of polyphenols, anthocyanins and vitamins, and possess high total antioxidant capacity. We also showed that strawberry extracts (25 μg/mL–1 mg/mL) exert a noticeable photoprotection in HuDe, increasing cell viability in a dose-dependent way, and that these effects are potentiated by the presence of CoQ10red (100 μg/mL). We have demonstrated for the first time that the topical use of strawberry extract may provide good photoprotection, even if more in-depth studies are strongly encouraged in order to evaluate the cellular and molecular effects of strawberry protection. PMID:26247940

  17. A prospective, multicentre, randomised controlled study of human fibroblast-derived dermal substitute (Dermagraft) in patients with venous leg ulcers.

    PubMed

    Harding, Keith; Sumner, Michael; Cardinal, Matthew

    2013-04-01

    This was an open-label, prospective, multicentre, randomised controlled study to evaluate the efficacy and safety of human fibroblast-derived dermal substitute (HFDS) plus four-layer compression therapy compared with compression therapy alone in the treatment of venous leg ulcers. The primary outcome variable was the proportion of patients with completely healed study ulcers by 12 weeks. The number healed was further summarised by ulcer duration and baseline ulcer size. Sixty-four (34%) of 186 patients in the HFDS group experienced healing by week 12 compared with 56 (31%) of 180 patients in the control group (P = 0·235). For ulcers ≤ 12 months duration, 49 (52%) of 94 patients in the HFDS group versus 36 (37%) of 97 patients in the control group healed at 12 weeks (P = 0·029). For ulcers ≤ 10 cm(2), complete healing at week 12 was observed in 55 (47%) of 117 patients in the HFDS group compared with 47 (39%) of 120 patients in the control group (P = 0·223). The most common adverse events (AEs) were wound infection, cellulitis and skin ulcer. The frequency of AEs did not markedly differ between the treatment and control groups.

  18. Matrix metalloproteinase-1 inhibitory activities of Morinda citrifolia seed extract and its constituents in UVA-irradiated human dermal fibroblasts.

    PubMed

    Masuda, Megumi; Murata, Kazuya; Naruto, Shunsuke; Uwaya, Akemi; Isami, Fumiyuki; Matsuda, Hideaki

    2012-01-01

    The objective of this study was to examine whether a 50% ethanolic extract (MCS-ext) of the seeds of Morinda citrifolia (noni) and its constituents have matrix metalloproteinase-1 (MMP-1) inhibitory activity in UVA-irradiated normal human dermal fibroblasts (NHDFs). The MCS-ext (10 μg/mL) inhibited MMP-1 secretion from UVA-irradiated NHDFs, without cytotoxic effects, at 48 h after UV exposure. The ethyl acetate-soluble fraction of MCS-ext was the most potent inhibitor of MMP-1 secretion. Among the constituents of the fraction, a lignan, 3,3'-bisdemethylpinoresinol (1), inhibited the MMP-1 secretion at a concentration of 0.3 μM without cytotoxic effects. Furthermore, 1 (0.3 μM) reduced the level of intracellular MMP-1 expression. Other constituents, namely americanin A (2), quercetin (3) and ursolic acid (4), were inactive. To elucidate inhibition mechanisms of MMP-1 expression and secretion, the effect of 1 on mitogen-activated protein kinases (MAPKs) phosphorylation was examined. Western blot analysis revealed that 1 (0.3 μM) reduced the phosphorylations of p38 and c-Jun-N-terminal kinase (JNK). These results suggested that 1 suppresses intracellular MMP-1 expression, and consequent secretion from UVA-irradiated NHDFs, by down-regulation of MAPKs phosphorylation.

  19. Boron nitride nanotube-mediated stimulation modulates F/G-actin ratio and mechanical properties of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Ricotti, Leonardo; das Neves, Ricardo Pires; Ciofani, Gianni; Canale, Claudio; Nitti, Simone; Mattoli, Virgilio; Mazzolai, Barbara; Ferreira, Lino; Menciassi, Arianna

    2014-02-01

    F/G-actin ratio modulation is known to have an important role in many cell functions and in the regulation of specific cell behaviors. Several attempts have been made in the latest decades to finely control actin production and polymerization, in order to promote certain cell responses. In this paper we demonstrate the possibility of modulating F/G-actin ratio and mechanical properties of normal human dermal fibroblasts by using boron nitride nanotubes dispersed in the culture medium and by stimulating them with ultrasound transducers. Increasing concentrations of nanotubes were tested with the cells, without any evidence of cytotoxicity up to 10 μg/ml concentration of nanoparticles. Cells treated with nanoparticles and ultrasound stimulation showed a significantly higher F/G-actin ratio in comparison with the controls, as well as a higher Young's modulus. Assessment of Cdc42 activity revealed that actin nucleation/polymerization pathways, involving Rho GTPases, are probably influenced by nanotube-mediated stimulation, but they do not play a primary role in the significant increase of F/G-actin ratio of treated cells, such effect being mainly due to actin overexpression.

  20. Glycyrrhizic acid (GA), a triterpenoid saponin glycoside alleviates ultraviolet-B irradiation-induced photoaging in human dermal fibroblasts.

    PubMed

    Afnan, Quadri; Adil, Mushtaq Dar; Nissar-Ul, Ashraf; Rafiq, Ahmad Rather; Amir, Hussian Faridi; Kaiser, Peerzada; Gupta, Vijay Kumar; Vishwakarma, Ram; Tasduq, Sheikh Abdullah

    2012-05-15

    Glycyrrhizic acid (GA), a triterpenoid saponin glycoside from the roots and rhizomes of licorice is used in traditional and modern medicine for the treatment of numerous medical conditions including skin diseases and beauty care product. In the present study, we investigated the effect of GA against ultraviolet B (UVB) irradiation-induced photoaging in human dermal fibroblasts (HDFs) and its possible mechanism of action. HDFs were subjected to photoaging by sub-toxic dose of UVB (10 mj/cm(2)) irradiation. Cell viability, matrix metalloproteinase 1 (MMP1), pro-collagen 1, cellular and nuclear morphology, cell cycle, intracellular reactive oxygen species (ROS), caspase 3 and hyaluronidase inhibition assays were performed. Western blotting was used to evaluate the expression of NF-kappa B (NF-κB) and cytochrome-C proteins. GA treatment significantly inhibited photoaging. It achieved this by reducing ROS, NF-κB, cytochrome c, caspase 3 levels and inhibiting hyaluronidase enzyme. The main mechanism seems to be, most likely by blocking MMP1 activation by modulating NF-κB signaling. These findings may be useful for development of natural and safe photoprotective agents against UVB irradiation. Copyright © 2012 Elsevier GmbH. All rights reserved.

  1. Effects of fiber orientation and diameter on the behavior of human dermal fibroblasts on electrospun PMMA scaffolds.

    PubMed

    Liu, Ying; Ji, Yuan; Ghosh, Kaustabh; Clark, Richard A F; Huang, Lei; Rafailovich, Miriam H

    2009-09-15

    We used the electrospinning technique to produce fibrous scaffolds of poly(methyl methacrylate) (PMMA). Using a rotating drum, we aligned the fibers and formed multilayered structures where both the fiber spacing and pore size could be varied. We then plated adult human dermal fibroblasts and studied the effect of fiber diameter and orientation on the cell conformation, integrin receptor expression, proliferation, and migration. We found that a critical diameter minimum diameter existed, D0 = 0.97 microm for cell orientation to occur. For D < D0, no big difference in aspect ratio was observed relative to the control samples on PMMA thin film. Hence, we could fabricate substrate patterned with fibers of different diameters where different cell conformations coexisted on the same scaffold. On the other hand, staining for vinculin proteins in the cells indicated that on large diameter fibers and on flat surfaces, the integrin receptors followed the cell perimeter. On the very small diameter surfaces, the receptors were distributed uniformly along the cell. Cell dynamics studies indicated that the proliferation and migration were also affected by the fiber orientation.

  2. A Pilot Study of the Photoprotective Effects of Strawberry-Based Cosmetic Formulations on Human Dermal Fibroblasts.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Yuliett; Afrin, Sadia; Alvarez-Suarez, José Miguel; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Bompadre, Stefano; Quiles, José Luis; Mezzetti, Bruno; Giampieri, Francesca

    2015-08-04

    Strawberry polyphenols have been extensively studied over the last two decades for their beneficial properties. Recently, their possible use in ameliorating skin conditions has also been proposed; however, their role in preventing UVA-induced damage in cosmetic formulation has not yet been investigated. Skin is constantly exposed to several environmental stressors, such as UVA radiation, that induce oxidative stress, inflammation and cell death via the production of reactive oxygen species (ROS). In the present study, we assessed the potential photoprotective capacity of different strawberry-based formulations, enriched with nanoparticles of Coenzyme Q10 and with sun protection factor 10 (SPF10), in human dermal fibroblasts (HuDe) exposed to UVA radiation. We confirmed that strawberries are a very rich source of polyphenols, anthocyanins and vitamins, and possess high total antioxidant capacity. We also showed that strawberry extracts (25 μg/mL-1 mg/mL) exert a noticeable photoprotection in HuDe, increasing cell viability in a dose-dependent way, and that these effects are potentiated by the presence of CoQ10red (100 μg/mL). We have demonstrated for the first time that the topical use of strawberry extract may provide good photoprotection, even if more in-depth studies are strongly encouraged in order to evaluate the cellular and molecular effects of strawberry protection.

  3. UVA-induced ROS generation inhibition by Oenothera paradoxa defatted seeds extract and subsequent cell death in human dermal fibroblasts.

    PubMed

    Jaszewska, Edyta; Soin, Magdalena; Filipek, Agnieszka; Naruszewicz, Marek

    2013-09-05

    UVA radiation stimulates the production of reactive oxygen species (ROS), which react with lipids, proteins and other intracellular molecules leading to oxidative stress, cellular damage and ultimately cell death. There is, therefore, a growing need for substances exhibiting antioxidant activity, which may support repair mechanisms of the skin. This study evaluates the protective effect of the aqueous Oenothera paradoxa Hudziok defatted seeds extract, rich in polyphenolic compounds, against UVA (25 and 50J/cm(2))-induced changes in normal human dermal fibroblasts (NHDFs). The tested extract (0.1-10μg/ml) has decreased, in a concentration-dependent fashion, the UVA-induced release of lactate dehydrogenase (LDH) into the culture medium, the ROS production (with the use of 2',7'-dichlorodihydrofluorescein diacetate) and lipid peroxidation (utilizing redox reactions with ferrous ions) as compared to the control cells (incubated without the extract). Moreover, the extract increased the number of viable (calcein positive) cells decreasing the number of cells in late apoptosis (annexin V-FITC and propidium iodide positive). Thus our results show that O. paradoxa defatted seeds extract may be beneficial for the prevention of UVA skin damage.

  4. Photoprotective Potential of Anthocyanins Isolated from Acanthopanax divaricatus Var. albeofructus Fruits against UV Irradiation in Human Dermal Fibroblast Cells.

    PubMed

    Lyu, Su-Yun; Park, Won-Bong

    2012-03-01

    Ultraviolet (UV) A penetrates deeply into the skin and induces the generation of reactive oxygen species (ROS) causing damage to fibroblasts, which leads to aging of the skin. However, the body has developed an antioxidant defence system against the harmful effects of ROS. Enzymes such as superoxide dismutase (SOD) and catalase (CAT) play critical roles on the removal of excess ROS in living organisms. In this study, the antioxidant activities of anthocyanins (cyanidin 3-galactoside and cyanidin 3-lathyroside) from Acanthopanax divaricatus var. albeofructus (ADA) fruits were investigated by xylenol orange, thiobarbituric acid reactive substances (TBARS), and antioxidant enzyme assay. As a result, generation of H2O2 and lipid peroxide induced by UVA-irradiation in human dermal fibroblast (HDF-N) cells was reduced by treatment of anthocyanins. Also, augmented enzyme (SOD and CAT) activities were observed in UVA-irradiated cells when treated with anthocyanin. In conclusion, the results obtained show that anthocyanins from ADA fruits are potential candidates for the protection of fibroblast against the damaging effects of UVA irradiation. Furthermore, anthocyanin may be a good candidate for antioxidant agent development.

  5. Influence of flavonoids and vitamins on the MMP- and TIMP-expression of human dermal fibroblasts after UVA irradiation.

    PubMed

    Hantke, Bernd; Lahmann, Christine; Venzke, Kirsten; Fischer, Tim; Kocourek, Andreas; Windsor, L Jack; Bergemann, Jörg; Stäb, Franz; Tschesche, Harald

    2002-10-01

    UV irradiation leads to distinct changes in skin connective tissue by degradation of collagen, for example. Many of these alterations in the extracellular matrix are mediated by MMPs (matrix metalloproteinases) with reduced content of their antagonist TIMPs (tissue inhibitors of metalloproteinases). Potential candidates to reduce MMP activity in the skin after solar stimulation were examined. The influence of vitamin C, vitamin E and the flavonoids AGR (alpha-glucosylrutin) and 8-prenylnaringenine on the MMP and TIMP expression was investigated. Human dermal fibroblasts were incubated with these additives and irradiated with UVA [10 J cm(-2)]. The gene expression of MMP-1 (collagenase-1) and TIMP-1, the protein expression of MMP-1, MMP-2 (gelatinase-A), TIMP-1 and TIMP-2 as well as the enzyme activity of MMP-1 and MMP-2 were examined. AGR and vitamins C and E were shown to reduce MMP expression and activity, whereas 8-prenylnaringenine appeared to be responsible for the opposite effect. None of the substances considerably influenced the TIMP levels. AGR represented the most effective additive in reducing the collagenase protein expression to 60% and may be useful to level out the MMP activity in the skin after sun exposure. Furthermore, no protein expression of MMP-8, MMP-9, MMP-12 and MMP-13 could be detected.

  6. Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide.

    PubMed Central

    van der Vlies, Dennis; Pap, Eward H W; Post, Jan Andries; Celis, Julio E; Wirtz, Karel W A

    2002-01-01

    The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H(2)O(2) (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [(35)S]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the (35)S-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the (35)S-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed co-localization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum. PMID:12071860

  7. Polyphenol-rich strawberry extract protects human dermal fibroblasts against hydrogen peroxide oxidative damage and improves mitochondrial functionality.

    PubMed

    Giampieri, Francesca; Alvarez-Suarez, José M; Mazzoni, Luca; Forbes-Hernandez, Tamara Y; Gasparrini, Massimiliano; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Quiles, José L; Bompadre, Stefano; Mezzetti, Bruno; Battino, Maurizio

    2014-06-11

    Strawberry bioactive compounds are widely known to be powerful antioxidants. In this study, the antioxidant and anti-aging activities of a polyphenol-rich strawberry extract were evaluated using human dermal fibroblasts exposed to H2O2. Firstly, the phenol and flavonoid contents of strawberry extract were studied, as well as the antioxidant capacity. HPLC-DAD analysis was performed to determine the vitamin C and β-carotene concentration, while HPLC-DAD/ESI-MS analysis was used for anthocyanin identification. Strawberry extract presented a high antioxidant capacity, and a relevant concentration of vitamins and phenolics. Pelargonidin- and cyanidin-glycosides were the most representative anthocyanin components of the fruits. Fibroblasts incubated with strawberry extract and stressed with H2O2 showed an increase in cell viability, a smaller intracellular amount of ROS, and a reduction of membrane lipid peroxidation and DNA damage. Strawberry extract was also able to improve mitochondrial functionality, increasing the basal respiration of mitochondria and to promote a regenerative capacity of cells after exposure to pro-oxidant stimuli. These findings confirm that strawberries possess antioxidant properties and provide new insights into the beneficial role of strawberry bioactive compounds on protecting skin from oxidative stress and aging.

  8. Modulation of heat shock protein 90 affects TGF-β-induced collagen synthesis in human dermal fibroblast cells.

    PubMed

    Lee, Sae Bin; Lim, A-Ram; Rah, Dong Kyun; Kim, Kyung Soo; Min, Hyun Jin

    2016-12-01

    Heat shock protein 90 is a chaperone molecule that aids in proper folding of target proteins. Recently, heat shock protein 90 was found to play a role in would healing through regulation of fibroblast functions. The aim of the present study was to investigate the role of heat shock protein 90 in collagen synthesis in human dermal fibroblasts. The effects of transforming growth factor-β, 17-N-allylamino-17-demethoxygeldanamycin, and transfection of heat shock protein 90 were evaluated by real-time PCR, western blot, and immunofluorescence assays. The Smad 2/3 and Akt pathways were evaluated to identify the signaling pathways involved in collagen synthesis. Heat shock protein 90 and collagen levels were compared in keloid and control tissues by immunohistochemical analysis. The expression of collagen was significantly increased after treatment with transforming growth factor-β, while 17-N-allylamino-17-demethoxygeldanamycin inhibited transforming growth factor-β-induced collagen synthesis. Overexpression of heat shock protein 90 itself with or without transforming growth factor-β increased collagen synthesis. These effects were dependent on Smad 2/3 pathway signaling. Finally, expression of heat shock protein 90 was increased in keloid tissue compared with control tissues. Taken together, these results demonstrate that modulation of heat shock protein 90 influences transforming growth factor-β-induced collagen synthesis via regulation of Smad 2/3 phosphorylation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Inhibition of UV-induced ROS and collagen damage by Phyllanthus emblica extract in normal human dermal fibroblasts.

    PubMed

    Majeed, Muhammed; Bhat, Beena; Anand, Susmitha; Sivakumar, A; Paliwal, Pritee; Geetha, K G

    2011-01-01

    As a part of ongoing research for novel natural cosmeceutical actives from plant extracts, this study demonstrates that Phyllanthus emblica fruit extract has shown its efficacy in protection against ultraviolet-B (UVB) irradiation-induced reactive oxygen species (ROS) and collagen damage in normal human dermal fibroblasts. At a concentration of 0.5 mg/ml, emblica extract showed a significant response of 9.5 ± 0.28-fold protection from UVB induced-collagen damage as compared to untreated cells. A known active, ascorbic acid, at a concentration of 0.5 mg/ml, showed 3.7 ± 0.07-fold protection from UVB-induced collagen damage. While the untreated cells showed 84 ± 1.4% induction in ROS on UVB irradiation as compared to the non-irradiated cells, emblica extract treatment inhibited the induction of ROS to 15 ± 4% at a concentration of 0.5 mg/ml. Ascorbic acid inhibited the induction in ROS to 64 ± 2% at a concentration of 0.5 mg/ml. Emblica extract is a significantly better natural active, with promising cosmeceutical benefits against photoaging.

  10. Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration.

    PubMed

    Cox, Steve; Cole, Marietta; Tawil, Bill

    2004-01-01

    Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal fibroblasts. Fibroblast proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. Fibroblasts proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-17 mg/mL) and thrombin (1-167 U/mL). These fibroblasts also retained good morphology and growth characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of growth factors and interleukins in the entire fibroblast-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which fibroblasts showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects fibroblast behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for fibroblasts that could be used for the treatment of chronic wounds.

  11. Camphor Induces Proliferative and Anti-senescence Activities in Human Primary Dermal Fibroblasts and Inhibits UV-Induced Wrinkle Formation in Mouse Skin.

    PubMed

    Tran, Thao Anh; Ho, Manh Tin; Song, Yeon Woo; Cho, Moonjae; Cho, Somi Kim

    2015-12-01

    Camphor ((1R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-one), a bicyclic monoterpene, is one of the major constituents of essential oils from various herbs such as rosemary, lavender, and sage. In this study, we investigated the beneficial effects of camphor as a botanical ingredient in cosmetics. Camphor induced the proliferation of human primary dermal fibroblasts in a dose-dependent manner via the PI3K/AKT and ERK signaling pathways. Camphor attenuated the elevation of senescence associated with β-galactosidase (SA-β-gal) activity. Elastase activity decreased, while the total amount of collagen increased, in a dose- and time-dependent manner in human primary dermal fibroblasts treated with camphor. Camphor induced the expression of collagen IA, collagen IIIA, collagen IVA, and elastin in human primary dermal fibroblasts. In addition, posttreatment with 26 and 52 mM camphor for 2 weeks led to a significant reduction in the expression of MMP1 but increases in the expression of collagen IA, IIIA, and elastin in mouse skin exposed to UV for 4 weeks. These posttreatments also reduced the depths of the epidermis and subcutaneous fat layer in UV-exposed mouse skin. Taken together, these findings suggest camphor to be a potent wound healing and antiwrinkle agent with considerable potential for use in cosmeceuticals. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

    PubMed Central

    Tanaka, Miyuki; Misawa, Eriko; Yamauchi, Koji; Abe, Fumiaki; Ishizaki, Chiaki

    2015-01-01

    Background Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods First, we investigated the capability of Aloe sterols (cycloartenol and lophenol) to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP) containing 40 μg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake period. Conclusion The present study confirms that daily oral Aloe sterol-containing AVGP significantly reduced facial wrinkles in women aged ≥40 years, and Aloe sterols stimulate collagen and hyaluronic acid production by human dermal fibroblasts. PMID:25759593

  13. Relevant In Vitro Predictors of Human Acellular Dermal Matrix-Associated Inflammation and Capsule Formation in a Nonhuman Primate Subcutaneous Tissue Expander Model.

    PubMed

    Sandor, Maryellen; Leamy, Patrick; Assan, Pearl; Hoonjan, Amardeep; Huang, Li-Ting; Edwards, Marianne; Zuo, Wenqi; Li, Hui; Xu, Hui

    2017-01-01

    Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam-sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam-sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix

  14. Relevant In Vitro Predictors of Human Acellular Dermal Matrix-Associated Inflammation and Capsule Formation in a Nonhuman Primate Subcutaneous Tissue Expander Model

    PubMed Central

    Leamy, Patrick; Assan, Pearl; Hoonjan, Amardeep; Huang, Li-Ting; Edwards, Marianne; Zuo, Wenqi; Li, Hui; Xu, Hui

    2017-01-01

    Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam–sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam–sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix

  15. bFGF Promotes the Migration of Human Dermal Fibroblasts under Diabetic Conditions through Reactive Oxygen Species Production via the PI3K/Akt-Rac1- JNK Pathways

    PubMed Central

    Shi, Hongxue; Cheng, Yi; Ye, Jingjing; Cai, Pingtao; Zhang, Jinjing; Li, Rui; Yang, Ying; Wang, Zhouguang; Zhang, Hongyu; Lin, Cai; Lu, Xianghong; Jiang, Liping; Hu, Aiping; Zhu, Xinbo; Zeng, Qiqiang; Fu, Xiaobing; Li, Xiaokun; Xiao, Jian

    2015-01-01

    Fibroblasts play a pivotal role in the process of cutaneous wound repair, whereas their migratory ability under diabetic conditions is markedly reduced. In this study, we investigated the effect of basic fibroblast growth factor (bFGF) on human dermal fibroblast migration in a high-glucose environment. bFGF significantly increased dermal fibroblast migration by increasing the percentage of fibroblasts with a high polarity index and reorganizing F-actin. A significant increase in intracellular reactive oxygen species (ROS) was observed in dermal fibroblasts under diabetic conditions following bFGF treatment. The blockage of bFGF-induced ROS production by either the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) almost completely neutralized the increased migration rate of dermal fibroblasts promoted by bFGF. Akt, Rac1 and JNK were rapidly activated by bFGF in dermal fibroblasts, and bFGF-induced ROS production and promoted dermal fibroblast migration were significantly attenuated when suppressed respectively. In addition, bFGF-induced increase in ROS production was indispensable for the activation of focal adhesion kinase (FAK) and paxillin. Therefore, our data suggested that bFGF promotes the migration of human dermal fibroblasts under diabetic conditions through increased ROS production via the PI3K/Akt-Rac1-JNK pathways. PMID:26078726

  16. Morphology of the lingual papillae in the Japanese marten.

    PubMed

    Emura, Shoichi; Okumura, Toshihiko; Chen, Huayue

    2007-08-01

    The dorsal lingual surfaces of two adult Japanese marten (Martes melampus) were examined by scanning electron microscopy (SEM). Filiform, fungiform, vallate and foliate papillae were observed. A small filiform papilla on the apical surface of the tongue had several pointed processes. A small filiform papilla contained the connective tissue core consisting of several small processes. A large filiform papilla of the lingual body consisted of a main papilla and some secondary papillae. A large filiform papilla contained the connective tissue core consisting of processes of various size. The fungiform papillae are round in shape. The connective tissue core of the fungiform papilla had a top with several round depressions. The four vallate papillae were located on both sides of the posterior end of the lingual body and each papilla was surrounded by groove and crescent pad. A zigzag surface structure appeared on the connective tissue core of the vallate papilla. The foliate papillae were seen on the dorsolateral aspect of the tongue and some ridges and grooves were exposed reciprocally. A zigzag surface structures appeared on the connective tissue cores of the ridges of the foliate papillae.

  17. The pharmacokinetics and metabolism of 14C/13C-labeled ortho-phenylphenol formation following dermal application to human volunteers.

    PubMed

    Timchalk, C; Selim, S; Sangha, G; Bartels, M J

    1998-08-01

    1. The pharmacokinetics and metabolism of uniformly labeled 14C/13C-ortho-phenylphenol (OPP) were followed in six human male volunteers given a single 8 h dermal dose of 6 microg OPP/kg body weight formulated as a 0.4% (w/v) solution in isopropyl alcohol. The application site was covered with a non-occlusive dome allowing free movement of air, but preventing the loss of radioactivity due to physical contact. At 8 h post-exposure the non-occlusive dome was removed, the dose site was wiped with isopropyl alcohol containing swabs and the skin surface repeatedly stripped with tape. Blood specimens, urine, and feces were collected from each volunteer over a 5 day post-exposure period and were analyzed for radioactivity and metabolites (urine only). 2. Following dermal application, peak plasma levels of radioactivity were obtained within 4 h post-exposure and rapidly declined with virtually all of the absorbed dose rapidly excreted into the urine within 24 h post-exposure. A one-compartment pharmacokinetic model was used to describe the time-course of OPP absorption and clearance in male human volunteers. Approximately 43% of the dermally applied dose was absorbed through the skin with an average absorption half-life of 10 h. Once absorbed the renal clearance of OPP was rapid with an average half-life of 0.8 h. The rate limiting step for renal clearance was the relatively slower rate of dermal absorption; therefore the pharmacokinetics of OPP in humans was described by a 'flip-flop' single compartment model. Overall, the pharmacokinetics were similar between individuals, and the model parameters were in excellent agreement with the experimental data. 3. Approximately 73% of the total urinary radioactivity was accounted for as free OPP, OPP-sulfate and OPP-glucuronide conjugates. The sulfate conjugate was the major metabolite (approximately 69%). Therefore, total urinary OPP equivalents (acid-labile conjugates+free OPP) can be used to estimate the systemically absorbed

  18. Disseminated Autochthonous Dermal Leishmaniasis Caused by Leishmania siamensis (PCM2 Trang) in a Patient from Central Thailand Infected with Human Immunodeficiency Virus.

    PubMed

    Supsrisunjai, Chavalit; Kootiratrakarn, Tanawatt; Puangpet, Pailin; Bunnag, Thareena; Chaowalit, Prapaipit; Wessagowit, Vesarat

    2017-05-01

    AbstractSeveral case reports of autochthonous leishmaniasis in Thailand have been published since 1996. Most of the previous cases presented with visceral leishmaniasis (VL) and were mostly reported in southern part of Thailand. Recently, it has been evident that Leishmania martiniquensis is the main cause of Leishmania infection in Thailand. However, Leishmania siamensis (PCM2 Trang isolate) was found to be of a separate lineage with restricted distribution in southern Thailand and also a cause of disseminated dermal and visceral leishmaniasis in one published case. Here we report the first patient from central Thailand with human immunodeficiency virus infection presenting with disseminated dermal leishmaniasis. Polymerase chain reaction and DNA sequencing analysis (large subunit of RNA polymerase II and 18S ribosomal RNA internal transcribed spacer 1) from the tissue biopsy sample revealed the pathogen sequences to be highly homologous to PCM2 Trang strain previously reported from southern Thailand.

  19. Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato

    PubMed Central

    Meddeb, Mariam; Carpentier, Wassila; Cagnard, Nicolas; Nadaud, Sophie; Grillon, Antoine; Barthel, Cathy; De Martino, Sylvie Josiane; Jaulhac, Benoît; Boulanger, Nathalie

    2016-01-01

    In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether “species-related” inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved. PMID:27706261

  20. Simultaneous and multi-point measurement of ammonia emanating from human skin surface for the estimation of whole body dermal emission rate.

    PubMed

    Furukawa, Shota; Sekine, Yoshika; Kimura, Keita; Umezawa, Kazuo; Asai, Satomi; Miyachi, Hayato

    2017-05-15

    Ammonia is one of the members of odor gases and a possible source of odor in indoor environment. However, little has been known on the actual emission rate of ammonia from the human skin surface. Then, this study aimed to estimate the whole-body dermal emission rate of ammonia by simultaneous and multi-point measurement of emission fluxes of ammonia employing a passive flux sampler - ion chromatography system. Firstly, the emission fluxes of ammonia were non-invasively measured for ten volunteers at 13 sampling positions set in 13 anatomical regions classified by Kurazumi et al. The measured emission fluxes were then converted to partial emission rates using the surface body areas estimated by weights and heights of volunteers and partial rates of 13 body regions. Subsequent summation of the partial emission rates provided the whole body dermal emission rate of ammonia. The results ranged from 2.9 to 12mgh(-1) with an average of 5.9±3.2mgh(-1) per person for the ten healthy young volunteers. The values were much greater than those from human breath, and thus the dermal emission of ammonia was found more significant odor source than the breath exhalation in indoor environment. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts

    PubMed Central

    Baek, Beomyeol; Lee, Su Hee; Lim, Hye-Won

    2016-01-01

    Ellagic acid (EA), an antioxidant polyphenolic constituent of plant origin, has been reported to possess diverse pharmacological properties, including anti-inflammatory, anti-tumor and immunomodulatory activities. This work aimed to clarify the skin anti-photoaging properties of EA in human dermal fibroblasts. The skin anti-photoaging activity was evaluated by analyzing the reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity levels as well as cell viability in dermal fibroblasts under UV-B irradiation. When fibroblasts were exposed to EA prior to UV-B irradiation, EA suppressed UV-B-induced ROS and proMMP-2 elevation. However, EA restored total GSH and SOD activity levels diminished in fibroblasts under UV-B irradiation. EA had an up-regulating activity on the UV-B-reduced Nrf2 levels in fibroblasts. EA, at the concentrations used, was unable to interfere with cell viabilities in both non-irradiated and irradiated fibroblasts. In human dermal fibroblasts, EA plays a defensive role against UV-B-induced oxidative stress possibly through an Nrf2-dependent pathway, indicating that this compound has potential skin antiphotoaging properties. PMID:27162481

  2. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  3. Preventive effects of epigallocatechin-3-O-gallate against replicative senescence associated with p53 acetylation in human dermal fibroblasts.

    PubMed

    Han, Dong-Wook; Lee, Mi Hee; Kim, Bongju; Lee, Jun Jae; Hyon, Suong-Hyu; Park, Jong-Chul

    2012-01-01

    Considering the various pharmacological activities of epigallocatechin-3-O-gallate (EGCG) including anticancer, and anti-inflammatory, antidiabetic, and so forth, relatively less attention has been paid to the antiaging effect of EGCG on primary cells. In this study, the preventive effects of EGCG against serial passage-induced senescence were investigated in primary cells including rat vascular smooth muscle cells (RVSMCs), human dermal fibroblasts (HDFs), and human articular chondrocytes (HACs). The involvement of Sirt1 and acetylated p53 was examined as an underlying mechanism for the senescence preventive activity of EGCG in HDFs. All cells were employed with the initial passage number (PN) between 3 and 7. For inducing senescence, the cells were serially passaged at the predetermined times and intervals in the absence or presence of EGCG (50 or 100 μM). Serial passage-induced senescence in RVSMCs and HACs was able to be significantly prevented at 50 μM EGCG, while in HDFs, 100 μM EGCG could significantly prevent senescence and recover their cell cycle progression close to the normal level. Furthermore, EGCG was found to prevent serial passage- and H(2)O(2)-induced senescence in HDFs by suppressing p53 acetylation, but the Sirt1 activity was unaffected. In addition, proliferating HDFs showed similar cellular uptake of FITC-conjugated EGCG into the cytoplasm with their senescent counterparts but different nuclear translocation of it from them, which would partly account for the differential responses to EGCG in proliferating versus senescent cells. Taking these results into consideration, it is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent.

  4. Deletion of Porcn in Mice Leads to Multiple Developmental Defects and Models Human Focal Dermal Hypoplasia (Goltz Syndrome)

    PubMed Central

    Liu, Wei; Shaver, Timothy M.; Balasa, Alfred; Ljungberg, M. Cecilia; Wang, Xiaoling; Wen, Shu; Nguyen, Hoang; Van den Veyver, Ignatia B.

    2012-01-01

    Background Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated. Results We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion. Conclusions These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures. PMID:22412863

  5. Dermal uptake of Tetrabromobisphenol A TBBPA by female Wistar Han rat and human skin

    EPA Science Inventory

    TBBPA, a brominated analog of Bisphenol A, is the highest production volume brominated flame retardant in production and human exposure is ubiquitous. Although the major route of exposure to TBBPA is oral uptake, skin penetration is possible. In the studies presented here, the de...

  6. EXHALED HUMAN BREATH MEASUREMENT OF JET FUEL CONSTITUENTS: DISTINGUISHING BETWEEN INHALATION AND DERMAL EXPOSURE ROUTES

    EPA Science Inventory

    In response to anecdotal reports, perceived health issues, and widespread complaints, the U.S. military launched an investigation into the occupational and environmental human exposure to jet fuel. The work described in the presentation assesses the correlation between two breat...

  7. EXHALED HUMAN BREATH MEASUREMENT OF JET FUEL CONSTITUENTS: DISTINGUISHING BETWEEN INHALATION AND DERMAL EXPOSURE ROUTES

    EPA Science Inventory

    In response to anecdotal reports, perceived health issues, and widespread complaints, the U.S. military launched an investigation into the occupational and environmental human exposure to jet fuel. The work described in the presentation assesses the correlation between two breat...

  8. Dermal uptake of Tetrabromobisphenol A TBBPA by female Wistar Han rat and human skin

    EPA Science Inventory

    TBBPA, a brominated analog of Bisphenol A, is the highest production volume brominated flame retardant in production and human exposure is ubiquitous. Although the major route of exposure to TBBPA is oral uptake, skin penetration is possible. In the studies presented here, the de...

  9. Differential Response of Human Adipose Tissue-Derived Mesenchymal Stem Cells, Dermal Fibroblasts, and Keratinocytes to Burn Wound Exudates: Potential Role of Skin-Specific Chemokine CCL27

    PubMed Central

    van den Broek, Lenie J.; Kroeze, Kim L.; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C.; Niessen, Frank B.; Middelkoop, Esther; Scheper, Rik J.

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell–cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006–9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue

  10. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  11. The influence of transferrin stabilised magnetic nanoparticles on human dermal fibroblasts in culture.

    PubMed

    Berry, Catherine C; Charles, Stuart; Wells, Stephen; Dalby, Matthew J; Curtis, Adam S G

    2004-01-09

    Magnetic nanoparticles have been used for bio-medical purposes including drug delivery, cell destruction and as MRI contrast agents for several years. A more recent biological application has focused on targeted drug delivery. To this end, a wide variety of iron oxide nanoparticles have been synthesised. This study involves the use of magnetic nanoparticles synthesised and derivatised with human transferrin, compared to identical underivatised particles. Human fibroblasts were used, representative of a tissue cell-type. The influence in vitro was determined using light and fluorescence microscopy, scanning and transmission electron microscopy, and 1718 gene microarray. The results indicate that the transferrin derivatised particles appear to localise to the cell membrane without instigating receptor-mediated endocytosis, and also induce up-regulation in the cells for many genes, particularly in the area of cytoskeleton and cell signalling. The microscopy results further support these findings.

  12. Azelaic acid reduced senescence-like phenotype in photo-irradiated human dermal fibroblasts: possible implication of PPARγ.

    PubMed

    Briganti, Stefania; Flori, Enrica; Mastrofrancesco, Arianna; Kovacs, Daniela; Camera, Emanuela; Ludovici, Matteo; Cardinali, Giorgia; Picardo, Mauro

    2013-01-01

    Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long-time treatment with AzA. We previously unrevealed that anti-inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo-aggravated disease, we investigated the ability of AzA to counteract stress-induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8-methoxypsoralen (PUVA), previously reported to activate a senescence-like phenotype, including long-term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence-associated β-galactosidase (SA-β-gal). We found that PUVA-treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP-1 release and SA-β-galactosidase-positive cells. Moreover, AzA induced a reduction in ROS generation, an up-modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA-treated HDFs. Further evidences of AzA anti-senescence effect were repression of p53 and p21, increase in type I pro-collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA-SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA-induced senescence-like phenotype and its ability to activate PPAR-γ provides relevant insights into the anti-senescence mechanism. © 2013 John Wiley & Sons A/S.