Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds.

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Law, Jia Xian; Chowdhury, Shiplu Roy; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

2017-08-01

Advances in tissue engineering led to the development of various tissue-engineered skin substitutes (TESS) for the treatment of skin injuries. The majority of the autologous TESS required lengthy and costly cell expansion process to fabricate. In this study, we determine the possibility of using a low density of human skin cells suspended in platelet-rich plasma (PRP)-enriched medium to promote the healing of full-thickness skin wounds. To achieve this, full-thickness wounds of size 1.767 cm 2 were created at the dorsum part of nude mice and treated with keratinocytes (2 × 10 4  cells/cm 2 ) and fibroblasts (3 × 10 4  cells/cm 2 ) suspended in 10% PRP-enriched medium. Wound examination was conducted weekly and the animals were euthanized after 2 weeks. Gross examination showed that re-epithelialization was fastest in the PRP+cells group at both day 7 and 14, followed by the PRP group and NT group receiving no treatment. Only the PRP+cells group achieved complete wound closure by 2 weeks. Epidermal layer was presence in the central region of the wound of the PRP+cells and PRP groups but absence in the NT group. Comparison between the PRP+cells and PRP groups showed that the PRP+cells-treated wound was more mature as indicated by the presence of thinner epidermis with single cell layer thick basal keratinocytes and less cellular dermis. In summary, the combination of low cell density and diluted PRP creates a synergistic effect which expedites the healing of full-thickness wounds. This combination has the potential to be developed as a rapid wound therapy via the direct application of freshly harvested skin cells in diluted PRP. Copyright © 2017 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

Gelatin for purification and proliferation of primary keratinocyte culture for use in chronic wounds and burns.

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Rahsaz, Marjan; Geramizadeh, Bita; Kaviani, Maryam; Marzban, Saeed

2015-04-01

Human epidermal keratinocytes are currently established as a treatment for burns and wounds and have laboratory applications. Keratinocyte culture contamination by unwanted cells and inhibition of cell proliferation are barriers in primary keratinocyte culture. According to the recent literature, these cells are hard to culture. The present study was conducted to evaluate the efficacy of gelatin-coated surfaces in keratinocyte cultures. After enzymatic isolation of keratinocytes from normal epidermis by trypsin, the cells were cultured on gelatin-coated flasks in serum-free medium. Another group of cells were cultured as a control group without gelatin coating. We showed positive effects of surface coating with gelatin on the primary culture of keratinocytes. Culture of these cells on a gelatincoated surface showed better proliferation with suitable morphology. By using gelatin, adhesion of these cells to the surface was more efficient and without contamination by small round cells. Successful primary culture of keratinocytes on a gelatin-coated surface may provide better yield and optimal number of cells for research and clinical applications.

Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

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Dongyuan Lü

Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers.

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Aschermann, Ilknur; Noor, Seema; Venturelli, Sascha; Sinnberg, Tobias; Mnich, Christian D; Busch, Christian

2017-01-01

Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred "from bench to bedside", and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial) were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the treatment of chronic, therapy-refractory ulcers. © 2017 The Author

Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers

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Ilknur Aschermann

2017-02-01

Full Text Available Background/Aims: Chronic leg ulcers (CLUs are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. Methods: We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Results: Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred “from bench to bedside”, and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. Conclusions: The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the

Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes

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Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

2017-01-01

Background: Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. Objective: To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. Materials and Methods: We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Results: Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. Conclusion: These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. SUMMARY Hisbiscus syriacus L. flower absolute increases HaCaT cell migration and proliferation.Hisbiscus syriacus L. flower absolute regulates phosphorylation of ERK 1/2 and Akt in HaCaT cell.Treatment with Hisbiscus syriacus L. flower induced sprout outgrowth.The wound in the tail-skin of rat was reduced by Hisbiscus syriacus

Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: bilayered versus single-layered substitute.

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Idrus, Ruszymah Bt Hj; Rameli, Mohd Adha bin P; Low, Kiat Cheong; Law, Jia Xian; Chua, Kien Hui; Latiff, Mazlyzam Bin Abdul; Saim, Aminuddin Bin

2014-04-01

Split-skin grafting (SSG) is the gold standard treatment for full-thickness skin defects. For certain patients, however, an extensive skin lesion resulted in inadequacies of the donor site. Tissue engineering offers an alternative approach by using a very small portion of an individual's skin to harvest cells for propagation and biomaterials to support the cells for implantation. The objective of this study was to determine the effectiveness of autologous bilayered tissue-engineered skin (BTES) and single-layer tissue-engineered skin composed of only keratinocytes (SLTES-K) or fibroblasts (SLTES-F) as alternatives for full-thickness wound healing in a sheep model. Full-thickness skin biopsies were harvested from adult sheep. Isolated fibroblasts were cultured using medium Ham's F12: Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, whereas the keratinocytes were cultured using Define Keratinocytes Serum Free Medium. The BTES, SLTES-K, and SLTES-F were constructed using autologous fibrin as a biomaterial. Eight full-thickness wounds were created on the dorsum of the body of the sheep. On 4 wounds, polyvinyl chloride rings were used as chambers to prevent cell migration at the edge. The wounds were observed at days 7, 14, and 21. After 3 weeks of implantation, the sheep were euthanized and the skins were harvested. The excised tissues were fixed in formalin for histological examination via hematoxylin-eosin, Masson trichrome, and elastin van Gieson staining. The results showed that BTES, SLTES-K, and SLTES-F promote wound healing in nonchambered and chambered wounds, and BTES demonstrated the best healing potential. In conclusion, BTES proved to be an effective tissue-engineered construct that can promote the healing of full-thickness skin lesions. With the support of further clinical trials, this procedure could be an alternative to SSG for patients with partial- and full-thickness burns.

Thalidomide increases human keratinocyte migration and proliferation.

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Nasca, M R; O'Toole, E A; Palicharla, P; West, D P; Woodley, D T

1999-11-01

Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behçet's disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 microM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2. 5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2-4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway.

Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

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Khadjavi, Amina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Magnetto, Chiara [Istituto Nazionale di Ricerca Metrologica (INRIM), Torino (Italy); Panariti, Alice [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Argenziano, Monica [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Gulino, Giulia Rossana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Rivolta, Ilaria [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Cavalli, Roberta [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Giribaldi, Giuliana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Guiot, Caterina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Prato, Mauro, E-mail: mauro.prato@unito.it [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Università di Torino, Torino (Italy)

2015-08-01

Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

International Nuclear Information System (INIS)

Khadjavi, Amina; Magnetto, Chiara; Panariti, Alice; Argenziano, Monica; Gulino, Giulia Rossana; Rivolta, Ilaria; Cavalli, Roberta; Giribaldi, Giuliana; Guiot, Caterina; Prato, Mauro

2015-01-01

Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

Comparative transcriptomic profiling of hydrogen peroxide signaling networks in zebrafish and human keratinocytes: Implications toward conservation, migration and wound healing.

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Lisse, Thomas S; King, Benjamin L; Rieger, Sandra

2016-02-05

Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H2O2) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H2O2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H2O2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H2O2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H2O2-dependent pathways, EGF, FOXO1, and IKKα. This study expands on our current understanding of the clinical potential of low-level H2O2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits.

Cultivation and grafting of human keratinocytes on a poly(hydroxyethyl methacrylate) support to the wound bed: a clinical study.

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Dvoránková, B; Smetana, K; Königová, R; Singerová, H; Vacík, J; Jelínková, M; Kapounková, Z; Zahradník, M

1998-01-01

Cultured epithelial sheets on a textile support are used for the treatment of seriously burned patients. In this study we demonstrate a new procedure for the grafting of keratinocytes directly on a polymer cultivation support. This procedure is much easier in comparison with classical techniques, and encouraging results of clinical trials demonstrate the improved healing of the wound bed after the use of this procedure. There is no difference in the cytokeratine pattern (LP-34, cytokeratin-10) of the reconstructed epidermis and normal human skin.

Early Gene Expression in Wounded Human Keratinocytes Revealed by DNA Microarray Analysis

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Pascal Barbry

2006-04-01

Full Text Available Wound healing involves several steps: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical ‘scratch’ method. The two aims of the present study were: (a to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor α-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.

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

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Yun-Hee Choi

2015-11-01

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

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

Science.gov (United States)

Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

2015-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

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Paes, Camila, E-mail: camilaquinetti@gmail.com [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nagase, Takashi, E-mail: tnagase@fb3.so-net.ne.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Huang, Lijuan, E-mail: koureikenhlj@gmail.com [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Sari, Yunita, E-mail: yunita-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Sanada, Hiromi, E-mail: hsanada-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

2012-10-19

Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalian cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration

Mesenchymal stem cells ameliorate impaired wound healing through enhancing keratinocyte functions in diabetic foot ulcerations on the plantar skin of rats.

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Kato, Jiro; Kamiya, Hideki; Himeno, Tatsuhito; Shibata, Taiga; Kondo, Masaki; Okawa, Tetsuji; Fujiya, Atsushi; Fukami, Ayako; Uenishi, Eita; Seino, Yusuke; Tsunekawa, Shin; Hamada, Yoji; Naruse, Keiko; Oiso, Yutaka; Nakamura, Jiro

2014-01-01

Although the initial healing stage involves a re-epithelialization in humans, diabetic foot ulceration (DFU) has been investigated using rodent models with wounds on the thigh skin, in which a wound contraction is initiated. In this study, we established a rodent model of DFU on the plantar skin and evaluated the therapeutic efficacy of bone-marrow-derived mesenchymal stem cells (BM-MSCs) in this model. The wounds made on the hind paws or thighs of streptozotocin induced diabetic or control rats were treated with BM-MSCs. Expression levels of phosphorylated focal adhesion kinase (pFAK), matrix metaroprotease (MMP)-2, EGF, and IGF-1, were evaluated in human keratinocytes, which were cultured in conditioned media of BM-MSCs (MSC-CM) with high glucose levels. Re-epithelialization initiated the healing process on the plantar, but not on the thigh, skin. The therapy utilizing BM-MSCs ameliorated the delayed healing in diabetic rats. In the keratinocytes cultured with MSC-CM, the decreased pFAK levels in the high glucose condition were restored, and the MMP2, EGF, and IGF-1 levels increased. Our study established a novel rat DFU model. The impaired healing process in diabetic rats was ameliorated by transplantation of BM-MSCs. This amelioration might be accounted for by the modification of keratinocyte functions. Copyright © 2014 Elsevier Inc. All rights reserved.

Cell motility in models of wounded human skin is improved by Gap27 despite raised glucose, insulin and IGFBP-5

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Wright, Catherine S.; Berends, Rebecca F. [Department of Life Sciences, School of Health and Life Sciences, Glasgow Caledonian University, 70 Cowcaddens Road, Glasgow G4 0BA (United Kingdom); Flint, David J. [Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE (United Kingdom); Martin, Patricia E.M., E-mail: Patricia.Martin@gcu.ac.uk [Department of Life Sciences, School of Health and Life Sciences, Glasgow Caledonian University, 70 Cowcaddens Road, Glasgow G4 0BA (United Kingdom)

2013-02-15

Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance. - Highlights: ► Human organotypic and keratinocyte ‘diabetic’ skin models were used to demonstrate the ability of Gap27 to improve scrape-wound closure. ► Gap27 enhanced scrape-wound closure by reducing Cx43-mediated communication, whereas IGFBP-5 retarded cell migration. ► IGF-I and IGFBP-5 did not affect connexin-mediated pathways. ► Gap27 can override altered glucose, insulin, IGF-I, and IGFBP-5 in ‘diabetic’ skin models and thus has therapeutic potential.

Platelet-released growth factors inhibit proliferation of primary keratinocytes in vitro.

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Bayer, Andreas; Tohidnezhad, Mersedeh; Berndt, Rouven; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Simanski, Maren; Gläser, Regine; Harder, Jürgen

2018-01-01

Autologous thrombocyte concentrate lysates as platelet-released growth factors (PRGF) or Vivostat Platelet Rich Fibrin (PRF ® ) represent important tools in modern wound therapy, especially in the treatment of chronic, hard-to-heal or infected wounds. Nevertheless, underlying cellular and molecular mechanisms of the beneficial clinical effects of a local wound therapy with autologous thrombocyte concentrate lysates are poorly understood. Recently, we have demonstrated that PRGF induces antimicrobial peptides in primary keratinocytes and accelerates keratinocytes' differentiation. In the present study we analyzed the influence of PRGF on primary human keratinocytes' proliferation. Using the molecular proliferation marker Ki-67 we observed a concentration- and time dependent inhibition of Ki-67 gene expression in PRGF treated primary keratinocytes. These effects were independent from the EGFR- and the IL-6-R pathway. Inhibition of primary keratinocytes' proliferation by PRGF treatment was confirmed in colorimetric cell proliferation assays. Together, these data indicate that the clinically observed positive effects of autologous thrombocytes concentrates in the treatment of chronic, hard-to-heal wounds are not based on an increased keratinocytes proliferation. Copyright © 2017 Elsevier GmbH. All rights reserved.

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

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Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo. PMID:28808357

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

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Andreas Bayer

2017-01-01

Full Text Available Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs or their clinically related formulations (e.g., Vivostat PRF® came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10 and late (transglutaminase-1 and involucrin differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR- dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

Lysophosphatidic acid induces expression of genes in human oral keratinocytes involved in wound healing.

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Thorlakson, Hong Huynh; Engen, Stian Andre; Schreurs, Olav; Schenck, Karl; Blix, Inger Johanne Schytte

2017-08-01

Epithelial cells participate in wound healing by covering wounds, but also as important mediators of wound healing processes. Topical application of the phospholipid growth factor lysophosphatidic acid (LPA) accelerates dermal wound healing and we hypothesized that LPA can play a role in human oral wound healing through its effects on human oral keratinocytes (HOK). HOK were isolated from gingival biopsies and exposed to LPA. The LPA receptor profile, signal transduction pathways, gene expression and secretion of selected cytokines were analyzed. HOK expressed the receptors LPA 1 , LPA 5 and LPA 6 and LPA activated the ERK1/2, JNK and p38 intracellular pathways, substantiated by secretion of IL-6 and IL-8. The early (2h) and intermediate (6h) gene expression profiles of HOK after LPA treatment showed a wide array of regulated genes. The majority of the strongest upregulated genes were related to chemotaxis and inflammation, and became downregulated after 6h. At 6h, genes coding for factors involved in extracellular matrix remodeling and re-epithelialization became highly expressed. IL-36γ, not earlier known to be regulated by LPA, was strongly transcribed and translated but not secreted. After stimulation with LPA, HOK responded by regulating factors and genes that are essential in wound healing processes. As LPA is found in saliva and is released by activated cells after wounding, our results indicate that LPA has a favorable physiological role in oral wound healing. This may further point towards a beneficial role for application of LPA on oral surgical or chronic wounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

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Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

2016-07-01

The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication. Copyright © 2016 Elsevier B.V. All rights reserved.

Loss of the Desmosomal Component Perp Impairs Wound Healing In Vivo

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Veronica G. Beaudry

2010-01-01

Full Text Available Epithelial wound closure is a complex biological process that relies on the concerted action of activated keratinocytes and dermal fibroblasts to resurface and close the exposed wound. Modulation of cell-cell adhesion junctions is thought to facilitate cellular proliferation and migration of keratinocytes across the wound. In particular, desmosomes, adhesion complexes critical for maintaining epithelial integrity, are downregulated at the wound edge. It is unclear, however, how compromised desmosomal adhesion would affect wound reepithelialization, given the need for a delicate balance between downmodulating adhesive strength to permit changes in cellular morphology and maintaining adhesion to allow coordinated migration of keratinocyte sheets. Here, we explore the contribution of desmosomal adhesion to wound healing using mice deficient for the desmosomal component Perp. We find that Perp conditional knockout mice display delayed wound healing relative to controls. Furthermore, we determine that while loss of Perp compromises cell-cell adhesion, it does not impair keratinocyte proliferation and actually enhances keratinocyte migration in in vitro assays. Thus, Perp's role in promoting cell adhesion is essential for wound closure. Together, these studies suggest a role for desmosomal adhesion in efficient wound healing.

Low levels of glutathione are sufficient for survival of keratinocytes after UV irradiation and for healing of mouse skin wounds.

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Telorack, Michèle; Abplanalp, Jeannette; Werner, Sabine

2016-08-01

Reduced levels of the cellular antioxidant glutathione are associated with premature skin aging, cancer and impaired wound healing, but the in vivo functions of glutathione in the skin remain largely unknown. Therefore, we analyzed mice lacking the modifier subunit of the glutamate cysteine ligase (Gclm), the enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Glutathione levels in the skin of these mice were reduced by 70 %. However, neither skin development and homeostasis, nor UVA- or UVB-induced apoptosis in the epidermis were affected. Histomorphometric analysis of excisional wounds did not reveal wound healing abnormalities in young Gclm-deficient mice, while the area of hyperproliferative epithelium as well as keratinocyte proliferation were affected in aged mice. These findings suggest that low levels of glutathione are sufficient for wound repair in young mice, but become rate-limiting upon aging.

The role of PRP and adipose tissue-derived keratinocytes on burn wound healing in diabetic rats.

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Hosseini Mansoub, Navid; Gürdal, Mehmet; Karadadaş, Elif; Kabadayi, Hilal; Vatansever, Seda; Ercan, Gulinnaz

2018-01-01

Introduction: Diabetic burn wounds and ulcers are significant complications of diabetic patients. The aim of this study is to investigate the use of platelet rich-plasma (PRP) and/or keratinocyte-like cells (KLCs) in diabetic thermal wound rat model and to evaluate EGF, FGF-2, TGF-β1, COL1α2, MCP-1 and VEGF-α as wound healing markers at gene expression level. Method: In this study, we used adipose tissue as the source of mesenchymal stem cells (MSCs) and differentiated MSCs into KLCs. KLCs were characterized and transferred to the burn areas on the dorsum of streptozotocine (STZ)-induced diabetic rats. We prepared PRP from rat blood and evaluated its effect alone or in combination with KLCs. On 3 rd , 7 th , 10 th and 14 th days after treatment, wound areas were measured and biopsy samples were excised from the wound areas of the KLCs and/or PRP-treated and untreated diabetic rats to analyze gene expression levels of wound healing markers by qPCR. Results: We observed that, wound contraction started earlier in the PRP and/or KLCs-treated groups in comparison to the control group. However, PRP and KLCs when applied in combination showed additive affect in wound healing. In all groups treated with KLCs and/or PRP, the gene expression levels of evaluated growth factors and COL1α2 increased, while MCP-1 levels decreased when compared to the untreated diabetic rats. In addition, the most prominent difference in qPCR results belongs to combined PRP and KLCs-treated group. Conclusion: We demonstrated that applying PRP and KLCs in combination has a greater potential for treatment of diabetic burn wounds.

Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing.

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Seo, Eunhui; Lim, Jae Soo; Jun, Jin-Bum; Choi, Woohyuk; Hong, In-Sun; Jun, Hee-Sook

2017-02-15

Diminished wound healing is a major complication of diabetes mellitus and can lead to foot ulcers. However, there are limited therapeutic methods to treat this condition. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, is known to have many beneficial effects on diabetes. In addition, mesenchymal stem cells are known to have wound healing effects. We investigated the effects of Ex-4 in combination with human adipose tissue-derived stem cells (ADSCs) on diabetic wound healing in a diabetic animal model. Diabetic db/db (blood glucose levels, >500 mg/dl) or C57BL/6 mice were subjected to wounding on the skin of the back. One day after wounding, each wound received ADSCs (2.5 × 10 5 cells) injected intradermally around the wound and/or Ex-4 (50 μl of 100 nM Ex-4) topically applied on the wound with a fine brush daily. Wound size was monitored and wound histology was examined. Human endothelial cells and keratinocyte cells were used to assess angiogenesis and vascular endothelial growth factor expression in vitro. Topical administration of Ex-4 or injection of ADSCs resulted in a rapid reduction of wound size in both diabetic and normoglycemic animals compared with vehicle treatment. Histological analysis also showed rapid skin reconstruction in Ex-4-treated or ADSC-injected wounds. A combination of Ex-4 and ADSCs showed a significantly better therapeutic effect over either treatment alone. In vitro angiogenesis assays showed that both Ex-4 and ADSC-conditioned media (CM) treatment improved migration, invasion and proliferation of human endothelial cells. ADSC-CM also increased migration and proliferation of human keratinocytes. In addition, both Ex-4 and ADSC-CM increased the expression of vascular endothelial growth factor. Co-culture with ADSCs increased migration and proliferation of these cells similar to that found after ADSC-CM treatment. We suggest that Ex-4 itself is effective for the treatment of diabetic skin wounds, and a combination of

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

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Gonzalez-Curiel, Irma; Trujillo, Valentin; Montoya-Rosales, Alejandra; Rincon, Kublai; Rivas-Calderon, Bruno; deHaro-Acosta, Jeny; Marin-Luevano, Paulina; Lozano-Lopez, Daniel; Enciso-Moreno, Jose A; Rivas-Santiago, Bruno

2014-01-01

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

The Benefit of Microskin in Combination With Autologous Keratinocyte Suspension to Treat Full Skin Loss In Vivo.

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Yuru, Shang; Dawei, Li; Chuanan, Shen; Kai, Yin; Li, Ma; Longzhu, Li; Dongxu, Zhao; Wenfeng, Cheng

Patients with extensive deep burns often lack enough autologous skin to cover the wounds. This study explores a new method using microskin in combination with autologous keratinocytes in the treatment of extensive deep burn. Wounds in the combination group were treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area) and autologous keratinocyte suspension, which were compared with the following treatments: no autotransplant, only allografts (control group); autologous keratinocyte suspension only (keratinocyte only group); automicroskin at an area expansion ratio of 20:1 (20:1 group); and automicroskin at an area expansion ratio of 10:1 (10:1 group, positive control). The authors used epithelialization rate (epithelialized area on day 21 divided by original wound area), hematoxylin and eosin staining, laminin, and type IV collagen immunohistochemistry to assess wound healing. The epithelialization rate of combination group (74.2% ± 8.0%) was similar to that of 10: 1 group (84.3% ± 11.9%, P = .085) and significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). The hematoxylin and eosin staining and immunohistochemistry showed the epithelialization in the combination group was better than that in the keratinocyte only group and control group. Microskin in combination with autologous keratinocyte suspension can promote the reepithelialization of full-thickness wounds and reduce the requirements for automircoskin, and it is a useful option in the treatment of extensive deep burns.

FOXO1 expression in keratinocytes promotes connective tissue healing

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Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

2017-01-01

Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

International Nuclear Information System (INIS)

Paes, Camila; Nakagami, Gojiro; Minematsu, Takeo; Nagase, Takashi; Huang, Lijuan; Sari, Yunita; Sanada, Hiromi

2012-01-01

Highlights: ► An evidence of the positive effect of AHL on epithelialization process is provided. ► AHL enhances keratinocyte’s ability to migrate in an in vitro scratch wound model. ► AHL induces the expression of Mmp13. ► Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalian cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte’s activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte’s ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.

Far infrared promotes wound healing through activation of Notch1 signaling.

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Hsu, Yung-Ho; Lin, Yuan-Feng; Chen, Cheng-Hsien; Chiu, Yu-Jhe; Chiu, Hui-Wen

2017-11-01

The Notch signaling pathway is critically involved in cell proliferation, differentiation, development, and homeostasis. Far infrared (FIR) has an effect that promotes wound healing. However, the underlying molecular mechanisms are unclear. In the present study, we employed in vivo and HaCaT (a human skin keratinocyte cell line) models to elucidate the role of Notch1 signaling in FIR-promoted wound healing. We found that FIR enhanced keratinocyte migration and proliferation. FIR induced the Notch1 signaling pathway in HaCaT cells and in a microarray dataset from the Gene Expression Omnibus database. We next determined the mRNA levels of NOTCH1 in paired normal and wound skin tissues derived from clinical patients using the microarray dataset and Ingenuity Pathway Analysis software. The result indicated that the Notch1/Twist1 axis plays important roles in wound healing and tissue repair. In addition, inhibiting Notch1 signaling decreased the FIR-enhanced proliferation and migration. In a full-thickness wound model in rats, the wounds healed more rapidly and the scar size was smaller in the FIR group than in the light group. Moreover, FIR could increase Notch1 and Delta1 in skin tissues. The activation of Notch1 signaling may be considered as a possible mechanism for the promoting effect of FIR on wound healing. FIR stimulates keratinocyte migration and proliferation. Notch1 in keratinocytes has an essential role in FIR-induced migration and proliferation. NOTCH1 promotes TWIST1-mediated gene expression to assist wound healing. FIR might promote skin wound healing in a rat model. FIR stimulates keratinocyte migration and proliferation. Notch1 in keratinocytes has an essential role in FIR-induced migration and proliferation. NOTCH1 promotes TWIST1-mediated gene expression to assist wound healing. FIR might promote skin wound healing in a rat model.

Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

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Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

2015-11-05

A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

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Rasmussen, Cathy A; Allen-Hoffmann, B Lynn

2012-04-01

For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss. Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites. Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures. Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application. This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

Delivery of Flightless I Neutralizing Antibody from Porous Silicon Nanoparticles Improves Wound Healing in Diabetic Mice.

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Turner, Christopher T; McInnes, Steven J P; Melville, Elizabeth; Cowin, Allison J; Voelcker, Nicolas H

2017-01-01

Flightless I (Flii) is elevated in human chronic wounds and is a negative regulator of wound repair. Decreasing its activity improves healing responses. Flii neutralizing antibodies (FnAbs) decrease Flii activity in vivo and hold significant promise as healing agents. However, to avoid the need for repeated application in a clinical setting and to protect the therapeutic antibody from the hostile environment of the wound, suitable delivery vehicles are required. In this study, the use of porous silicon nanoparticles (pSi NPs) is demonstrated for the controlled release of FnAb to diabetic wounds. We achieve FnAb loading regimens exceeding 250 µg antibody per mg of vehicle. FnAb-loaded pSi NPs increase keratinocyte proliferation and enhance migration in scratch wound assays. Release studies confirm the functionality of the FnAb in terms of Flii binding. Using a streptozotocin-induced model of diabetic wound healing, a significant improvement in healing is observed for mice treated with FnAb-loaded pSi NPs compared to controls, including FnAb alone. FnAb-loaded pSi NPs treated with proteases show intact and functional antibody for up to 7 d post-treatment, suggesting protection of the antibodies from proteolytic degradation in wound fluid. pSi NPs may therefore enable new therapeutic approaches for the treatment of diabetic ulcers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model.

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Irma Gonzalez-Curiel

Full Text Available Diabetic foot ulcers (DFU are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH2 D3 and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

In vitro induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 expression in keratinocytes by boron and manganese.

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Chebassier, Nathalie; El Houssein, Ouijja; Viegas, Isabelle; Dréno, Brigitte

2004-08-01

Matrix metalloproteinase (MMP)-2 and MMP-9 are involved in keratinocyte migration and granulation tissue remodeling during wound healing. Thermal water cures are sometimes proposed as complementary treatment for accelerating healing of wounds resulting from burns and/or surgery, but their mechanisms of action remain unknown. Some thermal waters are rich in trace elements such as boron and manganese. Interestingly, clinical studies have shown the beneficial effects of trace elements such as boron and manganese for human wound healing. To try to specify the role of trace elements in cutaneous healing, the present study investigated the effects of these trace elements on the production of MMP-2 and MMP-9 by normal human keratinocytes cultured in vitro. Immunohistochemistry and Western blot showed that intracellular MMP-9 expression in keratinocytes was induced when incubated for 6 h with boron at 10 micro g/ml or manganese at 0.2 micro g/ml. Moreover, gelatin zymography on keratinocyte supernatants showed an increase of gelatinase secretion after 24 h of incubation of keratinocytes with boron or manganese, regardless of concentration. Gelatinase secretion was not associated with keratinocyte proliferation induced by trace elements. Thus, our results suggest that boron and manganese could play a role in the clinical efficiency of thermal water on wound healing.

Leptin promotes wound healing in the skin.

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Susumu Tadokoro

Full Text Available Leptin, a 16 kDa anti-obesity hormone, exhibits various physiological properties. Interestingly, skin wound healing was proven to delay in leptin-deficient ob/ob mice. However, little is known on the mechanisms of this phenomenon. In this study, we attempted to elucidate a role of leptin in wound healing of skin.Immunohistochemical analysis was performed to confirm the expression of the leptin receptor (Ob-R in human and mouse skin. Leptin was topically administered to chemical wounds created in mouse back skin along with sustained-release absorbable hydrogel. The process of wound repair was histologically observed and the area of ulceration was measured over time. The effect of leptin on the proliferation, differentiation and migration of human epidermal keratinocytes was investigated.Ob-R was expressed in epidermal cells of human and mouse skin. Topical administration of leptin significantly promoted wound healing. Histological analysis showed more blood vessels in the dermal connective tissues in the leptin-treated group. The proliferation, differentiation/function and migration of human epidermal keratinocytes were enhanced by exogenous leptin.Topically administered leptin was proven to promote wound healing in the skin by accelerating proliferation, differentiation/function and migration of epidermal keratinocytes and enhancing angiogenesis around the wounded area. These results strongly suggest that topical administration of leptin may be useful as a treatment to promote wound healing in the skin.

Treatment of burn injuries with keratinocyte cultures

International Nuclear Information System (INIS)

Syring, C.; Maenig, H.J.; Von Versen, R.; Bruck, J.

1999-01-01

The German Institute for Cell and Tissue Replacement (DIZG) provides burned patients with skin and amnion for a temporary wound closure. Severely burned patients (>60% BSA for adults, >40% BSA for children) were supplied with autologous and allogenic grafts from cultured keratinocytes. The keratinocyte culture is done under GMP-conditions using the method of Rheinwald and Green. The 3T3 fibroblasts were irradiated with 60 Gy and used as feeder cells to produce keratinocyte sheets within 3 weeks. In this time up to 6.000 cm are available. The sheets were harvested by detachment with dispase (1,2 U/ml), fixed to gauze and transported to the hospital. The DIZG has a 3 years experience in the treatment of burns with keratinocyte sheets. The sheets were transplanted to patients in different hospitals, the total transplanted area is about 30.000 cm. This paper describes the experiences with ten severely burned patients treated with keratinocyte sheet

Studies on wound healing potential of polyherbal formulation using in vitro and in vivo assays

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Yogesh P. Talekar

2017-04-01

Conclusion: Polyherbal formulation prepared from the plant extracts accelerates wound healing process by proliferation and mobilization of fibroblast and keratinocytes, and angiogenesis at the site of injury. It also shows fast contraction of wound with its beneficial improvement in tissue biochemical and antioxidant parameters.

Neurohumoral mechanisms of keratinocytes regulation in diabetes mellitus

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Ekaterina Viktorovna Artemova

2016-12-01

Full Text Available The extent of damage to the nervous, vascular and microcirculatory systems in diabetic patients determine the regulation of physiological events that lead to the formation of chronic wounds, reduction of patient quality of life and increase of the financial value of medical care. Successful physiological repair is impossible without the successive phases of inflammation, proliferation and wound healing. Keratinocytes are the major cellular barrier components of the epidermis. These cells play an important role in physiological repair, as suggested by recent research, with many cells able to secrete steroid hormones de novo. Damage to the integrity of the skin leads to keratinocyte activation, triggering a cascade of reactions that contribute to changes in epidermal cell phenotype and lead to their proliferation and migration, analogous to changes in cellular adhesion and configuration of the cytoskeleton. An open question remains as to how the keratinocyte cell cycle, which is altered under conditions of hyperglycemia, and neurotransmitter metabolism during different stages of physiological repair are regulated. Understanding these processes will provide a scientific basis for the development of new targets for pharmacotherapies.

The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization

DEFF Research Database (Denmark)

Kruse, Carla R; Singh, Mansher; Targosinski, Stefan

2017-01-01

primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5-12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full-thickness wound model was used...... to investigate the effect of pH (5.5-9.5) on wound healing in vivo. The effect of pH on inflammation was monitored by measuring IL-1 α concentrations from wounds and cell cultures exposed to different pH environments. Our results showed that both skin cell types tolerated wide range of pH very well. They further...... demonstrated that both acidic and alkaline environments decelerated cell migration in comparison to neutral environments and interestingly alkaline conditions significantly enhanced cell proliferation. Results from the in vivo experiments indicated that a prolonged, strongly acidic wound environment prevents...

Evaluation of dermal-epidermal skin equivalents ('composite-skin') of human keratinocytes in a collagen-glycosaminoglycan matrix(Integra artificial skin).

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Kremer, M; Lang, E; Berger, A C

2000-09-01

Integra artificial skin (Integra LifeSciences Corp., Plainsboro, NJ, USA) is a dermal template consisting of bovine collagen, chondroitin-6-sulphate and a silastic membrane manufactured as Integra. This product has gained widespread use in the clinical treatment of third degree burn wounds and full thickness skin defects of different aetiologies. The product was designed to significantly reduce the time needed to achieve final wound closure in the treatment of major burn wounds, to optimise the sparse autologous donor skin resources and to improve the durable mechanical quality of the skin substitute. The clinical procedure requires two stages. The first step creates a self neodermis, the second creates a self epidermis on the neodermis. However, it is desirable to cover major burn wounds early in a single step by a skin substitute consisting of a dermal equivalent seeded in vitro with autologous keratinocytes ('composite-skin') out of which a full thickness skin develops in vivo.The goal of this experimental study was to develop a method to integrate human keratinocytes in homogeneous distribution and depth into Integra Artificial Skin. The seeded cell-matrix composites were grafted onto athymic mice in order to evaluate their potential to reconstitute a human epidermis in vivo. We were able to demonstrate that the inoculated human keratinocytes reproducibly displayed a homogeneous pattern of distribution, adherence, proliferation and confluence. The cell-matrix composites grafted in this model exhibited good wound adherence, complete healing, minor wound contraction and had the potential to reconstitute an elastic, functional and durable human skin. Histologically we were able to show that the inoculated human keratinocytes in vivo colonised the matrix in a histomorphologically characteristic epidermal pattern (keratomorula, keratinocyte bubbling) and developed a persisting, stratified, keratinising epidermis which immunohistologically proved to be of human

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

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Andreas Bayer

2017-01-01

Full Text Available Platelet-released growth factors (PRGF and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF® contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3 is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR. In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

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Lammel, Justus; Tohidnezhad, Mersedeh; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Cremer, Jochen; Jahr, Holger; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds. PMID:28811680

Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

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Moura, Liane I F; Dias, Ana M A; Suesca, Edward; Casadiegos, Sergio; Leal, Ermelindo C; Fontanilla, Marta R; Carvalho, Lina; de Sousa, Hermínio C; Carvalho, Eugénia

2014-01-01

Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (phealing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (pdiabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone. © 2013.

In vitro human keratinocyte migration rates are associated with SNPs in the KRT1 interval.

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Heng Tao

Full Text Available Efforts to develop effective therapeutic treatments for promoting fast wound healing after injury to the epidermis are hindered by a lack of understanding of the factors involved. Re-epithelialization is an essential step of wound healing involving the migration of epidermal keratinocytes over the wound site. Here, we examine genetic variants in the keratin-1 (KRT1 locus for association with migration rates of human epidermal keratinocytes (HEK isolated from different individuals. Although the role of intermediate filament genes, including KRT1, in wound activated keratinocytes is well established, this is the first study to examine if genetic variants in humans contribute to differences in the migration rates of these cells. Using an in vitro scratch wound assay we observe quantifiable variation in HEK migration rates in two independent sets of samples; 24 samples in the first set and 17 samples in the second set. We analyze genetic variants in the KRT1 interval and identify SNPs significantly associated with HEK migration rates in both samples sets. Additionally, we show in the first set of samples that the average migration rate of HEK cells homozygous for one common haplotype pattern in the KRT1 interval is significantly faster than that of HEK cells homozygous for a second common haplotype pattern. Our study demonstrates that genetic variants in the KRT1 interval contribute to quantifiable differences in the migration rates of keratinocytes isolated from different individuals. Furthermore we show that in vitro cell assays can successfully be used to deconstruct complex traits into simple biological model systems for genetic association studies.

Astragaloside IV Downregulates β-Catenin in Rat Keratinocytes to Counter LiCl-Induced Inhibition of Proliferation and Migration

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Fu-Lun Li

2012-01-01

Full Text Available Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compound in this drug as astragaloside IV (AS-IV, but the underlying molecular mechanisms of its therapeutic action on keratinocytes remain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl-induced cultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA expression. AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediates mouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin to increase keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

Abnormalities in the basement membrane structure promote basal keratinocytes in the epidermis of hypertrophic scars to adopt a proliferative phenotype.

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Yang, Shaowei; Sun, Yexiao; Geng, Zhijun; Ma, Kui; Sun, Xiaoyan; Fu, Xiaobing

2016-05-01

The majority of studies on scar formation have mainly focused on the dermis and little is known of the involvement of the epidermis. Previous research has demonstrated that the scar tissue-derived keratinocytes are different from normal cells at both the genetic and cell biological levels; however, the mechanisms responsible for the fundamental abnormalities in keratinocytes during scar development remain elusive. For this purpose, in this study, we used normal, wound edge and hypertrophic scar tissue to examine the morphological changes which occur during epidermal regeneration as part of the wound healing process and found that the histological structure of hypertrophic scar tissues differed from that of normal skin, with a significant increase in epidermal thickness. Notably, staining of the basement membrane (BM) appeared to be absent in the scar tissues. Moreover, immunofluorescence staining for cytokeratin (CK)10, CK14, CK5, CK19 and integrin-β1 indicated the differential expression of cell markers in the epidermal keratinocytes among the normal, wound edge and hypertrophic scar tissues, which corresponded with the altered BM structures. By using a panel of proteins associated with BM components, we validated our hypothesis that the BM plays a significant role in regulating the cell fate decision of epidermal keratinocytes during skin wound healing. Alterations in the structure of the BM promote basal keratinocytes to adopt a proliferative phenotype both in vivo and in vitro.

[Cultivated keratinocytes on micro-carriers: in vitro studies of a new carrier system].

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Hecht, J; Hoefter, E A; Hecht, J; Haraida, S; Nerlich, A; Hartinger, A; Mühlbauer, W; Dimoudis, N

1997-03-01

Epidermal grafts from confluently cultivated keratinocytes have been used since the early eighties for the treatment of severe burns, where the shortage of donor sites for split-thickness skin grafts did not allow for adequate wound coverage. The difficult handling of these grafts as well as the advanced differentiation of their epithelial cells into a multilayer sheet poses a problem for their clinical application. The aim of the study was to characterize cultivated keratinocytes, as well as to observe their migration and proliferation from the MC onto a surface. Keratinocytes were isolated from human foreskin and cultivated in serum-free and serum-containing medium according to a modified method by Rheinwald and Green. Collagen-coated Dextran beads were used as MC. The MC were colonized with keratinocytes using the Spinner culture technique. After seeding the colonized MC into culture flasks, their migration and proliferation was monitored regularly through immunohistochemical studies and measurement of the metabolic cell activity. Immunohistological staining proved that the cells isolated from human foreskin represent keratinocytes of the basal type. Keratinocytes, cultivated with serum-containing and serum free medium, both adhered to the surface of the MC, then migrated onto the surface of the flasks and proliferated to form a multilayer of epithelial cells. In the long-term, a flexible epithelial graft consisting of poorly differentiated keratinocytes should be available, which is simple to produce and easy to handle. This would be an alternative method for treating wounds, where the conventional multilayer epithelial graft (ET) is insufficient.

Allogeneic cultured keratinocytes vs. cadaveric skin to cover wide-mesh autogenous split-thickness skin grafts.

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Monstrey, S; Beele, H; Kettler, M; Van Landuyt, K; Blondeel, P; Matton, G; Naeyaert, J M

1999-09-01

Improved shock therapy has extended the limits of survival in patients with massive burns, and nowadays skin coverage has become the major problem in burn management. The use of mesh skin grafts is still the simplest technique to expand the amount of available donor skin. However, very wide-mesh skin grafts take a very long time to heal, often resulting in unaesthetic scar formation. On the other hand, allogeneic cultured keratinocytes have been reported as a natural source of growth factors and thus could be useful to improve wound healing of these wide-mesh grafts. A clinical study was performed to compare the use of cryopreserved allogeneic cultured keratinocytes vs. the traditional cadaveric skin as a double layer over widely expanded autogenous skin grafts. This procedure was performed in 18 pairs of full-thickness burn wounds (with similar depth and location) in 11 severely burned patients. Early clinical evaluation was made at 2, 3, and 4 to 5 weeks. Parameters such as epithelialization, granulation tissue formation, infection, and scar formation were evaluated. Biopsies were taken to compare the histological characteristics of the epidermis, the epidermal-dermal junction, and the dermis. Late evaluations were performed at 6 and 12 months regarding color, softness, thickness, and subjective feeling of the scar tissue. Aside from a faster (p keratinocyte group at 2 weeks, there were no statistically different results in any of the early evaluated parameters, neither clinically nor histologically. At long-term follow-up, clinical results and scar characteristics were not significantly different in the two compared groups. It is concluded from the results of this study that, during the early phase, epithelialization was faster with allogeneic cultured keratinocytes compared with cadaveric skin. However, taking into account the substantial difference in costs, the described use of cryopreserved allogeneic cultured keratinocytes as a double layer on meshed

T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

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Cécilia Brun

Full Text Available Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte

Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

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Bonnie H Y Yeung

Full Text Available Stanniocalcin-1 (STC1, a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam formation, followed by cell migration. In this study, staurosporine (STS treatment induced human keratinocyte (HaCaT e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK, the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

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Yeung, Bonnie H Y; Wong, Chris K C

2011-01-01

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

OpenAIRE

Salonurmi, T.; Parikka, M.; Kontusaari, S.; Pirila, E.; Munaut, Carine; Salo, T.; Tryggvason, K.

2004-01-01

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymograph...

The heme-heme oxygenase system in wound healing; implications for scar formation.

NARCIS (Netherlands)

Wagener, F.A.D.T.G.; Scharstuhl, A.; Tyrrell, R.M.; Hoff, J.W. Von den; Jozkowicz, A.; Dulak, J.; Russel, F.G.M.; Kuijpers-Jagtman, A.M.

2010-01-01

Wound healing is an intricate process requiring the concerted action of keratinocytes, fibroblasts, endothelial cells, and macrophages. Here, we review the literature on normal wound healing and the pathological forms of wound healing, such as hypertrophic or excessive scar formation, with special

Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs

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Ayesha Bhatia

2016-01-01

Full Text Available Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5–treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing.

Dual therapeutic functions of F-5 fragment in burn wounds: preventing wound progression and promoting wound healing in pigs.

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Bhatia, Ayesha; O'Brien, Kathryn; Chen, Mei; Wong, Alex; Garner, Warren; Woodley, David T; Li, Wei

2016-01-01

Burn injuries are a leading cause of morbidity including prolonged hospitalization, disfigurement, and disability. Currently there is no Food and Drug Administration-approved burn therapeutics. A clinical distinction of burn injuries from other acute wounds is the event of the so-called secondary burn wound progression within the first week of the injury, in which a burn expands horizontally and vertically from its initial boundary to a larger area. Therefore, an effective therapeutics for burns should show dual abilities to prevent the burn wound progression and thereafter promote burn wound healing. Herein we report that topically applied F-5 fragment of heat shock protein-90α is a dual functional agent to promote burn wound healing in pigs. First, F-5 prevents burn wound progression by protecting the surrounding cells from undergoing heat-induced caspase 3 activation and apoptosis with increased Akt activation. Accordingly, F-5-treated burn and excision wounds show a marked decline in inflammation. Thereafter, F-5 accelerates burn wound healing by stimulating the keratinocyte migration-led reepithelialization, leading to wound closure. This study addresses a topical agent that is capable of preventing burn wound progression and accelerating burn wound healing.

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds.

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Teddy Léguillier

Full Text Available Calophyllum inophyllum L. (Calophyllaceae is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO. In this work, five CIO from Indonesia (CIO1, Tahiti (CIO2, 3, Fiji islands (CIO4 and New Caledonia (CIO5 were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results

CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.

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Dahlhoff, Maik; Gaborit, Nadège; Bultmann, Sebastian; Leonhardt, Heinrich; Yarden, Yosef; Schneider, Marlon R

2017-10-01

While the epidermal growth factor receptor (EGFR) is an established regulator of skin development and homeostasis, the functions of the related tyrosine kinase receptors ERBB2 and ERBB3 in this tissue have only recently been examined. Previously reported, skin-specific deletion of each of these receptors in mice resulted in similar defects in keratinocyte proliferation and migration, resulting in impaired wound healing and tumorigenesis. Because both ERBB2 and ERBB3 are targets for treating an array of cancer types, it is important to examine the consequences of receptor inhibition in human keratinocytes. Here, we employed the CRISPR/Cas9 technology to generate HaCaT cells (an established human keratinocyte cell line) lacking ERBB2 or ERBB3. HaCaT clones lacking ERBB2 or ERBB3 showed comparable reductions in cell proliferation as assessed by BrdU staining. Apoptosis, in contrast, was reduced in ERBB3-deficient HaCaT cells only. Assessment of cell migration using a wound healing (scratch) assay showed that the closure of the wound gaps was completed by 48 h in mock and in ERBB3 knockout clones. In contrast, this process was considerably delayed in ERBB2 knockout clones, and a complete closure of the gap in the latter cells did not occur before 72 h. In conclusion, both ERBB2 and ERBB3 are essential for normal proliferation of skin keratinocytes, but in contrast to ERBB3, ERBB2 is essential for migration of human keratinocytes. These observations might bear significance to patient adverse effects of therapeutic agents targeting ERBB2 and ERBB3. © 2017 Federation of European Biochemical Societies.

New Guar Biopolymer Silver Nanocomposites for Wound Healing Applications

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Runa Ghosh Auddy

2013-01-01

Full Text Available Wound healing is an innate physiological response that helps restore cellular and anatomic continuity of a tissue. Selective biodegradable and biocompatible polymer materials have provided useful scaffolds for wound healing and assisted cellular messaging. In the present study, guar gum, a polymeric galactomannan, was intrinsically modified to a new cationic biopolymer guar gum alkylamine (GGAA for wound healing applications. Biologically synthesized silver nanoparticles (Agnp were further impregnated in GGAA for extended evaluations in punch wound models in rodents. SEM studies showed silver nanoparticles well dispersed in the new guar matrix with a particle size of ~18 nm. In wound healing experiments, faster healing and improved cosmetic appearance were observed in the new nanobiomaterial treated group compared to commercially available silver alginate cream. The total protein, DNA, and hydroxyproline contents of the wound tissues were also significantly higher in the treated group as compared with the silver alginate cream (P<0.05. Silver nanoparticles exerted positive effects because of their antimicrobial properties. The nanobiomaterial was observed to promote wound closure by inducing proliferation and migration of the keratinocytes at the wound site. The derivatized guar gum matrix additionally provided a hydrated surface necessary for cell proliferation.

Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

International Nuclear Information System (INIS)

Zhang Yi; He Hong; Gao Wenjuan; Lu Shuangyun; Liu Yang; Gu Haiying

2009-01-01

The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.

A hyaluronic acid membrane delivery system for cultured keratinocytes: clinical "take" rates in the porcine kerato-dermal model.

Science.gov (United States)

Myers, S R; Grady, J; Soranzo, C; Sanders, R; Green, C; Leigh, I M; Navsaria, H A

1997-01-01

The clinical take rates of cultured keratinocyte autografts are poor on a full-thickness wound unless a dermal bed is provided. Even under these circumstances two important problems are the time delay in growing autografts and the fragility of the grafts. A laser-perforated hyaluronic acid membrane delivery system allows grafting at early confluence without requiring dispase digestion to release grafts from their culture dishes. We designed this study to investigate the influence of this membrane on clinical take rates in an established porcine kerato-dermal grafting model. The study demonstrated a significant reduction in take as a result of halving the keratinocyte seeding density onto the membrane. The take rates, however, of grafts grown on the membrane at half or full conventional seeding density and transplanted to a dermal wound bed were comparable, if not better, than those of keratinocyte sheet grafts.

IKKα regulates human keratinocyte migration through surveillance of the redox environment.

Science.gov (United States)

Lisse, Thomas S; Rieger, Sandra

2017-03-01

Although the functions of H 2 O 2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H 2 O 2 -dependent wound repair mechanisms. Scratch wounding led to H 2 O 2 production in two or three cell layers at the wound margin within ∼30 min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H 2 O 2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H 2 O 2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H 2 O 2 -dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK ), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells. © 2017. Published by The Company of Biologists Ltd.

Keratinocyte-derived growth factors play a role in the formation of hypertrophic scars

NARCIS (Netherlands)

Niessen, FB; Andriessen, MP; Schalkwijk, J; Visser, L; Timens, W

In predisposed individuals, wound healing can lead to hypertrophic scar or keloid formation, characterized by an overabundant extracellular matrix. It has recently been shown that hypertrophic scars are accompanied by abnormal keratinocyte differentiation and proliferation, and significantly

Komodo dragon-inspired synthetic peptide DRGN-1 promotes wound-healing of a mixed-biofilm infected wound.

Science.gov (United States)

M C Chung, Ezra; Dean, Scott N; Propst, Crystal N; Bishop, Barney M; van Hoek, Monique L

2017-01-01

Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon ( Varanus komodoensis) , called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus . DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm ( Pseudomonas aeruginosa and Staphylococcus aureus )-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial ( Pseudomonas aeruginosa & Staphylococcus aureus ) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide's ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was

The expression of proinflammatory genes in epidermal keratinocytes is regulated by hydration status.

Science.gov (United States)

Xu, Wei; Jia, Shengxian; Xie, Ping; Zhong, Aimei; Galiano, Robert D; Mustoe, Thomas A; Hong, Seok J

2014-04-01

Mucosal wounds heal more rapidly, exhibit less inflammation, and are associated with minimal scarring when compared with equivalent cutaneous wounds. We previously demonstrated that cutaneous epithelium exhibits an exaggerated response to injury compared with mucosal epithelium. We hypothesized that treatment of injured skin with a semiocclusive dressing preserves the hydration of the skin and results in a wound healing phenotype that more closely resembles that of mucosa. Here we explored whether changes in hydration status alter epidermal gene expression patterns in rabbit partial-thickness incisional wounds. Using microarray studies on injured epidermis, we showed that global gene expression patterns in highly occluded versus non-occluded wounds are distinct. Many genes including IL-1β, IL-8, TNF-α (tumor necrosis factor-α), and COX-2 (cyclooxygenase 2) are upregulated in non-occluded wounds compared with highly occluded wounds. In addition, decreased levels of hydration resulted in an increased expression of proinflammatory genes in human ex vivo skin culture (HESC) and stratified keratinocytes. Hierarchical analysis of genes using RNA interference showed that both TNF-α and IL-1β regulate the expression of IL-8 through independent pathways in response to reduced hydration. Furthermore, both gene knockdown and pharmacological inhibition studies showed that COX-2 mediates the TNF-α/IL-8 pathway by increasing the production of prostaglandin E2 (PGE2). IL-8 in turn controls the production of matrix metalloproteinase-9 in keratinocytes. Our data show that hydration status directly affects the expression of inflammatory signaling in the epidermis. The identification of genes involved in the epithelial hydration pathway provides an opportunity to develop strategies to reduce scarring and optimize wound healing.

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

Directory of Open Access Journals (Sweden)

Stefanie Löffek

Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

Science.gov (United States)

Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

2016-02-01

Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization*s

Science.gov (United States)

Sehgal, Bernd U.; DeBiase, Phillip J.; Matzno, Sumio; Chew, Teng-Leong; Claiborne, Jessica N.; Hopkinson, Susan B.; Russell, Alan; Marinkovich, M. Peter; Jones, Jonathan C. R.

2010-01-01

Whether α6β4 integrin regulates migration remains controversial. β4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express β4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type β4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with α6β4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the α6β4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells. PMID:16973601

Gallic Acid Promotes Wound Healing in Normal and Hyperglucidic Conditions

Directory of Open Access Journals (Sweden)

Dong Joo Yang

2016-07-01

Full Text Available Skin is the outermost layer of the human body that is constantly exposed to environmental stressors, such as UV radiation and toxic chemicals, and is susceptible to mechanical wounding and injury. The ability of the skin to repair injuries is paramount for survival and it is disrupted in a spectrum of disorders leading to skin pathologies. Diabetic patients often suffer from chronic, impaired wound healing, which facilitate bacterial infections and necessitate amputation. Here, we studied the effects of gallic acid (GA, 3,4,5-trihydroxybenzoic acid; a plant-derived polyphenolic compound on would healing in normal and hyperglucidic conditions, to mimic diabetes, in human keratinocytes and fibroblasts. Our study reveals that GA is a potential antioxidant that directly upregulates the expression of antioxidant genes. In addition, GA accelerated cell migration of keratinocytes and fibroblasts in both normal and hyperglucidic conditions. Further, GA treatment activated factors known to be hallmarks of wound healing, such as focal adhesion kinases (FAK, c-Jun N-terminal kinases (JNK, and extracellular signal-regulated kinases (Erk, underpinning the beneficial role of GA in wound repair. Therefore, our results demonstrate that GA might be a viable wound healing agent and a potential intervention to treat wounds resulting from metabolic complications.

Wound Care Center of Excellence: A Process for Continuous Monitoring and Improvement of Wound Care Quality.

Science.gov (United States)

Howell, Raelina S; Kohan, Lauren S; Woods, Jon S; Criscitelli, Theresa; Gillette, Brian M; Donovan, Virginia; Gorenstein, Scott

2018-05-01

To provide information about a study using a new process for continuous monitoring to improve chronic wound care quality.This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care.After completing this continuing education activity, you should be better able to:1. Recognize problems associated with chronic wound care.2. Identify methods used in this project to improve care.3. Illustrate the findings from this and similar projects and implications for providing improved wound care.Patients with chronic wounds require complex care because of comorbidities that can affect healing. Therefore, the goal of this project was to develop a system of reviewing all hospitalized patients seen by the study authors' wound care service on a weekly basis to decrease readmissions, morbidity, and mortality. Weekly multidisciplinary conferences were conducted to evaluate patient data and systematically assess for adherence to wound care protocols, as well as to create and modify patient care plans. This review of pathology and the performance of root-cause analyses often led to improved patient care.

Intermittent pressure decreases human keratinocyte proliferation in vitro.

Science.gov (United States)

Nasca, Maria R; Shih, Alan T; West, Dennis P; Martinez, Wanda M; Micali, Giuseppe; Landsman, Adam S

2007-01-01

The aim of this study was to investigate the correlation between pressure changes and keratinocyte proliferation by determining whether keratinocytes exposed to altered mechanical pressures would proliferate at different rates compared to control cells not subjected to pressure changes. Tissue culture flasks of human keratinocytes plated at an approximate density of 15,000 cells/cm(2) undergoing an intermittent cyclic pressure of 362 mm Hg at a frequency of 2.28 or 5.16 cycles/min (0.038 or 0.086 Hz) for 8 h were compared to control flasks grown at ambient room pressure. An in-line pressure transducer was used to monitor and adjust pressure within the cell chambers, using a solenoid valve. A thymidine incorporation assay assessed the amount of cell proliferation in each set of experiments. Differences in proliferation between keratinocytes subjected to cyclic pressure changes and control cells were found to be statistically significant (p < 0.05) in 4 out of 5 proliferation assays. Also, a higher frequency of pressure changes consistently generated a reduced proliferation rate compared to that seen in cells exposed to a lower frequency of pressure changes. These data indicate that keratinocytes undergoing intermittent pressure changes exhibit decreased proliferation rates compared to controls. Furthermore, an increased frequency rate seems to have a greater effect on proliferation than low-frequency rate pressure changes, suggesting that the stress caused by frequently changed pressure may play a greater role in reducing keratinocyte proliferation than the actual magnitude of load applied to the cells. Our results support the current treatment protocol of reducing speed and duration of walking on the site of the wound to promote healing of foot ulcers. (c) 2007 S. Karger AG, Basel.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

OpenAIRE

Andreas Bayer; Justus Lammel; Mersedeh Tohidnezhad; Sebastian Lippross; Peter Behrendt; Tim Klüter; Thomas Pufe; Jochen Cremer; Holger Jahr; Franziska Rademacher; Regine Gläser; Jürgen Harder

2017-01-01

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF?)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting acti...

Prevention of burn wound conversion by allogeneic keratinocytes cultured on acellular xenodermis

Czech Academy of Sciences Publication Activity Database

Matoušková, Eva; Brož, L.; Pokorná, Eva; Königová, R.

2002-01-01

Roč. 3, č. 1 (2002), s. 29-35 ISSN 1389-9333 Institutional research plan: CEZ:AV0Z5052915 Keywords : human keratinocytes * tissue engineered skin * dried porcine dermis Subject RIV: EB - Genetics ; Molecular Biology

Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds.

Science.gov (United States)

Galeano, Mariarosaria; Altavilla, Domenica; Bitto, Alessandra; Minutoli, Letteria; Calò, Margherita; Lo Cascio, Patrizia; Polito, Francesca; Giugliano, Giovanni; Squadrito, Giovanni; Mioni, Chiara; Giuliani, Daniela; Venuti, Francesco S; Squadrito, Francesco

2006-04-01

Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Randomized experiment. Research laboratory. C57BL/6 male mice weighing 25-30 g. Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 microL subcutaneously) or its vehicle alone (100 microl/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

In Vivo Assessment of Printed Microvasculature in a Bilayer Skin Graft to Treat Full-Thickness Wounds

Science.gov (United States)

Yanez, Maria; Rincon, Julio; Dones, Aracely; De Maria, Carmelo; Gonzales, Raoul

2015-01-01

Chronic wounds such as diabetic foot ulcers and venous leg ulcers are common problems in people suffering from type 2 diabetes. These can cause pain, and nerve damage, eventually leading to foot or leg amputation. These types of wounds are very difficult to treat and sometimes take months or even years to heal because of many possible complications during the process. Allogeneic skin grafting has been used to improve wound healing, but the majority of grafts do not survive several days after being implanted. We have been studying the behavior of fibroblasts and keratinocytes in engineered capillary-like endothelial networks. A dermo-epidermal graft has been implanted in an athymic nude mouse model to assess the integration with the host tissue as well as the wound healing process. To build these networks into a skin graft, a modified inkjet printer was used, which allowed the deposit of human microvascular endothelial cells. Neonatal human dermal fibroblast cells and neonatal human epidermal keratinocytes were manually mixed in the collagen matrix while endothelial cells printed. A full-thickness wound was created at the top of the back of athymic nude mice and the area was covered by the bilayered graft. Mice of the different groups were followed until completion of the specified experimental time line, at which time the animals were humanely euthanized and tissue samples were collected. Wound contraction improved by up to 10% when compared with the control groups. Histological analysis showed the neoskin having similar appearance to the normal skin. Both layers, dermis and epidermis, were present with thicknesses resembling normal skin. Immunohistochemistry analysis showed favorable results proving survival of the implanted cells, and confocal images showed the human cells' location in the samples that were collocated with the bilayer printed skin graft. PMID:25051339

Tilapia Piscidin 4 (TP4 Stimulates Cell Proliferation and Wound Closure in MRSA-Infected Wounds in Mice

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Hang-Ning Huang

2015-05-01

Full Text Available Antimicrobial peptides (AMPs are endogenous antibiotics that directly affect microorganisms, and also have a variety of receptor-mediated functions. One such AMP, Tilapia piscidin 4 (TP4, was isolated from Nile tilapia (Oreochromis niloticus; TP4 has antibacterial effects and regulates the innate immune system. The aim of the present study was to characterize the role of TP4 in the regulation of wound closure in mice and proliferation of a keratinocyte cell line (HaCaT and fibroblast cell line (Hs-68. In vitro, TP4 stimulated cell proliferation and activated collagen I, collagen III, and keratinocyte growth factor (KGF gene expression in Hs-68 cells, which induces keratin production by HaCaT cells. This effect was detectable at TP4 concentrations of 6.25 µg/mL in both cell lines. In vivo, TP4 was found to be highly effective at combating peritonitis and wound infection caused by MRSA in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that TP4 enhances the survival rate of mice infected with the bacterial pathogen MRSA through both antimicrobial and wound closure activities mediated by epidermal growth factor (EGF, transforming growth factor (TGF, and vascular endothelial growth factor (VEGF. The peptide is likely involved in antibacterial processes and regulation of tissue homeostasis in infected wounds in mice. Overall, these results suggest that TP4 may be suitable for development as a novel topical agent for wound dressing.

Toxicity of silver nanoparticles in monocytes and keratinocytes

DEFF Research Database (Denmark)

Orłowski, Piotr; Krzyzowska, Malgorzata; Winnicka, Anna

2012-01-01

Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The possible local inflammatory response to silver nanoparticles and the role of cell death in determining...... these effects are largely unknown. Effects of the mixture of silver nanoparticles of different sizes were compared in in vitro assays for cytotoxicity, caspase-1 and caspase-9 activity and bax expression. In all tested concentrations, silver nanoparticles were more toxic for RAW 264.7 monocytes than for 291.03C...... keratinocytes and induced significant caspase-1 activity and necrotic cell death. In keratinocytes, more significantly than in macrophages, silver nanoparticles led to increase of caspase-9 activity and apoptosis. These results indicate that effects of silver nanoparticles depend on the type of exposed cells...

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

OpenAIRE

Bayer, Andreas; Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF?) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiatio...

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

International Nuclear Information System (INIS)

Zhang Yueqing; Hu Xiaobo; Tian Ruiyang; Wei Wangui; Hu Wei; Chen Xia; Han Wei; Chen Huayou; Gong Yi

2006-01-01

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the α v -containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism

Role of adipose-derived stem cells in wound healing.

Science.gov (United States)

Hassan, Waqar Ul; Greiser, Udo; Wang, Wenxin

2014-01-01

Impaired wound healing remains a challenge to date and causes debilitating effects with tremendous suffering. Recent advances in tissue engineering approaches in the area of cell therapy have provided promising treatment options to meet the challenges of impaired skin wound healing such as diabetic foot ulcers. Over the last few years, stem cell therapy has emerged as a novel therapeutic approach for various diseases including wound repair and tissue regeneration. Several different types of stem cells have been studied in both preclinical and clinical settings such as bone marrow-derived stem cells, adipose-derived stem cells (ASCs), circulating angiogenic cells (e.g., endothelial progenitor cells), human dermal fibroblasts, and keratinocytes for wound healing. Adipose tissue is an abundant source of mesenchymal stem cells, which have shown an improved outcome in wound healing studies. ASCs are pluripotent stem cells with the ability to differentiate into different lineages and to secrete paracrine factors initiating tissue regeneration process. The abundant supply of fat tissue, ease of isolation, extensive proliferative capacities ex vivo, and their ability to secrete pro-angiogenic growth factors make them an ideal cell type to use in therapies for the treatment of nonhealing wounds. In this review, we look at the pathogenesis of chronic wounds, role of stem cells in wound healing, and more specifically look at the role of ASCs, their mechanism of action and their safety profile in wound repair and tissue regeneration. © 2014 by the Wound Healing Society.

Novel Anti-Melanogenesis Properties of Polydeoxyribonucleotide, a Popular Wound Healing Booster

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Tai Kyung Noh

2016-09-01

Full Text Available Polydeoxyribonucleotide (PDRN, a deoxyribonucleotide polymer, is popularly used for faster healing of cutaneous wounds and boosting of neocollagenesis of photoaged skin among current dermatologic practitioners. Some patients receiving PDRN injection treatment also reported improvement of photoaging-associated mottled pigmentation (PMP. To investigate the effect of PDRN on cutaneous melanogenesis, we examined the effect of PDRN and an available product (Placentex® containing PDRN on melanogenesis using human melanocytes-keratinocytes cocultures and mouse melanocytes. Melanin content, tyrosinase activity, and levels of microphthalmia-associated transcription factor (MITF, tyrosinase, and tyrosinase-related protein (TRP-1 were determined. Intracellular signaling pathways were assessed by Western blotting. PDRN and Placentex® led to decreases in melanin content, tyrosinase activity, and MITF and TRP-1 expression with concomitant increases in phosphorylated forms of extracellular signal-regulated protein kinase (ERK and AKT in mouse melanocytes. More importantly, both PDRN and Placentex® significantly suppressed the melanin content in human melanocyte–keratinocyte cocultures. Clinical evaluation of six female patients with facial hyperpigmentation after three sessions of intradermal PDRN injections using a 5-point scale revealed that PDRN led to more than noticeable improvements in hyperpigmented lesions. This is the first study to demonstrate that PDRN, which is known for its wound-healing properties, may have novel anti-melanogenesis and potential skin whitening properties.

Enhanced secretion of TIMP-1 by human hypertrophic scar keratinocytes could contribute to fibrosis.

Science.gov (United States)

Simon, Franck; Bergeron, Daniele; Larochelle, Sébastien; Lopez-Vallé, Carlos A; Genest, Hervé; Armour, Alexis; Moulin, Véronique J

2012-05-01

Hypertrophic scars are a pathological process characterized by an excessive deposition of extracellular matrix components. Using a tissue-engineered reconstructed human skin (RHS) method, we previously reported that pathological keratinocytes induce formation of a fibrotic dermal matrix. We further investigated keratinocyte action using conditioned media. Results showed that conditioned media induce a similar action on dermal thickness similar to when an epidermis is present. Using a two-dimensional electrophoresis technique, we then compared conditioned media from normal or hypertrophic scar keratinocytes and determined that TIMP-1 was increased in conditioned media from hypertrophic scar keratinocytes. This differential profile was confirmed using ELISA, assaying TIMP-1 presence on media from monolayer cultured keratinocytes and from RHS. The dermal matrix of these RHS was recreated using mesenchymal cells from three different origins (skin, wound and hypertrophic scar). The effect of increased TIMP-1 levels on dermal fibrosis was also validated independently from the mesenchymal cell origin. Immunodetection of TIMP-1 showed that this protein was increased in the epidermis of hypertrophic scar biopsies. The findings of this study represent an important advance in understanding the role of keratinocytes as a direct potent modulator for matrix degradation and scar tissue remodeling, possibly through inactivation of MMPs. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

Secretion of wound healing mediators by single and bi-layer skin substitutes.

Science.gov (United States)

Maarof, Manira; Law, Jia Xian; Chowdhury, Shiplu Roy; Khairoji, Khairul Anuar; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

2016-10-01

Limitations of current treatments for skin loss caused by major injuries leads to the use of skin substitutes. It is assumed that secretion of wound healing mediators by these skin substitutes plays a role in treating skin loss. In our previous study, single layer keratinocytes (SK), single layer fibroblast (SF) and bilayer (BL; containing keratinocytes and fibroblasts layers) skin substitutes were fabricated using fibrin that had shown potential to heal wounds in preclinical studies. This study aimed to quantify the secretion of wound healing mediators, and compare between single and bi-layer skin substitutes. Skin samples were digested to harvest fibroblasts and keratinocytes, and expanded to obtain sufficient cells for the construction of skin substitutes. Acellular fibrin (AF) construct was used as control. Substitutes i.e. AF, SK, SF and BL were cultured for 2 days, and culture supernatant was collected to analyze secretion of wound healing mediators via multiplex ELISA. Among 19 wound healing mediators tested, BL substitute secreted significantly higher amounts of CXCL1 and GCSF compared to SF and AF substitute but this was not significant with respect to SK substitute. The BL substitute also secreted significantly higher amounts of CXCL5 and IL-6 compared to other substitutes. In contrast, the SK substitute secreted significantly higher amounts of VCAM-1 compared to other substitutes. However, all three skin substitutes also secreted CCL2, CCL5, CCL11, GM-CSF, IL8, IL-1α, TNF-α, ICAM-1, FGF-β, TGF-β, HGF, VEGF-α and PDGF-BB factors, but no significant difference was seen. Secretion of these mediators after transplantation may play a significant role in promoting wound healing process for the treatment of skin loss.

Tissue-Engineered Skin Substitute Enhances Wound Healing after Radiation Therapy.

Science.gov (United States)

Busra, Mohd Fauzi bin Mh; Chowdhury, Shiplu Roy; bin Ismail, Fuad; bin Saim, Aminuddin; Idrus, Ruszymah Bt Hj

2016-03-01

When given in conjunction with surgery for treating cancer, radiation therapy may result in impaired wound healing, which, in turn, could cause skin ulcers. In this study, bilayer and monolayer autologous skin substitutes were used to treat an irradiated wound. A single dose of 30 Gy of linear electron beam radiation was applied to the hind limb of nude mice before creating the skin lesion (area of 78.6 mm). Monolayer tissue-engineered skin substitutes (MTESSs) were prepared by entrapping cultured keratinocytes in fibrin matrix, and bilayer tissue-engineered skin substitutes (BTESSs) were prepared by entrapping keratinocytes and fibroblasts in separate layers. Bilayer tissue-engineered skin substitute and MTESS were implanted to the wound area. Gross appearance and wound area were analyzed to evaluate wound healing efficiency. Skin regeneration and morphological appearance were observed via histological and electron microscopy. Protein expressions of transforming growth factor β1 (TGF-β1), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor (VEGF) in skin regeneration were evaluated by immunohistochemistry (IHC). Macroscopic observation revealed that at day 13, treatments with BTESS completely healed the irradiated wound, whereas wound sizes of 1.1 ± 0.05 and 6.8 ± 0.14 mm were measured in the MTESS-treated and untreated control groups, respectively. Hematoxylin-eosin (H&E) analysis showed formation of compact and organized epidermal and dermal layers in the BTESS-treated group, as compared with MTESS-treated and untreated control groups. Ultrastructural analysis indicates maturation of skin in BTESS-treated wound evidenced by formation of intermediate filament bundles in the dermal layer and low intercellular space in the epidermal layer. Expressions of TGF-β1, PDGF-BB, and VEGF were also higher in BTESS-treated wounds, compared with MTESS-treated wounds. These results indicate that BTESS is the preferred treatment for

Induction of PDGF-B in TCA-treated epidermal keratinocytes.

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Yonei, Nozomi; Kanazawa, Nobuo; Ohtani, Toshio; Furukawa, Fukumi; Yamamoto, Yuki

2007-11-01

Trichloroacetic acid (TCA) is one of the most widely used peeling agents, and induces full necrosis of the whole epidermis, followed by reconstitution of the epidermis and the matrix of the papillary dermis. The cytotoxic effects of TCA, such as suppressing proliferation of keratinocytes and fibroblasts and protein synthesis by fibroblasts, have already been reported. However, the entire biological mechanism responsible for TCA peeling has yet to be determined. Hypothetical activation effects of TCA treatment on epidermal cells to induce production of growth factors and cytokines are examined, and are compared with its cytotoxic effects in terms of time course and applied TCA concentrations. After various periods of incubation with TCA, viability of Pam212 murine keratinocytes was investigated with MTT assay and dye exclusion assay, and production of growth factors and cytokines with reverse transcription-polymerase chain reaction (RT-PCR). Changes in platelet-derived growth factor (PDGF)-B mRNA expression and protein production in the human skin specimens after TCA application were then examined by RT-PCR and immunohistochemistry, respectively. Incubation with TCA showed cytotoxicity and induced death of Pam212 cells, depending on the incubation period and the TCA concentration. In addition, expressions of PDGF-B, tumor growth factor (TGF)-alpha, TGF- beta1 and vascular endothelial growth factor, which are the growth factors reportedly secreted from keratinocytes during wound healing, were all detected in Pam212 cells after short-term treatment with TCA. Expressions of inflammatory cytokines such as interleukin (IL)-1 and IL-10 were also induced. In TCA-treated NIH-3T3 fibroblasts, in contrast, observed was upregulation of only keratinocyte growth factor, which is reportedly secreted from fibroblasts, as well as the similar cytotoxic effect. In human skin, PDGF-B mRNA expression became significantly upregulated after TCA application, and then immediately

Oral keratinocyte stem/progenitor cells: specific markers, molecular signaling pathways and potential uses.

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Calenic, Bogdan; Greabu, Maria; Caruntu, Constantin; Tanase, Cristiana; Battino, Maurizio

2015-10-01

Oral keratinocyte stem cells reside in the basal layers of the oral epithelium, representing a minor population of cells with a great potential to self-renew and proliferate over the course of their lifetime. As a result of the potential uses of oral keratinocyte stem cells in regenerative medicine and the key roles they play in tissue homeostasis, inflammatory conditions, wound healing and tumor initiation and progression, intense scientific efforts are currently being undertaken to identify, separate and reprogram these cells. Although currently there is no specific marker that can characterize and isolate oral keratinocyte stem cells, several suggestions have been made. Thus, different stem/progenitor-cell subpopulations have been categorized based on combinations of positive and/or negative membrane-surface markers, which include integrins, clusters of differentiation and cytokeratins. Important advances have also been made in understanding the molecular pathways that govern processes such as self-renewal, differentiation, proliferation, wound healing and programmed cell death. A thorough understanding of stem-cell biology and the molecular players that govern cellular fate is paramount in the quest for using stem-cell-derived therapies in the treatment of various oral pathologies. The current review focuses on recent advances in understanding the molecular signaling pathways coordinating the behavior of these cells and in identifying suitable markers used for their isolation and characterization. Special emphasis will also be placed on the roles played by oral keratinocyte stem and progenitor cells in normal and diseased oral tissues and on their potential uses in the fields of general medicine and dentistry. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Phenotypic overlap between MMP-13 and the plasminogen activation system during wound healing in mice

DEFF Research Database (Denmark)

Juncker-Jensen, Anna; Lund, Leif R

2011-01-01

combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases....

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

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Di Grazia, Antonio; Cappiello, Floriana; Imanishi, Akiko; Mastrofrancesco, Arianna; Picardo, Mauro; Paus, Ralf; Mangoni, Maria Luisa

2015-01-01

One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs) produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21)NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells) over a wide range of peptide concentrations (0.025-4 μM), and this notably more efficiently than human cathelicidin (LL-37). This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21)NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21)NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

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Antonio Di Grazia

Full Text Available One of the many functions of skin is to protect the organism against a wide range of pathogens. Antimicrobial peptides (AMPs produced by the skin epithelium provide an effective chemical shield against microbial pathogens. However, whereas antibacterial/antifungal activities of AMPs have been extensively characterized, much less is known regarding their wound healing-modulatory properties. By using an in vitro re-epithelialisation assay employing special cell-culture inserts, we detected that a derivative of the frog-skin AMP esculentin-1a, named esculentin-1a(1-21NH2, significantly stimulates migration of immortalized human keratinocytes (HaCaT cells over a wide range of peptide concentrations (0.025-4 μM, and this notably more efficiently than human cathelicidin (LL-37. This activity is preserved in primary human epidermal keratinocytes. By using appropriate inhibitors and an enzyme-linked immunosorbent assay we found that the peptide-induced cell migration involves activation of the epidermal growth factor receptor and STAT3 protein. These results suggest that esculentin-1a(1-21NH2 now deserves to be tested in standard wound healing assays as a novel candidate promoter of skin re-epithelialisation. The established ability of esculentin-1a(1-21NH2 to kill microbes without harming mammalian cells, namely its high anti-Pseudomonal activity, makes this AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas-infected, skin ulcers.

PLGA nanoparticles loaded with host defense peptide LL37 promote wound healing.

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Chereddy, Kiran Kumar; Her, Charles-Henry; Comune, Michela; Moia, Claudia; Lopes, Alessandra; Porporato, Paolo E; Vanacker, Julie; Lam, Martin C; Steinstraesser, Lars; Sonveaux, Pierre; Zhu, Huijun; Ferreira, Lino S; Vandermeulen, Gaëlle; Préat, Véronique

2014-11-28

Wound treatment remains one of the most prevalent and economically burdensome healthcare issues in the world. Poly (lactic-co-glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. LL37 is an endogenous human host defense peptide that modulates wound healing and angiogenesis and fights infection. Hence, we hypothesized that the administration of LL37 encapsulated in PLGA nanoparticles (PLGA-LL37 NP) promotes wound closure due to the sustained release of both LL37 and lactate. In full thickness excisional wounds, the treatment with PLGA-LL37 NP significantly accelerated wound healing compared to PLGA or LL37 administration alone. PLGA-LL37 NP-treated wounds displayed advanced granulation tissue formation by significant higher collagen deposition, re-epithelialized and neovascularized composition. PLGA-LL37 NP improved angiogenesis, significantly up-regulated IL-6 and VEGFa expression, and modulated the inflammatory wound response. In vitro, PLGA-LL37 NP induced enhanced cell migration but had no effect on the metabolism and proliferation of keratinocytes. It displayed antimicrobial activity on Escherichia coli. In conclusion, we developed a biodegradable drug delivery system that accelerated healing processes due to the combined effects of lactate and LL37 released from the nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Natural and synthetic polymers for wounds and burns dressing.

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Mogoşanu, George Dan; Grumezescu, Alexandru Mihai

2014-03-25

In the last years, health care professionals faced with an increasing number of patients suffering from wounds and burns difficult to treat and heal. During the wound healing process, the dressing protects the injury and contributes to the recovery of dermal and epidermal tissues. Because their biocompatibility, biodegradability and similarity to macromolecules recognized by the human body, some natural polymers such as polysaccharides (alginates, chitin, chitosan, heparin, chondroitin), proteoglycans and proteins (collagen, gelatin, fibrin, keratin, silk fibroin, eggshell membrane) are extensively used in wounds and burns management. Obtained by electrospinning technique, some synthetic polymers like biomimetic extracellular matrix micro/nanoscale fibers based on polyglycolic acid, polylactic acid, polyacrylic acid, poly-ɛ-caprolactone, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, exhibit in vivo and in vitro wound healing properties and enhance re-epithelialization. They provide an optimal microenvironment for cell proliferation, migration and differentiation, due to their biocompatibility, biodegradability, peculiar structure and good mechanical properties. Thus, synthetic polymers are used also in regenerative medicine for cartilage, bone, vascular, nerve and ligament repair and restoration. Biocompatible with fibroblasts and keratinocytes, tissue engineered skin is indicated for regeneration and remodeling of human epidermis and wound healing improving the treatment of severe skin defects or partial-thickness burn injuries. Copyright © 2013 Elsevier B.V. All rights reserved.

[A preliminary study of anti-aging and wound healing of recombination cytoglobin].

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Li, Zhao-Fa; Zhao, Xiao-Fang; Zhang, Ting-Ting

2012-01-01

In this paper, the preliminary study on antioxidant, enhancement of antioxidant enzymes activity, reducing the content of oxygen free radicals, delaying skin aging of the recombination cytoglobin (rCygb) purified by our lab were investigated through human keratinocyte cell line (HaCAT) H2O2 oxidative stress model, mouse skin aging model caused by continuous subcutaneous injection D-gal, rat acute liver injury model induced by CCl4 and rat skin wound healing model. The results showed that rCygb improved the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), reduced the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) as well as decreased the content of malondialdehyde (MDA). Skin biopsy showed that rCygb promoted angiogenesis, increased expression of collagen and improved the anti-inflammatory ability. All results displayed that rCygb improved the oxygen free radical scavenging ability, delayed skin aging and promoted wound healing.

Neutrophil depletion in the early inflammatory phase delayed cutaneous wound healing in older rats: improvements due to the use of un-denatured camel whey protein.

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Ebaid, Hossam

2014-03-04

While it is known that advanced age alters the recruitment of neutrophils during wound healing, thereby delaying the wound healing process, little is known about prolonged wound healing in advanced ages. Thus, we investigated the correlation of neutrophil recruitment with healing events, and the impact of whey protein (WP) on neutrophil activation. The animals were allocated into wounded young group, wounded older group and wounded older rats with daily treatment of WP at a dose of 100 mg/kg of body weight. Our results pointed to a marked deficiency in the number of neutrophils in the wounds of older rats, which was accompanied with impairment of the healing process. In the group of older rats, phagocytic activity, as tested by fluorescence microscopy, declined throughout the first 24 hours after wounding. Both the neutrophil number and the phagocytic activity recovered in older rats which received WP supplementation. Interestingly, WP was found to significantly up-regulate the MIP-1α and CINC-1 mRNA expression in old rats. On the other hand, the wound size in older rats was significantly higher than that in younger ones. Blood angiogenesis was also significantly delayed in the older group as opposed to the young rats. WP, however, was found to return these indices to normal levels in the older rats. Proliferation and epidermal migration of the keratinocytes and the collagen deposition were also returned to the normal rates. This data confirms the critical role of neutrophil recruitment in the early inflammatory phase of wound healing in older rats. In addition, WP protein was used to improve neutrophil function in older rats, healing events returned to a more normal profile. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2100966986117779.

Wound healing properties of jojoba liquid wax: an in vitro study.

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Ranzato, Elia; Martinotti, Simona; Burlando, Bruno

2011-03-24

The wound healing properties of jojoba (Simmondsia chinensis) liquid wax (JLW) were studied in vitro on HaCaT keratinocytes and human dermal fibroblasts, which are involved in wounded skin repair. JLW cytotoxicity was evaluated by the crystal violet staining and the neutral red uptake endpoint. Induction of wound healing by JLW was assessed by scratch wound assay on cell monolayers. The involvement of signaling pathways was evaluated by the use of the Ca(2+) chelator BAPTA and of kinase inhibitors, and by Western blot analysis of cell lysates using anti-phospho antibodies. Collagen and gelatinase secretion by cells were assayed by in-cell ELISA and zymography analysis, respectively. Cytotoxicity assays showed that the toxic effects of JLW to these cells are extremely low. Scratch wound experiments showed that JLW notably accelerates the wound closure of both keratinocytes and fibroblasts. The use of inhibitors and Western blot revealed that the mechanism of action of JLW is strictly Ca(2+) dependent and requires the involvement of the PI3K-Akt-mTOR pathway and of the p38 and ERK1/2 MAPKs. In addition, JLW was found to stimulate collagen I synthesis in fibroblasts, while no effect was detected on the secretion of MMP-2 and MMP-9 gelatinases by HaCaT or fibroblasts. Taken together, data provide a pharmacological characterization of JLW properties on skin cells and suggest that it could be used in the treatment of wounds in clinical settings. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Culture technique of rabbit primary epidermal keratinocytes

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Marini M

2012-10-01

Full Text Available The epidermis is the protective covering outer layer of the mammalian skin. The epidermal cells are stratified squamous epithelia which undergo continuous differentiation of loss and replacement of cells. Ninety per cent of epidermal cells consist of keratinocytes that are found in the basal layer of the stratified epithelium called epidermis. Keratinocytes are responsible for forming tight junctions with the nerves of the skin as well as in the process of wound healing. This article highlights the method of isolation and culture of rabbit primary epidermal keratinocytes in vitro. Approximately 2cm x 2cm oval shaped line was drawn on the dorsum of the rabbit to mark the surgical area. Then, the skin was carefully excised using a surgical blade and the target skin specimens harvested from the rabbits were placed in transport medium comprising of Dulbecco’s Modified Eagle Medium (DMEM and 1% of antibiotic-antimycotic solution. The specimens were transferred into a petri dish containing 70% ethanol and washed for 5 min followed by a wash in 1 x Dulbecco’s Phosphate Buffered Saline (DBPS. Then, the skin specimens were placed in DMEM and minced into small pieces using a scalpel. The minced pieces were placed in a centrifuge tube containing 0.6% Dispase and 1% antibiotic-antimycotic solution overnight at 4°C in a horizontal orientation. The epidermis layer (whitish, semi-transparent was separated from the dermis (pink, opaque, gooey with the aid of curved forceps by fixing the dermis with one pair of forceps while detaching the epidermis with the second pair. The cells were cultured at a density of 4 x 104 cells/cm2 in culture flask at 37°C and 5% CO2. The cell morphology of the keratinocytes was analyzed using inverted microscope.

Frozen cultured sheets of epidermal keratinocytes in reepithelialization and repair of the cornea after photorefractive keratectomy.

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Castro-Muñozledo, Federico; Ozorno-Zarate, Jorge; Naranjo-Tackman, Ramon; Kuri-Harcuch, Walid

2002-09-01

To determine whether frozen cultured sheets of human allogeneic epidermal keratinocytes (CEAK) improved wound repair after experimental corneal ablation by photorefractive keratectomy (PRK). Hospital "Luis Sanchez Bulnes" de la Asociación para Evitar la Ceguera en Mexico, I.A.P, and Department of Cell Biology, CINVESTAV-IPN, Mexico City, Mexico. Transepithelial PRK was performed in the right eye of male albino rabbits to obtain a 112 microm deep and 6.0 mm diameter ablation zone. In 17 eyes, the ablations were covered with frozen CEAK; in 11 eyes, the ablations were covered with a disposable contact lens without the cultured sheets; and in the control group (13 eyes), the ablations were not covered. Subepithelial fibrosis and reepithelialization of the ablated zone were evaluated in serial paraffin-embedded tissue sections from all wounds. Treatment with CEAK reduced fibroblast proliferation and the inflammatory response beneath the ablated zone and produced better organization of the newly formed epithelium by eliminating significant hyperplasia or discontinuities in the periodic acid Shiff-stained basement membrane. It also led to accelerated reepithelialization. The use of frozen CEAK as a biologically active wound dressing improved tissue repair at 1 month in corneas ablated by transepithelial PRK in the male albino rabbit model. Treatment with CEAK could improve the outcome of PRK in humans.

Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator

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Fibbi, G.; Magnelli, L.; Pucci, M.; Del Rosso, M. (Florence Univ. (Italy))

1990-03-01

On the basis of a fibrinolytic assay with {sup 125}I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, the authors have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, they hypothesize that this mechanism may be important in vivo during the process of wound repair.

Stem Cell Therapy to Improve Burn Wound Healing

Science.gov (United States)

2017-03-01

Award Number: W81XWH-13-2-0024 TITLE: Stem Cell Therapy to Improve Burn Wound Healing PRINCIPAL INVESTIGATOR: Carl Schulman, MD, PhD, MSPH...NUMBER Stem Cell Therapy to Improve Burn Wound Healing 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Carl Schulman, MD, PhD, MSPH...treatments, steroid injections, and compression garments. Mesenchymal stem cells (MSC’s) have been used in a variety of clinical applications to repair

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

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Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

2017-10-01

In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Non-invasive in vivo characterization of skin wound healing using label-free multiphoton microscopy (Conference Presentation)

Science.gov (United States)

Jones, Jake D.; Majid, Fariah; Ramser, Hallie; Quinn, Kyle P.

2017-02-01

Non-healing ulcerative wounds, such as diabetic foot ulcers, are challenging to diagnose and treat due to their numerous possible etiologies and the variable efficacy of advanced wound care products. Thus, there is a critical need to develop new quantitative biomarkers and diagnostic technologies that are sensitive to wound status in order to guide care. The objective of this study was to evaluate the utility of label-free multiphoton microscopy for characterizing wound healing dynamics in vivo and identifying potential differences in diabetic wounds. We isolated and measured an optical redox ratio of FAD/(NADH+FAD) autofluorescence to provide three-dimensional maps of local cellular metabolism. Using a mouse model of wound healing, in vivo imaging at the wound edge identified a significant decrease in the optical redox ratio of the epidermis (p≤0.0103) between Days 3 through 14 compared to Day 1. This decrease in redox ratio coincided with a decrease in NADH fluorescence lifetime and thickening of the epithelium, collectively suggesting a sensitivity to keratinocyte hyperproliferation. In contrast to normal wounds, we have found that keratinocytes from diabetic wounds remain in a proliferative state at later time points with a lower redox ratio at the wound edge. Microstructural organization and composition was also measured from second harmonic generation imaging of collagen and revealed differences between diabetic and non-diabetic wounds. Our work demonstrates label-free multiphoton microscopy offers potential to provide non-invasive structural and functional biomarkers associated with different stages of skin wound healing, which may be used to detect delayed healing and guide treatment.

Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

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Yasuaki Kobayashi

Full Text Available Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

In vivo relative quantitative proteomics reveals HMGB1 as a downstream mediator of oestrogen-stimulated keratinocyte migration.

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Shin, Jung U; Noh, Ji Yeon; Lee, Ju Hee; Lee, Won Jai; Yoo, Jong Shin; Kim, Jin Young; Kim, Hyeran; Jung, Inhee; Jin, Shan; Lee, Kwang Hoon

2015-06-01

It is known that oestrogen influences skin wound healing by modulating the inflammatory response, cytokine expression and extracellular matrix deposition; accelerating re-epithelialization; and stimulating angiogenesis. To identify novel proteins associated with effects of oestrogen on keratinocyte, stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry was performed. Using SILAC, quantification of 1085 proteins was achieved. Among these proteins, 60 proteins were upregulated and 32 proteins were downregulated. Among significantly upregulated proteins, high-mobility group protein B1 (HMGB1) has been further evaluated for its role in the effect of oestrogen on keratinocytes. HMGB1 expression was strongly induced in oestrogen-treated keratinocytes in dose- and time-dependent manner. Further, HMGB1 was able to significantly accelerate the rate of HaCaT cell migration. To determine whether HMGB1 is involved in E2-induced HaCaT cell migration, cells were transfected with HMGB1 siRNA. Knockdown of HMGB1 blocked oestrogen-induced keratinocyte migration. Collectively, these experiments demonstrate that HMGB1 is a novel downstream mediator of oestrogen-stimulated keratinocyte migration. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

In vitro cytotoxity of silver: implication for clinical wound care.

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Poon, Vincent K M; Burd, Andrew

2004-03-01

In this study, we look at the cytotoxic effects of silver on keratinocytes and fibroblasts. We have assessed the viability of monolayer cultures using the MTT and BrdU assays. The composition of the culture medium and also the culture technique were modified to assess the effects of culture 'environment' on the susceptibility of the cells to the toxic action of silver. Further in vitro, experiments were performed using tissue culture models to allow cellular behavior in three dimensional planes which more closely simulated in vivo behavior. The silver source was both silver released from silver nitrate solution but also nanocrystalline silver released from a commercially available dressing. The results show that silver is highly toxic to both keratinocytes and fibroblasts in monolayer culture. When using optimized and individualized culture the fibroblasts appear to be more sensitive to silver than keratinocytes. However, when both cell types were grown in the same medium their viability was the same. Using tissue culture models again indicated an 'environmental effect' with decreased sensitivity of the cells to the cytotoxic effects of the silver. Nevertheless in these studies the toxic dose of skin cells ranging from 7 x 10(-4) to 55 x 10(-4)% was similar to that of bacteria. These results suggest that consideration of the cytotoxic effects of silver and silver-based products should be taken when deciding on dressings for specific wound care strategies. This is important when using keratinocyte culture, in situ, which is playing an increasing role in contemporary wound and burn care.

Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

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Uchinaka, Ayako; Kawaguchi, Naomasa; Ban, Tsuyoshi; Hamada, Yoshinosuke; Mori, Seiji; Maeno, Yoshitaka; Sawa, Yoshiki; Nagata, Kohzo; Yamamoto, Hirofumi

2017-09-23

SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts. Copyright © 2017 Elsevier Inc. All rights reserved.

Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds

OpenAIRE

Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determine...

Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.

Science.gov (United States)

Naaldijk, Yahaira; Johnson, Adiv A; Friedrich-Stöckigt, Annett; Stolzing, Alexandra

2016-12-01

Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

Improved wound care product

DEFF Research Database (Denmark)

2012-01-01

The present invention pertains to use of sodium diacetate (NaHAc 2) as an antimicrobial agent against bacteria growing in biofilms. The aspects of the invention include a wound care product comprising sodium diacetate, a kit comprising a wound care product,and a methodof treating an infected wound....

Aloe vera and Vitis vinifera improve wound healing in an in vivo rat burn wound model.

Science.gov (United States)

Lin, Li-Xin; Wang, Peng; Wang, Yu-Ting; Huang, Yong; Jiang, Lei; Wang, Xue-Ming

2016-02-01

Aloe vera and Vitis vinifera have been traditionally used as wound healing agents. The present study aimed to investigate the effects of aloe emodin and resveratrol in the burn wound healing procedure. Burn wounds are common in developed and developing countries, however, in developing countries, the incidence of severe complications is higher and financial resources are limited. The results of the present study demonstrated that neither aloe emodin or resveratrol were cytotoxic to THP-1 macrophages at concentrations of 1, 100 and 500 ng/ml. A significant increase in wound-healing activity was observed in mice treated with the aloe emodin and resveratrol, compared with those which received control treatments. The levels of IL-1β in the exudates of the burn wound area of the treated mice increased in a time-dependent manner over 7 days following burn wound injury. At 10 days post-injury, steady and progressive wound healing was observed in the control animals. The present study confirmed that increased wound healing occurs following treatment with aloe emodin,, compared with resveratrol, providing support for the use of Aloe vera plants to improve burn wound healing.

Enhancement of keratinocyte performance in the production of tissue-engineered skin using a low-calcium medium.

Science.gov (United States)

Hernon, Catherine A; Harrison, Caroline A; Thornton, Daniel J A; MacNeil, Sheila

2007-01-01

The success of laboratory-expanded autologous keratinocytes for the treatment of severe burn injuries is often compromised by their lack of dermal remnants and failure to establish a secure dermo-epidermal junction on the wound bed. We have developed a tissue-engineered skin substitute for in vivo use, based on a sterilized donor human dermis seeded with autologous keratinocytes and fibroblasts. However, culture rates are currently too slow for clinical use in acute burns. Our aim in this study was to increase the rate of production of tissue-engineered skin. Two approaches were explored: one using a commercial low-calcium media and the other supplementing well-established media for keratinocyte culture with the calcium-chelating agent ethylene glutamine tetra-acetic acid (EGTA). Using commercial low-calcium media for both the initial cell culture and subsequent culture of tissue-engineered skin did not produce tissue suitable for clinical use. However, it was possible to enhance the initial proliferation of keratinocytes and to increase their horizontal migration in tissue-engineered skin by supplementing established culture medium with 0.04 mM EGTA without sacrificing epidermal attachment and differentiation. Enhancement of keratinocyte migration with EGTA was also maximal in the absence of fibroblasts or basement membrane.

Lumican as a multivalent effector in wound healing.

Science.gov (United States)

Karamanou, Konstantina; Perrot, Gwenn; Maquart, Francois-Xavier; Brézillon, Stéphane

2018-03-01

Wound healing, a complex physiological process, is responsible for tissue repair after exposure to destructive stimuli, without resulting in complete functional regeneration. Injuries can be stromal or epithelial, and most cases of wound repair have been studied in the skin and cornea. Lumican, a small leucine-rich proteoglycan, is expressed in the extracellular matrices of several tissues, such as the cornea, cartilage, and skin. This molecule has been shown to regulate collagen fibrillogenesis, keratinocyte phenotypes, and corneal transparency modulation. Lumican is also involved in the extravasation of inflammatory cells and angiogenesis, which are both critical in stromal wound healing. Lumican is the only member of the small leucine-rich proteoglycan family expressed by the epithelia during wound healing. This review summarizes the importance of lumican in wound healing and potential methods of lumican drug delivery to target wound repair are discussed. The involvement of lumican in corneal wound healing is described based on in vitro and in vivo models, with critical emphasis on its underlying mechanisms of action. Similarly, the expression and role of lumican in the healing of other tissues are presented, with emphasis on skin wound healing. Overall, lumican promotes normal wound repair and broadens new therapeutic perspectives for impaired wound healing. Copyright © 2018. Published by Elsevier B.V.

The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

Science.gov (United States)

Eisenbeis, Janina; Peisker, Henrik; Backes, Christian S; Bur, Stephanie; Hölters, Sebastian; Thewes, Nicolas; Greiner, Markus; Junker, Christian; Schwarz, Eva C; Hoth, Markus; Junker, Kerstin; Preissner, Klaus T; Jacobs, Karin; Herrmann, Mathias; Bischoff, Markus

2017-02-01

Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the

Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

Science.gov (United States)

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-01-01

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

Chitosan–aluminum monostearate composite sponge dressing containing asiaticoside for wound healing and angiogenesis promotion in chronic wound

International Nuclear Information System (INIS)

Phaechamud, Thawatchai; Yodkhum, Kotchamon; Charoenteeraboon, Juree; Tabata, Yasuhiko

2015-01-01

There are many factors that delay healing in chronic wounds including lowering level of growth factors and increasing exudate level comprising high amount of tissue destructive enzymes. Asiaticoside possesses interesting wound healing and angiogenic activities that are employed to stimulate tissue regeneration in wound healing application. This study attempted to develop chitosan–aluminum monostearate (Alst) composite sponge containing asiaticoside for use as an absorbent medical dressing in chronic wound. N-methyl-2-pyrrolidone (NMP) was used to enhance homogeneity of asiaticoside in the polymer composite matrix. The sponge dressings were prepared by lyophilization and dehydrothermal treatment (DHT). Functional group interaction, crystallinity, and morphology of the prepared sponges were investigated using FT-IR, PXRD, and SEM, respectively. Physicochemical properties, porosity, hydrophilic/hydrophobic properties and mechanical property, were evaluated. Wound dressing properties, water vapor transmission rate (WVTR), fluid absorbency, oxygen permeation (OP), and bio-adhesive property, were investigated. In vitro asiaticoside release study was conducted using immersion method. Cytotoxicity was studied in normal human dermal fibroblast (NHDF) and normal human epidermal keratinocyte (NHEK). Angiogenic activity of asiaticoside was evaluated using chick-chorioallantoic membrane (CAM) assay. FT-IR and PXRD results revealed the amidation after DHT to enhance the crystallinity of the prepared sponges. The prepared sponges had high porosity comprising high Alst-loaded amount that exhibited more compact structure. Alst enhanced hydrophobicity therefore it reduced the fluid absorption and WVTR together with bio-adhesion of the prepared sponge dressings. Porosity of all sponges was more than 85% therefore resulting in their high OP. Enhancing hydrophobicity of the material by Alst and more homogeneity caused by NMP eventually retarded the asiaticoside release for 7 days

Chitosan–aluminum monostearate composite sponge dressing containing asiaticoside for wound healing and angiogenesis promotion in chronic wound

Energy Technology Data Exchange (ETDEWEB)

Phaechamud, Thawatchai, E-mail: thawatchaienator@gmail.com [Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000 (Thailand); Yodkhum, Kotchamon, E-mail: marskotchamon@gmail.com [Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000 (Thailand); Charoenteeraboon, Juree, E-mail: juree@su.ac.th [Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000 (Thailand); Tabata, Yasuhiko, E-mail: yasuhiko@frontier.kyoto-u.ac.jp [Department of Biomaterials, Field of tissue engineering, Institute for Frontier Medical Science, Kyoto University, Kyoto 606-8507 (Japan)

2015-05-01

There are many factors that delay healing in chronic wounds including lowering level of growth factors and increasing exudate level comprising high amount of tissue destructive enzymes. Asiaticoside possesses interesting wound healing and angiogenic activities that are employed to stimulate tissue regeneration in wound healing application. This study attempted to develop chitosan–aluminum monostearate (Alst) composite sponge containing asiaticoside for use as an absorbent medical dressing in chronic wound. N-methyl-2-pyrrolidone (NMP) was used to enhance homogeneity of asiaticoside in the polymer composite matrix. The sponge dressings were prepared by lyophilization and dehydrothermal treatment (DHT). Functional group interaction, crystallinity, and morphology of the prepared sponges were investigated using FT-IR, PXRD, and SEM, respectively. Physicochemical properties, porosity, hydrophilic/hydrophobic properties and mechanical property, were evaluated. Wound dressing properties, water vapor transmission rate (WVTR), fluid absorbency, oxygen permeation (OP), and bio-adhesive property, were investigated. In vitro asiaticoside release study was conducted using immersion method. Cytotoxicity was studied in normal human dermal fibroblast (NHDF) and normal human epidermal keratinocyte (NHEK). Angiogenic activity of asiaticoside was evaluated using chick-chorioallantoic membrane (CAM) assay. FT-IR and PXRD results revealed the amidation after DHT to enhance the crystallinity of the prepared sponges. The prepared sponges had high porosity comprising high Alst-loaded amount that exhibited more compact structure. Alst enhanced hydrophobicity therefore it reduced the fluid absorption and WVTR together with bio-adhesion of the prepared sponge dressings. Porosity of all sponges was more than 85% therefore resulting in their high OP. Enhancing hydrophobicity of the material by Alst and more homogeneity caused by NMP eventually retarded the asiaticoside release for 7 days

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

International Nuclear Information System (INIS)

Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

2011-01-01

Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

Improving Outcomes Following Penetrating Colon Wounds

Science.gov (United States)

Miller, Preston R.; Fabian, Timothy C.; Croce, Martin A.; Magnotti, Louis J.; Elizabeth Pritchard, F.; Minard, Gayle; Stewart, Ronald M.

2002-01-01

. More destructive injuries were managed in the CP group (27% vs. 19%). Abscess rate was lower in the CP group (27% vs. 37%), as was suture line leak rate (7% vs. 14%). Colon related mortality in the CP group was 5% as compared with 12% in the PS group. Conclusions The clinical pathway for destructive colon wound management has improved outcomes as measured by anastomotic leak rates and colon related mortality. The data demonstrated the need for colostomy in the face of shock and comorbidities. Institution of this pathway results in colostomy for only 7% of all colon wounds. PMID:12035033

Effect of gamma-hydroxybutyrate on keratinocytes proliferation: A preliminary prospective controlled study in severe burn patients

Science.gov (United States)

Rousseau, Anne-Françoise; Bargues, Laurent; Bever, Hervé Le; Vest, Philippe; Cavalier, Etienne; Ledoux, Didier; Piérard, Gérald E.; Damas, Pierre

2014-01-01

Background: Hypermetabolism and hyposomatotropism related to severe burns lead to impaired wound healing. Growth hormone (GH) boosts wound healing notably following stimulation of the production of insulin-like growth factor-1 (IGF1), a mitogen factor for keratinocytes. Gamma-hydroxybutyrate (GHB) stimulates endogenous GH secretion. Aim: To assess effects of GHB sedation on keratinocytes proliferation (based on immunohistochemical techniques). Design: Monocentric, prospective, controlled trial. Materials and Methods: Patients (aging 18-65 years, burn surface area >30%, expected to be sedated for at least one month) were alternately allocated, at the 5th day following injury, in three groups according to the intravenous GHB dose administered for 21 days: Evening bolus of 50 mg/kg (Group B), continuous infusion at the rate of 10 mg/kg/h (Group C), or absence of GHB (Group P). They all received local standard cares. Immunohistochemistry (Ki67/MIB-1, Ulex europaeus agglutinin-1 and Mac 387 antibodies) was performed at D21 on adjacent unburned skin sample for assessing any keratinocyte activation. Serum IGF1 levels were measured at initiation and completion of the protocol. Statistical Analysis: Categorical variables were compared with Chi-square test. Comparisons of medians were made using Kruskal-Wallis test. Post hoc analyses were performed using Mann-Whitney test with Bonferroni correction for multiple comparisons. A P study (Group B: n = 5, Group C: n = 5, Group P: n = 4). Continuous administration of GHB was associated with a significant higher Ki67 immunolabeling at D21 (P = 0.049) and with a significant higher increase in the IGF1 concentrations at D21 (P = 0.024). No adverse effects were disclosed. Conclusions: Our preliminary data support a positive effect of GHB on keratinocyte proliferation and are encouraging enough to warrant large prospective studies. PMID:25024938

Upregulation of adhesion complex proteins and fibronectin by human keratinocytes treated with an aqueous extract from the leaves of Chromolaena odorata (Eupolin).

Science.gov (United States)

Phan, T T; Allen, J; Hughes, M A; Cherry, G; Wojnarowska, F

2000-01-01

The fresh leaves and extract of the plant Chromolaena odorata are a traditional herbal treatment in developing countries for burns, soft tissue wounds and skin infections. We have previously shown that the extract had an effect on the growth and proliferation of keratinocytes and fibroblasts in culture. This study has demonstrated that Eupolin extract increased expression of several components of the adhesion complex and fibronectin by human keratinocytes. Using indirect immunofluorescence we found increased expression (dose-dependent) of laminin 5, laminin 1, collagen IV, and fibronectin. The expression of the b1 and b4 integrins was upregulated by the extract at low concentrations (0.1 and 1 microg/ml), but the expression was decreased at higher doses of Eupolin (10 microg-150 microg/ml). A number of clinical studies carried out by Vietnamese and international medical investigators have demonstrated the efficacy of this extract on the wound healing process. In this study we have shown that Eupolin stimulated the expression of many proteins of the adhesion complex and fibronectin by human keratinocytes. The adhesion complex proteins are essential to stabilise epithelium and this effect could contribute to the clinical efficacy of Eupolin in healing.

Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management - prospects and limitations.

Science.gov (United States)

Fromm-Dornieden, Carolin; Rembe, Julian-Dario; Schäfer, Nadine; Böhm, Julia; Stuermer, Ewa K

2015-04-01

The antimicrobial activity of cetylpyridinium chloride (CPC) and miramistin (MST) solutions at different concentrations (5×10(-5) to 0.4%) and a dressing, containing 0.15% CPC, were tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli after 30 (solutions) and 60 min (fleece) incubation, respectively. Furthermore, the cytotoxic effects of CPC and MST were examined in human keratinocyte (HaCaT) and murine fibroblast (L929) cell lines. A dose of 3×10(-3)% CPC or MST was sufficient to entirely eradicate S. aureus after 30 min incubation. To achieve the same effect, higher concentrations were required against E. coli (0.025% CPC; 0.0125% MST) and P. aeruginosa (0.5% CPC; 0.05% MST). The CPC-fleece showed a high antiseptic effect against all three bacterial strains, although it did not completely eliminate P. aeruginosa. Both substances showed a high cytotoxic impact at higher tested concentrations (CPC >3×10(-3)%; MST >8×10(-4)%). CPC showed high antimicrobial potency at low concentrations against S. aureus, accompanied by low cytotoxic (side) effects at these concentrations, whilst the required minimal concentration to eradicate E. coli and P. aeruginosa was shown to be cytotoxic for keratinocytes and fibroblasts. The necessary antibacterial amounts of MST were lower, but also cytotoxic in direct contact with typical human wound cells. With regard to demographic changes and increasing bacterial resistance, new effective antiseptics, such as CPC and MST, incorporated in wound dressings without releasing an active substance could help to improve the treatment and healing rates of chronic wounds. © 2015 The Authors.

Enhanced Cutaneous Wound Healing In Vivo by Standardized Crude Extract of Poincianella pluviosa.

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Fernanda Giacomini Bueno

Full Text Available Wound healing is a complex process that involves several biological events, and a delay in this process may cause economic and social problems for the patient. The search continues for new alternative treatments to aid healing, including the use of herbal medicines. Members of the genus Caesalpinia are used in traditional medicine to treat wounds. The related species Poincianella pluviosa (DC. L.P. Queiroz increases the cell viability of keratinocytes and fibroblasts and stimulates the proliferation of keratinocytes in vitro. The crude extract (CE from bark of P. pluviosa was evaluated in the wound-healing process in vivo, to validate the traditional use and the in vitro activity. Standardized CE was incorporated into a gel and applied on cutaneous wounds (TCEG and compared with the formulation without CE (Control for 4, 7, 10, or 14 days of treatment. The effects of the CE on wound re-epithelialization; cell proliferation; permeation, using photoacoustic spectroscopy (PAS; and proteins, including vascular endothelial growth factor (VEGF, superoxide dismutase 2 (SOD-2 and cyclooxygenase 2 (COX-2 were evaluated. The TCEG stimulated the migration of keratinocytes at day 4 and proliferation on the following days, with a high concentration of cells in metaphase at 7 days. Type I collagen formed more rapidly in the TCEG. PAS showed that the CE had permeated through the skin. TCEG stimulated VEGF at day 4 and SOD-2 and COX-2 at day 7. The results suggest that the CE promoted the regulation of proteins and helped to accelerate the processes involved in healing, promoting early angiogenesis. This led to an increase in the re-epithelialized surface, with significant mitotic activity. Maturation of collagen fibers was also enhanced, which may affect the resistance of the extracellular matrix. PAS indicated a correlation between the rate of diffusion and biological events during the healing process. The CE from P. pluviosa appears promising as an aid in

Honey: an immunomodulator in wound healing.

Science.gov (United States)

Majtan, Juraj

2014-01-01

Honey is a popular natural product that is used in the treatment of burns and a broad spectrum of injuries, in particular chronic wounds. The antibacterial potential of honey has been considered the exclusive criterion for its wound healing properties. The antibacterial activity of honey has recently been fully characterized in medical-grade honeys. Recently, the multifunctional immunomodulatory properties of honey have attracted much attention. The aim of this review is to provide closer insight into the potential immunomodulatory effects of honey in wound healing. Honey and its components are able to either stimulate or inhibit the release of certain cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6) from human monocytes and macrophages, depending on wound condition. Similarly, honey seems to either reduce or activate the production of reactive oxygen species from neutrophils, also depending on the wound microenvironment. The honey-induced activation of both types of immune cells could promote debridement of a wound and speed up the repair process. Similarly, human keratinocytes, fibroblasts, and endothelial cell responses (e.g., cell migration and proliferation, collagen matrix production, chemotaxis) are positively affected in the presence of honey; thus, honey may accelerate reepithelization and wound closure. The immunomodulatory activity of honey is highly complex because of the involvement of multiple quantitatively variable compounds among honeys of different origins. The identification of these individual compounds and their contributions to wound healing is crucial for a better understanding of the mechanisms behind honey-mediated healing of chronic wounds. © 2014 by the Wound Healing Society.

Reduced FOXO1 expression accelerates skin wound healing and attenuates scarring.

Science.gov (United States)

Mori, Ryoichi; Tanaka, Katsuya; de Kerckhove, Maiko; Okamoto, Momoko; Kashiyama, Kazuya; Tanaka, Katsumi; Kim, Sangeun; Kawata, Takuya; Komatsu, Toshimitsu; Park, Seongjoon; Ikematsu, Kazuya; Hirano, Akiyoshi; Martin, Paul; Shimokawa, Isao

2014-09-01

The forkhead box O (FOXO) family has been extensively investigated in aging and metabolism, but its role in tissue-repair processes remains largely unknown. Herein, we clarify the molecular aspect of the FOXO family in skin wound healing. We demonstrated that Foxo1 and Foxo3a were both up-regulated during murine skin wound healing. Partial knockout of Foxo1 in Foxo1(+/-) mice throughout the body led to accelerated skin wound healing with enhanced keratinocyte migration, reduced granulation tissue formation, and decreased collagen density, accompanied by an attenuated inflammatory response, but we observed no wound phenotype in Foxo3a(-/-) mice. Fibroblast growth factor 2, adiponectin, and notch1 genes were significantly increased at wound sites in Foxo1(+/-) mice, along with markedly altered extracellular signal-regulated kinase 1/2 and AKT phosphorylation. Similarly, transient knockdown of Foxo1 at the wound site by local delivery of antisense oligodeoxynucleotides enhanced skin wound healing. The link between FOXO1 and scarring extends to patients, in particular keloid scars, where we see FOXO1 expression markedly increased in fibroblasts and inflammatory cells within the otherwise normal dermis. This occurs in the immediate vicinity of the keloid by comparison to the center of the mature keloid, indicating that FOXO1 is associated with the overgrowth of this fibrotic response into adjacent normal skin. Overall, our data indicate that molecular targeting of FOXO1 may improve the quality of healing and reduce pathological scarring. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

Science.gov (United States)

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-10-02

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

In-vitro- and in-vivo-studies on the application of an innovative wound healing system for burn patients on the basis of a spray-transplantation of primary isolated skin cells and an active wound dressing made of hollow fiber capillaries

OpenAIRE

Plettig, Jörn

2016-01-01

The treatment of acute burns, especially for patients with 2b degree wounds, is not resolved satisfactorily. The thesis evaluates an innovative therapeutic concept based on a primary skin cell isolation from fetal or adult tissue, an autologous spray-transplantation and a temporary wound supply via an active wound dressing based on hollow fiber capillaries. The aim is to increase the yield of regenerative basal keratinocyte precursor cells through a gentle isolation technique and to distribut...

The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts.

Science.gov (United States)

Varkey, Mathew; Ding, Jie; Tredget, Edward E

2014-12-01

Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Improving wound and pressure area care in a nursing home.

Science.gov (United States)

Sprakes, Kate; Tyrer, Julie

Wound and pressure ulcer prevention are key quality indicators of nursing care. This article describes a collaborative project between a community skin care service and a nursing home. The aim of the project was to establish whether the implementation of a wound and pressure ulcer management competency framework within a nursing home would improve patient outcomes and reduce the severity and number of wounds and pressure ulcers. Following the project's implementation, there was a reduction in the number of wounds and pressure ulcers, hospital admissions and district nursing visits. Nursing home staff also reported an increase in their knowledge and skills.

Anterior gradient 2 is induced in cutaneous wound and promotes wound healing through its adhesion domain.

Science.gov (United States)

Zhu, Qi; Mangukiya, Hitesh Bhagavanbhai; Mashausi, Dhahiri Saidi; Guo, Hao; Negi, Hema; Merugu, Siva Bharath; Wu, Zhenghua; Li, Dawei

2017-09-01

Anterior gradient 2 (AGR2), a member of protein disulfide isomerase (PDI) family, is both located in cytoplasm and secreted into extracellular matrix. The orthologs of AGR2 have been linked to limb regeneration in newt and wound healing in zebrafish. In mammals, AGR2 influences multiple cell signaling pathways in tumor formation and in normal cell functions related to new tissue formation like angiogenesis. However, the function of AGR2 in mammalian wound healing remains unknown. This study aimed to investigate AGR2 expression and its function during skin wound healing and the possible application of external AGR2 in cutaneous wound to accelerate the healing process. Our results showed that AGR2 expression was induced in the migrating epidermal tongue and hyperplastic epidermis after skin excision. Topical application of recombinant AGR2 significantly accelerated wound-healing process by increasing the migration of keratinocytes (Kera.) and the recruitment of fibroblasts (Fibro.) near the wounded area. External AGR2 also promoted the migration of Kera. and Fibro. in vitro in a dose-dependent manner. The adhesion domain of AGR2 was required for the formation of focal adhesions in migrating Fibro., leading to the directional migration along AGR2 gradient. These results indicate that recombinant AGR2 accelerates skin wound healing through regulation of Kera. and Fibro. migration, thus demonstrating its potential utility as an alternative strategy of the therapeutics to accelerate the healing of acute or chronic skin wounds. © 2017 Federation of European Biochemical Societies.

Development of a wide-field fluorescence imaging system for evaluation of wound re-epithelialization

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Franco, Walfre; Gutierrez-Herrera, Enoch; Purschke, Martin; Wang, Ying; Tam, Josh; Anderson, R. Rox; Doukas, Apostolos

2013-03-01

Normal skin barrier function depends on having a viable epidermis, an epithelial layer formed by keratinocytes. The transparent epidermis, which is less than a 100 mum thick, is nearly impossible to see. Thus, the clinical evaluation of re-epithelialization is difficult, which hinders selecting appropriate therapy for promoting wound healing. An imaging system was developed to evaluate epithelialization by detecting endogenous fluorescence emissions of cellular proliferation over a wide field of view. A custom-made 295 nm ultraviolet (UV) light source was used for excitation. Detection was done by integrating a near-UV camera with sensitivity down to 300 nm, a 12 mm quartz lens with iris and focus lock for the UV regime, and a fluorescence bandpass filter with 340 nm center wavelength. To demonstrate that changes in fluorescence are related to cellular processes, the epithelialization of a skin substitute was monitored in vitro. The skin substitute or construct was made by embedding microscopic live human skin tissue columns, 1 mm in diameter and spaced 1 mm apart, in acellular porcine dermis. Fluorescence emissions clearly delineate the extent of lateral surface migration of keratinocytes and the total surface covered by the new epithelium. The fluorescence image of new epidermis spatially correlates with the corresponding color image. A simple, user-friendly way of imaging the presence of skin epithelium would improve wound care in civilian burns, ulcers and surgeries.

The effect of wound dressings on a bio-engineered human dermo-epidermal skin substitute in a rat model

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Hüging, Martina; Biedermann, Thomas; Sobrio, Monia; Meyer, Sarah; Böttcher-Haberzeth, Sophie; Manuel, Edith; Horst, Maya; Hynes, Sally; Reichmann, Ernst; Schiestl, Clemens; Hartmann-Fritsch, Fabienne

2017-01-01

Autologous bio-engineered dermo-epidermal skin substitutes are a promising treatment for large skin defects such as burns. For their successful clinical application, the graft dressing must protect and support the keratinocyte layer and, in many cases, possess antimicrobial properties. However, silver in many antimicrobial dressings may inhibit keratinocyte growth and differentiation. The purpose of our study is to evaluate the effect of various wound dressings on the healing of a human hydro...

A small peptide with potential ability to promote wound healing.

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Jing Tang

Full Text Available Wound-healing represents a major health burden, such as diabetes-induced skin ulcers and burning. Many works are being tried to find ideal clinical wound-healing biomaterials. Especially, small molecules with low cost and function to promote production of endogenous wound healing agents (i.e. transforming growth factor beta, TGF-β are excellent candidates. In this study, a small peptide (tiger17, c[WCKPKPKPRCH-NH2] containing only 11 amino acid residues was designed and proved to be a potent wound healer. It showed strong wound healing-promoting activity in a murine model of full thickness dermal wound. Tiger17 exerted significant effects on three stages of wound healing progresses including (1 the induction of macrophages recruitment to wound site at inflammatory reaction stage; (2 the promotion of the migration and proliferation both keratinocytes and fibroblasts, leading to reepithelialization and granulation tissue formation; and (3 tissue remodeling phase, by promoting the release of transforming TGF-β1 and interleukin 6 (IL-6 in murine macrophages and activating mitogen-activated protein kinases (MAPK signaling pathways. Considering its easy production, store and transfer and function to promote production of endogenous wound healing agents (TGF-β, tiger17 might be an exciting biomaterial or template for the development of novel wound-healing agents.

Burn wound: Pathophysiology and its management by herbal plants

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Dhirender Kaushik

2013-01-01

Full Text Available In human body, wound healing is a normal biological phenomenon. Burns may be acute or chronic depending upon the source and its time of exposure. Burn wounds may be superficial, partial or full thickness wounds. When skin comes in contact with higher temperature, protein denaturation takes place due to which the plasma membrane integrity is lost. When skin is burned, a number of inflammatory mediators and releasing agents such as histamine, nitric oxide, oxygen free radicals, eicosanoid products, tumor necrosis factors, and interleukins etc., are released at the site. For wound healing mechanism, the keratinocytes has to move from uninjured site to the burned area. For deeper burns this process takes a long time. By some unknown mechanisms, burn wounds may convert from one form to another form. So burn wound depth must be accurately measured before starting the treatment to prevent the complications. Burns can be induced in experimental animals by using different models. Many treatments such as herbal drugs, topical agents, gene therapy, volume therapy, and rehabilitation can be employed. This review article mainly deals with the theoretical and practical aspects of burn wound healing. Some burn wound healing plants with their chemical constituents, plant part used, uses and animal models are described here.

Cold atmospheric plasma (CAP changes gene expression of key molecules of the wound healing machinery and improves wound healing in vitro and in vivo.

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Stephanie Arndt

Full Text Available Cold atmospheric plasma (CAP has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.

Leptin regulates the pro-inflammatory response in human epidermal keratinocytes.

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Lee, Moonyoung; Lee, Eunyoung; Jin, Sun Hee; Ahn, Sungjin; Kim, Sae On; Kim, Jungmin; Choi, Dalwoong; Lim, Kyung-Min; Lee, Seung-Taek; Noh, Minsoo

2018-05-01

The role of leptin in cutaneous wound healing process has been suggested in genetically obese mouse studies. However, the molecular and cellular effects of leptin on human epidermal keratinocytes are still unclear. In this study, the whole-genome-scale microarray analysis was performed to elucidate the effect of leptin on epidermal keratinocyte functions. In the leptin-treated normal human keratinocytes (NHKs), we identified the 151 upregulated and 53 downregulated differentially expressed genes (DEGs). The gene ontology (GO) enrichment analysis with the leptin-induced DEGs suggests that leptin regulates NHKs to promote pro-inflammatory responses, extracellular matrix organization, and angiogenesis. Among the DEGs, the protein expression of IL-8, MMP-1, fibronectin, and S100A7, which play roles in which is important in the regulation of cutaneous inflammation, was confirmed in the leptin-treated NHKs. The upregulation of the leptin-induced proteins is mainly regulated by the STAT3 signaling pathway in NHKs. Among the downregulated DEGs, the protein expression of nucleosome assembly-associated centromere protein A (CENPA) and CENPM was confirmed in the leptin-treated NHKs. However, the expression of CENPA and CENPM was not coupled with those of other chromosome passenger complex like Aurora A kinase, INCENP, and survivin. In cell growth kinetics analysis, leptin had no significant effect on the cell growth curves of NHKs in the normal growth factor-enriched condition. Therefore, leptin-dependent downregulation of CENPA and CENPM in NHKs may not be directly associated with mitotic regulation during inflammation.

In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities.

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Gunes, Seda; Tamburaci, Sedef; Dalay, Meltem Conk; Deliloglu Gurhan, Ismet

2017-12-01

Algae have gained importance in cosmeceutical product development due to their beneficial effects on skin health and therapeutical value with bioactive compounds. Spirulina platensis Parachas (Phormidiaceae) is renowned as a potential source of high-value chemicals and recently used in skincare products. This study develops and evaluates skin creams incorporated with bioactive S. platensis extract. Spirulina platensis was cultivated, the aqueous crude extract was prepared and in vitro cytotoxicity of S. platensis extract in the range of 0.001-1% concentrations for 1, 3 and 7 d on HS2 keratinocyte cells was determined. Crude extracts were incorporated in skin cream formulation at 0.01% (w/w) concentration and in vitro wound healing and genotoxicity studies were performed. Immunohistochemical staining was performed to determine the collagen activity. 0.1% S. platensis extract exhibited higher proliferation activity compared with the control group with 198% of cell viability after 3 d. Skin cream including 1.125% S. platensis crude extract showed enhanced wound healing effect on HS2 keratinocyte cell line and the highest HS2 cell viability % was obtained with this concentration. The micronucleus (MN) assay results indicated that S. platensis extract incorporated creams had no genotoxic effect on human peripheral blood cells. Immunohistochemical analysis showed that collagen 1 immunoreactivity was improved by increased extract concentration and it was strongly positive in cells treated with 1.125% extract incorporated skin cream. The cell viability, wound healing activity and genotoxicity results showed that S. platensis incorporated skin cream could be of potential value in cosmeceutical and biomedical applications.

A Conditioned Medium of Umbilical Cord Mesenchymal Stem Cells Overexpressing Wnt7a Promotes Wound Repair and Regeneration of Hair Follicles in Mice

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Liang Dong

2017-01-01

Full Text Available Mesenchymal stem cells (MSCs can affect the microenvironment of a wound and thereby accelerate wound healing. Wnt proteins act as key mediators of skin development and participate in the formation of skin appendages such as hair. The mechanisms of action of MSCs and Wnt proteins on skin wounds are largely unknown. Here, we prepared a Wnt7a-containing conditioned medium (Wnt-CM from the supernatant of cultured human umbilical cord-MSCs (UC-MSCs overexpressing Wnt7a in order to examine the effects of this CM on cutaneous healing. Our results revealed that Wnt-CM can accelerate wound closure and induce regeneration of hair follicles. Meanwhile, Wnt-CM enhanced expression of extracellular matrix (ECM components and cell migration of fibroblasts but inhibited the migratory ability and expression of K6 and K16 in keratinocytes by enhancing expression of c-Myc. However, we found that the CM of fibroblasts treated with Wnt-CM (HFWnt-CM-CM can also promote wound repair and keratinocyte migration; but there was no increase in the number of hair follicles of regeneration. These data indicate that Wnt7a and UC-MSCs have synergistic effects: they can accelerate wound repair and induce hair regeneration via cellular communication in the wound microenvironment. Thus, this study opens up new avenues of research on the mechanisms underlying wound repair.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

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Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

2015-03-01

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

International Nuclear Information System (INIS)

Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

2015-01-01

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

Clinical application of a tissue-cultured skin autograft: an alternative for the treatment of non-healing or slowly healing wounds?

Science.gov (United States)

Zöller, Nadja; Valesky, Eva; Butting, Manuel; Hofmann, Matthias; Kippenberger, Stefan; Bereiter-Hahn, Jürgen; Bernd, August; Kaufmann, Roland

2014-01-01

The treatment regime of non-healing or slowly healing wounds is constantly improving. One aspect is surgical defect coverage whereby mesh grafts and keratinocyte suspension are applied. Tissue-cultured skin autografts may be an alternative for the treatment of full-thickness wounds and wounds that cover large areas of the body surface. Autologous epidermal and dermal cells were isolated, expanded in vitro and seeded on collagen-elastin scaffolds. The developed autograft was immunohistochemically characterized and subsequently transplanted onto a facial chronic ulceration of a 71-year-old patient with vulnerable atrophic skin. Characterization of the skin equivalent revealed comparability to healthy human skin due to the epidermal strata, differentiation and proliferation markers. Within 138 days, the skin structure at the transplantation site closely correlated with the adjacent undisturbed skin. The present study demonstrates the comparability of the developed organotypic skin equivalent to healthy human skin and the versatility for clinical applications.

Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

Science.gov (United States)

Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

2015-03-01

Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. Copyright © 2015 Elsevier Inc. All rights reserved.

Three-Dimensional Human Tissue Models That Incorporate Diabetic Foot Ulcer-Derived Fibroblasts Mimic In Vivo Features of Chronic Wounds

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Maione, Anna G.; Brudno, Yevgeny; Stojadinovic, Olivera; Park, Lara K.; Smith, Avi; Tellechea, Ana; Leal, Ermelindo C.; Kearney, Cathal J.; Veves, Aristidis; Tomic-Canic, Marjana; Mooney, David J.

2015-01-01

Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell–cell and cell–matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications. PMID:25343343

The role of nuclear hormone receptors in cutaneous wound repair.

Science.gov (United States)

Rieger, Sandra; Zhao, Hengguang; Martin, Paige; Abe, Koichiro; Lisse, Thomas S

2015-01-01

The cutaneous wound repair process involves balancing a dynamic series of events ranging from inflammation, oxidative stress, cell migration, proliferation, survival and differentiation. A complex series of secreted trophic factors, cytokines, surface and intracellular proteins are expressed in a temporospatial manner to restore skin integrity after wounding. Impaired initiation, maintenance or termination of the tissue repair processes can lead to perturbed healing, necrosis, fibrosis or even cancer. Nuclear hormone receptors (NHRs) in the cutaneous environment regulate tissue repair processes such as fibroplasia and angiogenesis. Defects in functional NHRs and their ligands are associated with the clinical phenotypes of chronic non-healing wounds and skin endocrine disorders. The functional relationship between NHRs and skin niche cells such as epidermal keratinocytes and dermal fibroblasts is pivotal for successful wound closure and permanent repair. The aim of this review is to delineate the cutaneous effects and cross-talk of various nuclear receptors upon injury towards functional tissue restoration. Copyright © 2014 John Wiley & Sons, Ltd.

Non-thermal atmospheric-pressure plasma possible application in wound healing.

Science.gov (United States)

Haertel, Beate; von Woedtke, Thomas; Weltmann, Klaus-Dieter; Lindequist, Ulrike

2014-11-01

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

MODERN ASSORTMENT, PROPERTIES AND PERSPECTIVES OF MEDICAL DRESSINGS IMPROVEMENT OF WOUND TREATMENT

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A. V. Mayorova

2018-01-01

Full Text Available The effectiveness of reparation is now characterized not only in terms of wound healing. The aesthetic result is also of great significance. Besides, it is important to ensure the comfort of medication, in order to combine the optimal therapy and the quality of life of the patient. The aim of the study was the literature review on the effective wound treatment with the help of modern dressings, including their assortment and prospects for improvement. Materials and methods. The materials of the study were reliable literary sources containing information about wounds, pathogenesis, the stages of the wound process and its possible violations; the assortment of modern wound coverings and the composition of biologically active substances that have a pharmacological effect; the results of preclinical and clinical trials and their prospects of use in aesthetic medicine. The research was carried out using the information retrieval and library databases (eLIBRARY, Cyberleninka, technical information. The research methods used in the work are: informational, analytical, descriptive. Results and discussion. As a result of generalization and analysis of modern publications devoted to the effective wound treatment, modern views on the wound process, wound coverings, their assortment and compositions are described. The results of pharmacological tests as well as the properties of biologically active substances and carrier polymers used are also presented. It is noted that the prospective compounds for including with the wound coverings and external drugs affecting the repair process at different stages of the wound process are tizol, bischofite, peptides (1-β-interleukin, ectoin. Conclusion. Thus, it has been established that modern wound dressings and dressings containing the substances of different pharmacological groups are offered for effective therapy: antiseptics, anesthetics, repair stimulators, antioxidants that affect different stages and elements

A conducive bioceramic/polymer composite biomaterial for diabetic wound healing.

Science.gov (United States)

Lv, Fang; Wang, Jie; Xu, Peng; Han, Yiming; Ma, Hongshi; Xu, He; Chen, Shijie; Chang, Jiang; Ke, Qinfei; Liu, Mingyao; Yi, Zhengfang; Wu, Chengtie

2017-09-15

Diabetic wound is a common complication of diabetes. Biomaterials offer great promise in inducing tissue regeneration for chronic wound healing. Herein, we reported a conducive Poly (caprolactone) (PCL)/gelatin nanofibrous composite scaffold containing silicate-based bioceramic particles (Nagelschmidtite, NAGEL, Ca 7 P 2 Si 2 O 16 ) for diabetic wound healing. NAGEL bioceramic particles were well distributed in the inner of PCL/gelatin nanofibers via co-electrospinning process and the Si ions maintained a sustained release from the composite scaffolds during the degradation process. The nanofibrous scaffolds significantly promoted the adhesion, proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human keratinocytes (HaCaTs) in vitro. The in vivo study demonstrated that the scaffolds distinctly induced the angiogenesis, collagen deposition and re-epithelialization in the wound sites of diabetic mice model, as well as inhibited inflammation reaction. The mechanism for nanofibrous composite scaffolds accelerating diabetic wound healing is related to the activation of epithelial to mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT) pathway in vivo and in vitro. Our results suggest that the released Si ions and nanofibrous structure of scaffolds have a synergetic effect on the improved efficiency of diabetic wound healing, paving the way to design functional biomaterials for tissue engineering and wound healing applications. In order to stimulate tissue regeneration for chronic wound healing, a new kind of conducive nanofibrous composite scaffold containing silicate-based bioceramic particles (Nagelschmidtite, NAGEL, Ca 7 P 2 Si 2 O 16 ) were prepared via co-electrospinning process. Biological assessments revealed that the NAGEL bioceramic particles could active epithelial to mesenchymal transition (EMT) and endothelial to mesenchymal transition (EndMT) pathway in vitro and in vivo. The new composite scaffold

Expert advice provided through telemedicine improves healing of chronic wounds: prospective cluster controlled study.

Science.gov (United States)

Zarchi, Kian; Haugaard, Vibeke B; Dufour, Deirdre N; Jemec, Gregor B E

2015-03-01

Telemedicine is widely considered as an efficient approach to manage the growing problem of chronic wounds. However, to date, there is no convincing evidence to support the clinical efficacy of telemedicine in wound management. In this prospective cluster controlled study, we tested the hypothesis that advice on wound management provided by a team of wound-care specialists through telemedicine would significantly improve the likelihood of wound healing compared with the best available conventional practice. A total of 90 chronic wound patients in home care met all study criteria and were included: 50 in the telemedicine group and 40 in the conventional group. Patients with pressure ulcers, surgical wounds, and cancer wounds were excluded. During the 1-year follow-up, complete wound healing was achieved in 35 patients (70%) in the telemedicine group compared with 18 patients (45%) in the conventional group. After adjusting for important covariates, offering advice on wound management through telemedicine was associated with significantly increased healing compared with the best available conventional practice (telemedicine vs. conventional practice: adjusted hazard ratio 2.19; 95% confidence interval: 1.15-4.17; P=0.017). This study strongly supports the use of telemedicine to connect home-care nurses to a team of wound experts in order to improve the management of chronic wounds.

Hydrogen sulfide improves diabetic wound healing in ob/ob mice via attenuating inflammation.

Science.gov (United States)

Zhao, Huichen; Lu, Shengxia; Chai, Jiachao; Zhang, Yuchao; Ma, Xiaoli; Chen, Jicui; Guan, Qingbo; Wan, Meiyan; Liu, Yuantao

2017-09-01

The proposed mechanisms of impaired wound healing in diabetes involve sustained inflammation, excess oxidative stress and compromised agiogenesis. Hydrogen sulfide (H 2 S) has been reported to have multiple biological activities. We aim to investigate the role of H 2 S in impaired wound healing in ob/ob mice and explore the possible mechanisms involved. Full-thickness skin dorsal wounds were created on ob/ob mice and C57BL/6 mice. Cystathionine-γ-lyase (CSE) expression and H 2 S production were determined in granulation tissues of the wounds. Effects of NaHS on wound healing were evaluated. Inflammation and angiogenesis in granulation tissues of the wounds were examined. CSE expression, and H 2 S content were significantly reduced in granulation tissues of wounds in ob/ob mice compared with control mice. NaHS treatment significantly improved wound healing in ob/ob mice, which was associated with reduced neutrophil and macrophage infiltration, decreased production of tumor necrosis factor (TNF)-α, interleukin (IL)-6. NaHS treatment decreased metalloproteinase (MMP)-9, whereas increased collagen deposition and vascular-like structures in granulation tissues of wounds in ob/ob mice. CSE down-regulation may play a role in the pathogenesis of diabetic impaired wound healing. Exogenous H 2 S could be a potential agent to improve diabetic impaired wound healing by attenuating inflammation and increasing angiogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

Science.gov (United States)

Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

2016-09-01

The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo.

Reconstruction of epidermis by grafting of keratinocytes cultured on polymer support--clinical study.

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Dvoránková, Barbora; Holíková, Zuzana; Vacík, Jirí; Königová, Radana; Kapounková, Zuzana; Michálek, Jirí; Prádn, Martin; Smetana, Karel

2003-03-01

Extensive wound coverage still represents a challenge for contemporary medicine. We demonstrate the results of a clinical trial of the grafting of cultured keratinocytes directly on a polymer cultivation support in the treatment of skin defects in seriously burned patients and in patients with trophic ulcers. Wound closure was evaluated clinically. The morphology and phenotypic pattern of the reconstructed epidermis, including the basal lamina, as well as the presence of Langerhans cells, were evaluated immunocytochemically using a panel of monoclonal antibodies. All layers of the reconstructed epidermis were normally differentiated (cytokeratin immunocytochemistry). The basal lamina contained collagen type IV and laminin. The reconstructed epidermis was extensively colonized by Langerhans cells. The results of the described technology are encouraging, especially in patients after a burn injury. The described procedure is suitable for the treatment of skin defects in clinical practice.

Foxn1 Transcription Factor Regulates Wound Healing of Skin through Promoting Epithelial-Mesenchymal Transition.

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Barbara Gawronska-Kozak

Full Text Available Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.

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Nuccitelli, Richard; Nuccitelli, Pamela; Li, Changyi; Narsing, Suman; Pariser, David M; Lui, Kaying

2011-01-01

Due to the transepidermal potential of 15-50 mV, inside positive, an injury current is driven out of all human skin wounds. The flow of this current generates a lateral electric field within the epidermis that is more negative at the wound edge than at regions more lateral from the wound edge. Electric fields in this region could be as large as 40 mV/mm, and electric fields of this magnitude have been shown to stimulate human keratinocyte migration toward the wounded region. After flowing out of the wound, the current returns through the space between the epidermis and stratum corneum, generating a lateral field above the epidermis in the opposite direction. Here, we report the results from the first clinical trial designed to measure this lateral electric field adjacent to human skin wounds noninvasively. Using a new instrument, the Dermacorder®, we found that the mean lateral electric field in the space between the epidermis and stratum corneum adjacent to a lancet wound in 18-25-year-olds is 107-148 mV/mm, 48% larger on average than that in 65-80-year-olds. We also conducted extensive measurements of the lateral electric field adjacent to mouse wounds as they healed and compared this field with histological sections through the wound to determine the correlation between the electric field and the rate of epithelial wound closure. Immediately after wounding, the average lateral electric field was 122 ± 9 mV/mm. When the wound is filled in with a thick, disorganized epidermal layer, the mean field falls to 79 ± 4 mV/mm. Once this epidermis forms a compact structure with only three cell layers, the mean field is 59 ± 5 mV/mm. Thus, the peak-to-peak spatial variation in surface potential is largest in fresh wounds and slowly declines as the wound closes. The rate of wound healing is slightly greater when wounds are kept moist as expected, but we could find no correlation between the amplitude of the electric field and the rate of wound

Effects of UVB irradiation on keratinocyte growth factor (KGF) and receptor (KGFR) expression in cultured human keratinocytes

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Zhou, Y.; Lee, H.S.T.; Kooshesh, F.; Fujisawa, H.; Sauder, D.N.; Kondo, S. [Univ. of Toronto, Sunnybrook Health Science Centre, Div. of Dermatology, Toronto (Canada)

1996-06-01

Keratinocyte growth factor (KGF) and its receptor (KGFR) are thought to play important roles in normal keratinocyte growth and differentiation. Since UVB radiation is known to influence keratinocyte growth, we sought to determine whether UVB would alter the expression of KGF and KGFR. Using a reverse-transcription coupled polymerase chain reaction (RT-PCR), the present study examined the expression of KGF and KGFR mRNA in cultured normal human keratinocytes exposed to UVB irradiation. Total cellular RNA was extracted from cultured keratinocytes at various time points after irradiation, reverse transcribed and used for PCR amplification using primers specific for KGF and KGFR. Constitutive expression of KGFR mRNA, but not KGF mRNA, was detected in normal cultured human keratinocytes. After UVB irradiation at 300 J/m{sup 2}, the KGF mRNA remained undetectable while the KGFR mRNA level was significantly decreased. The down-regulation of KGFR mRNA expression was also confirmed by Northern blot analysis. Immunohistochemical studies demonstrated a decreased positive signal of KGFR in human keratinocytes after UVB irradiation. Our results suggest a possible role for the KGF-KGFR signalling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of keratinocytes in hyperproliferative skin disorders may be related to downregulation of KGFR. (au) 39 refs.

Co-delivery of a growth factor and a tissue-protective molecule using elastin biopolymers accelerates wound healing in diabetic mice.

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Devalliere, Julie; Dooley, Kevin; Hu, Yong; Kelangi, Sarah S; Uygun, Basak E; Yarmush, Martin L

2017-10-01

Growth factor therapy is a promising approach for chronic diabetic wounds, but strategies to efficiently and cost-effectively deliver active molecules to the highly proteolytic wound environment remain as major obstacles. Here, we re-engineered keratinocyte growth factor (KGF) and the cellular protective peptide ARA290 into a protein polymer suspension with the purpose of increasing their proteolytic resistance, thus their activity in vivo. KGF and ARA290 were fused with elastin-like peptide (ELP), a protein polymer derived from tropoelastin, that confers the ability to separate into a colloidal suspension of liquid-like coacervates. ELP fusion did not diminish peptides activities as demonstrated by ability of KGF-ELP to accelerate keratinocyte proliferation and migration, and ARA290-ELP to protect cells from apoptosis. We examined the healing effect of ARA290-ELP and KGF-ELP alone or in combination, in a full-thickness diabetic wound model. In this model, ARA290-ELP was found to accelerate healing, notably by increasing angiogenesis in the wound bed. We further showed that co-delivery of ARA290 and KGF, with the 1:4 KGF-ELP to ARA290-ELP ratio, was the most effective wound treatment with the fastest healing rate, the thicker granulation tissue and regenerated epidermis after 28 days. Overall, this study shows that ARA290-ELP and KGF-ELP constitute promising new therapeutics for treatment of chronic wounds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experimental model of cultured keratinocytes Modelo experimental de cultura de queratinócitos

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Alfredo Gragnani

2003-01-01

Full Text Available The bioengineering research is essential in the development of ideal combination of biomaterials and cultured cells to produce the permanent wound coverage. The experimental model of cultured keratinocytes presents all steps of the culture, since the isolation of the keratinocytes, preparation of the human acellular dermis, preparation of the composite skin graft and their elevation to the air-liquid interface. The research in cultured keratinocytes model advances in two main ways: 1. optimization of the methods in vitro to the skin cells culture and proliferation and 2. developing biomaterials that present similar skin properties.A pesquisa em bioengenharia é primordial no desenvolvimento da combinação ideal de biomateriais e células cultivadas para produzir a cobertura definitiva das lesões. O modelo experimental da cultura de queratinócitos apresenta toda as etapas do cultivo, desde o isolamento dos queratinócitos, preparação da derme acelular humana, do enxerto composto e da sua elevação à interface ar-líquido. A pesquisa em modelo de cultura de queratinócitos desenvolve-se em duas vias principais: 1. otimização dos métodos in vitro para cultivo e proliferação de células da pele e 2. desenvolvimento de biomateriais que mimetizem as propriedades da pele.

Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately.

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Shi, Yan; Shu, Bin; Yang, Ronghua; Xu, Yingbin; Xing, Bangrong; Liu, Jian; Chen, Lei; Qi, Shaohai; Liu, Xusheng; Wang, Peng; Tang, Jinming; Xie, Julin

2015-06-16

Wnt and Notch signaling pathways are critically involved in relative cell fate decisions within the development of cutaneous tissues. Moreover, several studies identified the above two pathways as having a significant role during wound healing. However, their biological effects during cutaneous tissues repair are unclear. We employed a self-controlled model (Sprague-Dawley rats with full-thickness skin wounds) to observe the action and effect of Wnt/β-catenin and Notch signalings in vivo. The quality of wound repair relevant to the gain/loss-of-function Wnt/β-catenin and Notch activation was estimated by hematoxylin-and-eosin and Masson staining. Immunofluorescence analysis and Western blot analysis were used to elucidate the underlying mechanism of the regulation of Wnt and Notch signaling pathways in wound healing. Meanwhile, epidermal stem cells (ESCs) were cultured in keratinocyte serum-free medium with Jaggedl or in DAPT (N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl) to investigate whether the interruption of Notch signaling contributes to the expression of Wnt/β-catenin signaling. The results showed that in vivo the gain-of-function Wnt/β-catenin and Notch activation extended the ability to promote wound closure. We further determined that activation or inhibition of Wnt signaling and Notch signaling can affect the proliferation of ESCs, the differentiation and migration of keratinocytes, and follicle regeneration by targeting c-Myc and Hes1, which ultimately lead to enhanced or delayed wound healing. Furthermore, Western blot analysis suggested that the two pathways might interact in vivo and in vitro. These results suggest that Wnt and Notch signalings play important roles in cutaneous repair by targeting c-Myc and Hes1 separately. What's more, interaction between the above two pathways might act as a vital role in regulation of wound healing.

Adenoviral gene delivery to primary human cutaneous cells and burn wounds.

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Hirsch, Tobias; von Peter, Sebastian; Dubin, Grzegorz; Mittler, Dominik; Jacobsen, Frank; Lehnhardt, Markus; Eriksson, Elof; Steinau, Hans-Ulrich; Steinstraesser, Lars

2006-01-01

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has

Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

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Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

2016-08-11

The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections.

A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures

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Lee, Jee Yong; Kim, Hyun Ji; Yi, Jae Youn

2016-01-01

A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allows communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation-induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-(PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. Control of inflammatory waves, improved wound healing, and stabilization of the skin barrier are imperative for minimizing such injuries. Therefore, PPARα agonists and antagonists have the potential to become important therapeutic agents for the treatment of γ-irradiation induced skin damage. Specifically, our analysis suggests that the undesirable consequences of long-term exposure to ionizing radiation could be alleviated by PPARα agonists

A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures

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Lee, Jee Yong; Kim, Hyun Ji; Yi, Jae Youn [Korea Institute of Radiation and Medical Sciences, Daejeon (Korea, Republic of)

2016-05-15

A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allows communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation-induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes in genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-(PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. Control of inflammatory waves, improved wound healing, and stabilization of the skin barrier are imperative for minimizing such injuries. Therefore, PPARα agonists and antagonists have the potential to become important therapeutic agents for the treatment of γ-irradiation induced skin damage. Specifically, our analysis suggests that the undesirable consequences of long-term exposure to ionizing radiation could be alleviated by PPARα agonists.

Wound Healing Potential of Chlorogenic Acid and Myricetin-3-O-β-Rhamnoside Isolated from Parrotia persica

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Sara E. Moghadam

2017-09-01

Full Text Available Wound healing is a complex physiological process that is controlled by a well-orchestrated cascade of interdependent biochemical and cellular events, which has spurred the development of therapeutics that simultaneously target these active cellular constituents. We assessed the potential of Parrotia persica (Hamamelidaceae in wound repair by analyzing the regenerative effects of its two main phenolic compounds, myricetin-3-O-β-rhamnoside and chlorogenic acid. To accomplish this, we performed phytochemical profiling and characterized the chemical structure of pure compounds isolated from P. persica, followed by an analysis of the biological effects of myricetin-3-O-β-rhamnoside and chlorogenic acid on three cell types, including keratinocytes, fibroblasts, and endothelial cells. Myricetin-3-O-β-rhamnoside and chlorogenic acid exhibited complementary pro-healing properties. The percentage of keratinocyte wound closure as measured by a scratch assay was four fold faster in the presence of 10 µg/mL chlorogenic acid, as compared to the negative control. On the other hand, myricetin-3-O-β-rhamnoside at 10 µg/mL was more effective in promoting fibroblast migration, demonstrating a two-fold higher rate of closure compared to the negative control group. Both compounds enhanced the capillary-like tube formation of endothelial cells in an in vitro angiogenesis assay. Our results altogether delineate the potential to synergistically accelerate the fibroblastic and remodelling phases of wound repair by administering appropriate amounts of myricetin-3-O-β-rhamnoside and chlorogenic acid.

Polydeoxyribonucleotide improves wound healing of fractional laser resurfacing in rat model.

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Yu, Mi; Lee, Jun Young

2017-02-01

Polydeoxyribonucleotide (PDRN) is an active compound that can promote wound healing. PDRN stimulates wound healing by enhancing angiogenesis and increasing fibroblast growth rates. Laser skin resurfacing is a popular cosmetic procedure for skin rejuvenation. Despite excellent improvement of photo-damaged skin and acne scarring, it is accompanied with drawbacks, such as prolonged erythema and crusting. This study was designed to assess the effect of PDRN on wounds induced by fractional laser resurfacing. Twelve male rats aged 8 weeks were randomly assigned to the PDRN treatment group and the control group. Wounds were induced using a fractional ablative CO 2 laser. The treatment group received daily injections of PDRN and the control group received injections of the vehicle. Wound healing assessed by clinical features and histopathologic findings. The process of wound healing was faster in the treatment group than in the control group. In the histopathological examination, the granulation tissue thickness score of the treatment group was significantly higher than that of the control group. Results of immunohistochemical staining showed a marked increase of VEGF-positive cells and PECAM-1/CD31-positive microvessels in the treatment group. PDRN may be a beneficial option to promote wound healing after laser treatment.

Antioxidant Sol-Gel Improves Cutaneous Wound Healing in Streptozotocin-Induced Diabetic Rats

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Lee, Yen-Hsien; Chang, Jung-Jhih; Chien, Chiang-Ting; Yang, Ming-Chien; Chien, Hsiung-Fei

2012-01-01

We examined the effects of vitamin C in Pluronic F127 on diabetic wound healing. Full-thickness excision skin wounds were made in normal and diabetic Wistar rats to evaluate the effect of saline, saline plus vitamin C (antioxidant sol), Pluronic F127, or Pluronic F127 plus vitamin C (antioxidant sol-gel). The rate of wound contraction, the levels of epidermal and dermal maturation, collagen synthesis, and apoptosis production in the wound tissue were determined. In vitro data showed that after 6 hours of air exposure, the order of the scavenging abilities for HOCl, H2O2, and O2  − was antioxidant sol-gel > antioxidant saline > Pluronic F127 = saline. After 7 and 14 days of wound injury, the antioxidant sol-gel improved wound healing significantly by accelerated epidermal and dermal maturation, an increase in collagen content, and a decrease in apoptosis formation. However, the wounds of all treatments healed mostly at 3 weeks. Vitamin C in Pluronic F127 hastened cutaneous wound healing by its antioxidant and antiapoptotic mechanisms through a good drug delivery system. This study showed that Pluronic F127 plus vitamin C could potentially be employed as a novel wound-healing enhancer. PMID:22919368

Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

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Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

2010-10-01

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Developmental Biologists.

Interactions of methicillin resistant Staphylococcus aureus USA300 and Pseudomonas aeruginosa in polymicrobial wound infection.

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Irena Pastar

Full Text Available Understanding the pathology resulting from Staphylococcus aureus and Pseudomonas aeruginosa polymicrobial wound infections is of great importance due to their ubiquitous nature, increasing prevalence, growing resistance to antimicrobial agents, and ability to delay healing. Methicillin-resistant S. aureus USA300 is the leading cause of community-associated bacterial infections resulting in increased morbidity and mortality. We utilized a well-established porcine partial thickness wound healing model to study the synergistic effects of USA300 and P. aeruginosa on wound healing. Wound re-epithelialization was significantly delayed by mixed-species biofilms through suppression of keratinocyte growth factor 1. Pseudomonas showed an inhibitory effect on USA300 growth in vitro while both species co-existed in cutaneous wounds in vivo. Polymicrobial wound infection in the presence of P. aeruginosa resulted in induced expression of USA300 virulence factors Panton-Valentine leukocidin and α-hemolysin. These results provide evidence for the interaction of bacterial species within mixed-species biofilms in vivo and for the first time, the contribution of virulence factors to the severity of polymicrobial wound infections.

Keratinocyte Motility Is Affected by UVA Radiation—A Comparison between Normal and Dysplastic Cells

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Cristina M. Niculiţe

2018-06-01

Full Text Available UVA radiation induces multiple and complex changes in the skin, affecting epidermal cell behavior. This study reports the effects of UVA exposure on normal (HaCaT and dysplastic (DOK keratinocytes. The adherence, spreading and proliferation were investigated by time-lapse measurement of cell layer impedance on different matrix proteins. Prior to UVA exposure, the time required for adherence and spreading did not differ significantly for HaCaT and DOK cells, while spreading areas were larger for HaCaT cells. Under UVA exposure, HaCaT and DOK cells behavior differed in terms of movement and proliferation. The cells’ ability to cover the denuded surface and individual cell trajectories were recorded by time-lapse videomicroscopy, during wound healing experiments. Dysplastic keratinocytes showed more sensitivity to UVA, exhibiting transient deficiencies in directionality of movement and a delay in re-coating the denuded area. The actin cytoskeleton displayed a cortical organization immediately after irradiation, in both cell lines, similar to mock-irradiated cells. Post-irradiation, DOK cells displayed a better organization of stress fibers, persistent filopodia, and new, stronger focal contacts. In conclusion, after UVA exposure HaCaT and DOK cells showed a different behavior in terms of adherence, spreading, motility, proliferation, and actin cytoskeleton dynamics, with the dyplastic keratinocytes being more sensitive.

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

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Sunita Nayak

Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Improved Clinical Efficacy with Wound Support Network Between Hospital and Home Care Service.

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Bergersen, Tone Kristin; Storheim, Elisabeth; Gundersen, Stina; Kleven, Linn; Johnson, Maria; Sandvik, Leiv; Kvaerner, Kari Jorunn; Ørjasæter, Nils-Otto

2016-11-01

The aim of this study was to test the efficacy of a wound support network model between the primary home care service and the hospital. The impact on wound healing rate, cost benefit, and transfer of knowledge was investigated. The intervention group was exposed to a wound support network (n = 32), and the control group continued standard organization of treatment (n = 21). Nonrandomized controlled study; observations were made before (baseline) and after the implementation of the intervention (12 weeks). Patients with chronic wounds (lasting >6 weeks and with wound area >1 cm) in Oslo, Norway. Closure of the observation wound; wound size; total number of wounds; presence of eczema, edema, and pain; number of dressings per week; time spent per dressing; and number of control appointments at the hospital. The economic impact is calculated for the hospital and for the community of Oslo, Norway. The number of control appointments (t = 3.80, P home care service and the hospital is cost-effective, improves clinical efficacy of the home care services' work, and reduces the need for consultations at the hospital.

In-vitro Wound Healing Effect of 15-Hydroxyprostaglandin Dehydrogenase Inhibitor from Plant.

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Karna, Sandeep

2017-01-01

Prostaglandins (PGs) have short existence in vivo because they are rapidly metabolized by NAD + -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to 15-ketoprostaglandins. Inhibition of 15-PGDH causes elevated level of PGE 2 in cellular system. It will be valuable for the therapeutic management of diseases requiring elevated PGE 2 levels, like wound healing. Ninety-eight plant samples were screened for the discovery of potent 15-PGDH inhibitor. Among them, top five plant extracts as potent 15-PGDH inhibitor were chosen to determine PGE 2 release from HaCaT (Keratinocyte cell line) cell line. Finally, top 15-PGDH inhibitor was selected to evaluate in vitro wound healing effect on HaCaT scratch model. The inhibitory activity for 15-PGDH inhibitors was evaluated using fluorescence spectrophotometer by measuring the formation of NADH at 468 nm following excitation at 340 nm. Cell viability assay and PGE 2 release was evaluated in HaCaT cell line after treatment of 15-PGDH inhibitors. Scratches were made using sterile 200 μL on HaCaT cell and wound-healing effect was evaluated after treatment of 15-PGDH inhibitor. 15-PGDH inhibitors elevated PGE 2 levels in concentration-dependent manner. Ethanol extract of Artocarpus heterophyllus (EEAH), the most potent 15-PGDH inhibitor (IC 50 = 0.62 µg/mL) with least cytotoxicity (IC 50 = 670 µg/ml), elevated both intracellular and extracellular PGE 2 levels. EEAH facilitated in-vitro wound healing in a HaCaT (Keratinocyte cell line) scratch model. EEAH might apply to treat dermal wounds by elevating PGE 2 levels via COX-1 induction and 15-PGDH inhibition. Biological inactivation of 15-PGDH causes elevated level of PGE 2 which will be useful for the management of disease that requires elevated level of PGE 2 . Abbreviations used: 15-PGDH: 15-hydroxyprostaglandin dehydrogenase, COX: Cyclooxygenase, DTT: Dithiothreitol, DMEM: Dulbecco's modified Eagle's media, EEAH: Ethanol extract of Artocarpus heterophyllus, MRP4

Fibroblast Senescence and Squamous Cell Carcinoma: How wounding therapies could be protective

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Travers, Jeffrey B.; Spandau, Dan F; Lewis, Davina A.; Machado, Christiane; Kingsley, Melanie; Mousdicas, Nico; Somani, Ally-Khan

2014-01-01

Background Squamous cell carcinoma (SCC), which has one of the highest incidences of all cancers in the United States, is an age-dependent disease as the majority of these cancers are diagnosed in people over 70 years of age. Recent findings have led to a new hypothesis on the pathogenesis of SCC. Objectives To evaluate the potential of preventive therapies to reduce the incidence of SCC in at-risk geriatric patients. Materials and Methods Survey of current literature on wounding therapies to prevent SCCs. Results This new hypothesis of SCC photocarcinogenesis states that senescent fibroblasts accumulate in geriatric dermis resulting in a reduction in dermal insulin-like growth factor-1 (IGF-1) expression. This lack of IGF-1 expression sensitizes epidermal keratinocytes to fail to suppress UVB-induced mutations leading to increased proclivity to photocarcinogenesis. Recent evidence suggests that dermal wounding therapies, specifically dermabrasion and fractionated laser resurfacing, can decrease the proportion of senescent dermal fibroblasts, increase dermal IGF-1 expression, and correct the inappropriate UVB response found in geriatric skin, thus protecting geriatric keratinocytes from UVB-induced SCC initiation. Conclusions In this review, we will discuss the translation of pioneering basic science results implicating commonly used dermal fibroblast rejuvenation procedures as preventative treatments for SCC. PMID:23437969

Evaluation of the effect of radiation levels and dose rates in irradiation of murine fibroblasts used as a feeder layer in the culture of human keratinocytes

International Nuclear Information System (INIS)

Yoshito, Daniele; Almeida, Tiago L.; Santin, Stefany Plumeri; Somessari, Elizabeth S.R.; Silveira, Carlos G. da; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

2009-01-01

In 1975, Rheinwald and Green published an effective methodology for obtaining and cultivating human keratinocytes. This methodology consisted of seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate of which is then controlled through the action of ionizing radiation. The presence of the feeder layer encourages the development of keratinocyte colonies and their propagation in similar cultures, becoming possible several clinical applications as skin substitutes or wound dressings in situations such as post burn extensive skin loss and other skin disorders. However, good development of these keratinocytes depends on a high quality feeder layer among other factors. In the present work, we evaluated the relationship between radiation levels and dose rates applied to fibroblasts used in construction of feeder layers and the radiation effect on keratinocytes colonies forming efficiency. Results indicate 3T3 lineage murine fibroblasts irradiated with doses varying between 60 and 100 Gy can be used as a feeder layer immediately after irradiation or storage of the irradiated cells in suspension at 4 g C for 24 hours with similar results. The exception is when the irradiation dose rate is 2.75 Gyh -1 ; in this case, results suggested that the fibroblasts should be used immediately after irradiation. (author)

Bioinspired porous membranes containing polymer nanoparticles for wound healing.

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Ferreira, Ana M; Mattu, Clara; Ranzato, Elia; Ciardelli, Gianluca

2014-12-01

Skin damages covering a surface larger than 4 cm(2) require a regenerative strategy based on the use of appropriate wound dressing supports to facilitate the rapid tissue replacement and efficient self-healing of the lost or damaged tissue. In the present work, A novel biomimetic approach is proposed for the design of a therapeutic porous construct made of poly(L-lactic acid) (PLLA) fabricated by thermally induced phase separation (TIPS). Biomimicry of ECM was achieved by immobilization of type I collagen through a two-step plasma treatment for wound healing. Anti-inflammatory (indomethacin)-containing polymeric nanoparticles (nps) were loaded within the porous membranes in order to minimize undesired cell response caused by post-operative inflammation. The biological response to the scaffold was analyzed by using human keratinocytes cell cultures. In this work, a promising biomimetic construct for wound healing and soft tissue regeneration with drug-release properties was fabricated since it shows (i) proper porosity, pore size, and mechanical properties, (ii) biomimicry of ECM, and (iii) therapeutic potential. © 2014 Wiley Periodicals, Inc.

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling.

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Singkhorn, Sawana; Tantisira, Mayuree H; Tanasawet, Supita; Hutamekalin, Pilaiwanwadee; Wongtawatchai, Tulaporn; Sukketsiri, Wanida

2018-03-13

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration- and time-dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1-100 μg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound. Copyright © 2018 John Wiley & Sons, Ltd.

Protocol for a systematic review of the efficacy of fat grafting and platelet-rich plasma for wound healing.

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Smith, Oliver J; Kanapathy, Muholan; Khajuria, Ankur; Prokopenko, Max; Hachach-Haram, Nadine; Mann, Haroon; Mosahebi, Ash

2017-06-06

The use of fat grafting as a reconstructive surgical option is becoming much more common. Adipose-derived stem cells found in fat grafts are believed to facilitate wound healing via differentiation into fibroblasts and keratinocytes and the release of pro-healing growth factors. Several small studies have shown a positive effect of fat grafting in healing of wounds of a variety of aetiologies. When fat is combined with autologous platelet-rich plasma (PRP), there may be enhanced healing effects. This may be due to the pro-angiogenic and anti-inflammatory effects of PRP. We aim to synthesise the current evidence on combination fat grafting and PRP for wound healing to establish the efficacy of this technique. We will conduct a comprehensive literature search in the MEDLINE, EMBASE, CENTRAL, Science Citation Index, and Google Scholar databases (up to July 2017) to identify studies on fat grafting and PRP for wound healing. All primary studies and systematic reviews of these studies will be included, except case reports and case series with fewer than three patients, to evaluate the outcome of fat grafting and PRP on wound healing either on its own or when compared to other studies. Primary outcome measures are expected to be the proportion of total wounds healed at 12 weeks and the average wound healing time (time for 100% re-epithelialisation). Expected secondary outcome measures are the proportion of wounds achieving 50% wound healing, the type of wound benefitting most from fat grafting, economic evaluation, health-related quality of life, and adverse events. Subgroup analysis will be performed for the proportions of wounds healed based on wound aetiology. This review will provide robust evidence of the efficacy of fat grafting and PRP for wound healing. This is an emerging technique, and this review is expected to guide clinical practice and ongoing research aimed at improving wound care. PROSPERO CRD42016049881.

Thyrotropin-releasing hormone (TRH promotes wound re-epithelialisation in frog and human skin.

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Natalia T Meier

Full Text Available There remains a critical need for new therapeutics that promote wound healing in patients suffering from chronic skin wounds. This is, in part, due to a shortage of simple, physiologically and clinically relevant test systems for investigating candidate agents. The skin of amphibians possesses a remarkable regenerative capacity, which remains insufficiently explored for clinical purposes. Combining comparative biology with a translational medicine approach, we report the development and application of a simple ex vivo frog (Xenopus tropicalis skin organ culture system that permits exploration of the effects of amphibian skin-derived agents on re-epithelialisation in both frog and human skin. Using this amphibian model, we identify thyrotropin-releasing hormone (TRH as a novel stimulant of epidermal regeneration. Moving to a complementary human ex vivo wounded skin assay, we demonstrate that the effects of TRH are conserved across the amphibian-mammalian divide: TRH stimulates wound closure and formation of neo-epidermis in organ-cultured human skin, accompanied by increased keratinocyte proliferation and wound healing-associated differentiation (cytokeratin 6 expression. Thus, TRH represents a novel, clinically relevant neuroendocrine wound repair promoter that deserves further exploration. These complementary frog and human skin ex vivo assays encourage a comparative biology approach in future wound healing research so as to facilitate the rapid identification and preclinical testing of novel, evolutionarily conserved, and clinically relevant wound healing promoters.

Thyrotropin-Releasing Hormone (TRH) Promotes Wound Re-Epithelialisation in Frog and Human Skin

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Zhang, Guo-You; Emelianov, Vladimir; Paredes, Roberto; Debus, Sebastian; Augustin, Matthias; Funk, Wolfgang; Amaya, Enrique; Kloepper, Jennifer E.; Hardman, Matthew J.; Paus, Ralf

2013-01-01

There remains a critical need for new therapeutics that promote wound healing in patients suffering from chronic skin wounds. This is, in part, due to a shortage of simple, physiologically and clinically relevant test systems for investigating candidate agents. The skin of amphibians possesses a remarkable regenerative capacity, which remains insufficiently explored for clinical purposes. Combining comparative biology with a translational medicine approach, we report the development and application of a simple ex vivo frog (Xenopus tropicalis) skin organ culture system that permits exploration of the effects of amphibian skin-derived agents on re-epithelialisation in both frog and human skin. Using this amphibian model, we identify thyrotropin-releasing hormone (TRH) as a novel stimulant of epidermal regeneration. Moving to a complementary human ex vivo wounded skin assay, we demonstrate that the effects of TRH are conserved across the amphibian-mammalian divide: TRH stimulates wound closure and formation of neo-epidermis in organ-cultured human skin, accompanied by increased keratinocyte proliferation and wound healing-associated differentiation (cytokeratin 6 expression). Thus, TRH represents a novel, clinically relevant neuroendocrine wound repair promoter that deserves further exploration. These complementary frog and human skin ex vivo assays encourage a comparative biology approach in future wound healing research so as to facilitate the rapid identification and preclinical testing of novel, evolutionarily conserved, and clinically relevant wound healing promoters. PMID:24023889

Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice.

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Lei Chen

Full Text Available Growing evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs enhance wound repair via paracrine. Because the extent of environmental oxygenation affects the innate characteristics of BM-MSCs, including their stemness and migration capacity, the current study set out to elucidate and compare the impact of normoxic and hypoxic cell-culture conditions on the expression and secretion of BM-MSC-derived paracrine molecules (e.g., cytokines, growth factors and chemokines that hypothetically contribute to cutaneous wound healing in vivo. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA analyses of normoxic and hypoxic BM-MSCs and their conditioned medium fractions showed that the stem cells expressed and secreted significantly higher amounts of basic fibroblast growth factor (bFGF,vascular endothelial growth factor A (VEGF-A interleukin 6 (IL-6 and interleukin 8 (IL-8 under hypoxic conditions. Moreover, hypoxic BM-MSC-derived conditioned medium (hypoCM vs. normoxic BM-MSC-derived conditioned medium (norCM or vehicle control medium significantly enhanced the proliferation of keratinocytes, fibroblasts and endothelial cells, the migration of keratinocytes, fibroblasts, endothelial cells and monocytes, and the formation of tubular structures by endothelial cells cultured on Matrigel matrix. Consistent with these in vitro results, skin wound contraction was significantly accelerated in Balb/c nude mice treated with topical hypoCM relative to norCM or the vehicle control. Notably increased in vivo cell proliferation, neovascularization as well as recruitment of inflammatory macrophages and evidently decreased collagen I, and collagen III were also found in the hypoCM-treated group. These findings suggest that BM-MSCs promote murine skin wound healing via hypoxia-enhanced paracrine.

Comparison of In-Vitro and Ex-Vivo Wound Healing Assays for the Investigation of Diabetic Wound Healing and Demonstration of a Beneficial Effect of a Triterpene Extract.

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Christopher Ueck

Full Text Available Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH, in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05 compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in

Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

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Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A; Murdoch, Jennifer N; Humbert, Patrick O; Parekh, Vishwas; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M; Jane, Stephen M

2010-07-20

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair. (c) 2010 Elsevier Inc. All rights reserved.

Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells.

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Boink, Mireille A; van den Broek, Lenie J; Roffel, Sanne; Nazmi, Kamran; Bolscher, Jan G M; Gefen, Amit; Veerman, Enno C I; Gibbs, Susan

2016-01-01

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation. © 2015 by the Wound Healing Society.

Wound healing stimulation in mice by low-level light

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Demidova, Tatiana N.; Herman, Ira M.; Salomatina, Elena V.; Yaroslavsky, Anna N.; Hamblin, Michael R.

2006-02-01

It has been known for many years that low levels of laser or non-coherent light (LLLT) accelerate some phases of wound healing. LLLT can stimulate fibroblast and keratinocyte proliferation and migration. It is thought to work via light absorption by mitochondrial chromophores leading to an increase in ATP, reactive oxygen species and consequent gene transcription. However, despite many reports about the positive effects of LLLT on wound healing, its use remains controversial. Our laboratory has developed a model of a full thickness excisional wound in mice that allows quantitative and reproducible light dose healing response curves to be generated. We have found a biphasic dose response curve with a maximum positive effect at 2 J/cm2 of 635-nm light and successively lower beneficial effects from 3-25 J/cm2, the effect is diminished at doses below 2J/cm2 and gradually reaches control healing levels. At light doses above 25 J/cm2 healing is actually worse than controls. The two most effective wavelengths of light were found to be 635 and 820-nm. We found no difference between filtered 635+/-15-nm light from a lamp and 633-nm light from a HeNe laser. The strain and age of the mouse affected the magnitude of the effect. Light treated wounds start to contract after illumination while control wounds initially expand for the first 24 hours. Our hypothesis is that a single brief light exposure soon after wounding affects fibroblast cells in the margins of the wound. Cells may be induced to proliferate, migrate and assume a myofibroblast phenotype. Our future work will be focused on understanding the mechanisms underlying effects of light on wound healing processes.

Upregulation of cathepsin S in psoriatic keratinocytes.

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Schönefuss, Alexander; Wendt, Wiebke; Schattling, Benjamin; Schulten, Roxane; Hoffmann, Klaus; Stuecker, Markus; Tigges, Christian; Lübbert, Hermann; Stichel, Christine

2010-08-01

Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II-mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS-immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T- and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS-immunostaining is also detectable in keratinocytes. We show that cocultivation with T-cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T-cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II-associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

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Qian-Shi Zhang

Full Text Available Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1, also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5, after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1, the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93, or the downstream target, c-Jun N-terminal kinase (SP600125 also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580 had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration.

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration

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Zhang, Qian-Shi; Kurpad, Deepa S.; Mahoney, My G.; Steinbeck, Marla J.

2017-01-01

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration. PMID:29045420

Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

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Hou, Jingang; Kim, Sunchang

2018-05-05

Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Preliminary Characterization of Genipin-Cross-Linked Silk Sericin/Poly(vinyl alcohol Films as Two-Dimensional Wound Dressings for the Healing of Superficial Wounds

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Tippawan Siritientong

2013-01-01

Full Text Available The genipin-cross-linked silk sericin/poly(vinyl alcohol (PVA films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1% w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds.

Chitosan-aluminum monostearate composite sponge dressing containing asiaticoside for wound healing and angiogenesis promotion in chronic wound.

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Phaechamud, Thawatchai; Yodkhum, Kotchamon; Charoenteeraboon, Juree; Tabata, Yasuhiko

2015-05-01

There are many factors that delay healing in chronic wounds including lowering level of growth factors and increasing exudate level comprising high amount of tissue destructive enzymes. Asiaticoside possesses interesting wound healing and angiogenic activities that are employed to stimulate tissue regeneration in wound healing application. This study attempted to develop chitosan-aluminum monostearate (Alst) composite sponge containing asiaticoside for use as an absorbent medical dressing in chronic wound. N-methyl-2-pyrrolidone (NMP) was used to enhance homogeneity of asiaticoside in the polymer composite matrix. The sponge dressings were prepared by lyophilization and dehydrothermal treatment (DHT). Functional group interaction, crystallinity, and morphology of the prepared sponges were investigated using FT-IR, PXRD, and SEM, respectively. Physicochemical properties, porosity, hydrophilic/hydrophobic properties and mechanical property, were evaluated. Wound dressing properties, water vapor transmission rate (WVTR), fluid absorbency, oxygen permeation (OP), and bio-adhesive property, were investigated. In vitro asiaticoside release study was conducted using immersion method. Cytotoxicity was studied in normal human dermal fibroblast (NHDF) and normal human epidermal keratinocyte (NHEK). Angiogenic activity of asiaticoside was evaluated using chick-chorioallantoic membrane (CAM) assay. FT-IR and PXRD results revealed the amidation after DHT to enhance the crystallinity of the prepared sponges. The prepared sponges had high porosity comprising high Alst-loaded amount that exhibited more compact structure. Alst enhanced hydrophobicity therefore it reduced the fluid absorption and WVTR together with bio-adhesion of the prepared sponge dressings. Porosity of all sponges was more than 85% therefore resulting in their high OP. Enhancing hydrophobicity of the material by Alst and more homogeneity caused by NMP eventually retarded the asiaticoside release for 7 days. The

Different types of biotechnological wound coverages created with the application of alive human cells

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Papuga A. Ye.

2015-04-01

Full Text Available Currently, the development and the implementation of the new biotechnological wound coverings (skin equivalents designed for temporal or permanent replacement of damaged or destroyed areas of human skin remains extremely actual relevant in clinical practice. Skin equivalents or equivalents of individual skin layers which include alive cells of different types take a special place among the artificial wound coverings. They mostly contain two basic types of cells – fibroblasts and keratinocytes (together or separately. Such bioconstructions are usually served as temporary coverings, which supply the damaged skin by biologically active substances and stimulate the regeneration of the patient's own tissues. In this review we consider as commercially available wound coverings and those which are still studied in the laboratories. Until now ideal substitutes of natural skin have not yet created, so the efforts of many researchers are focusing on the solution of this problem.

Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

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Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

2012-04-10

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

Induction of proliferation of basal epidermal keratinocytes by cold atmospheric-pressure plasma.

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Hasse, S; Duong Tran, T; Hahn, O; Kindler, S; Metelmann, H-R; von Woedtke, T; Masur, K

2016-03-01

Over the past few decades, new cold plasma sources have been developed that have the great advantage of operating at atmospheric pressure and at temperatures tolerable by biological material. New applications for these have emerged, especially in the field of dermatology. Recently it was demonstrated that cold atmospheric-pressure plasma positively influences healing of chronic wounds. The potential of cold plasma lies in its capacity to reduce bacterial load in the wound while at the same time stimulating skin cells and therefore promoting wound closure. In recent years, there have been great advances in the understanding of the molecular mechanisms triggered by cold plasma involving signalling pathways and gene regulation in cell culture. To investigate cold plasma-induced effects in ex vivo treated human skin biopsies. Human skin tissue was exposed to cold plasma for different lengths of time, and analysed by immunofluorescence with respect to DNA damage, apoptosis, proliferation and differentiation markers. After cold plasma treatment, the epidermal integrity and keratin expression pattern remained unchanged. As expected, the results revealed an increase in apoptotic cells after 3 and 5 min of treatment. Strikingly, an induction of proliferating basal keratinocytes was detected after cold plasma exposure for 1 and 3 min. As these are the cells that regenerate the epidermis, this could indeed be beneficial for wound closure. We investigated the effect of cold plasma on human skin by detecting molecules for growth and apoptosis, and found that both processes are dependent on treatment time. Therefore, this approach offers promising results for further applications of cold plasma in clinical dermatology. © 2015 British Association of Dermatologists.

Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation.

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Roy, Sashwati; Driggs, Jason; Elgharably, Haytham; Biswas, Sabyasachi; Findley, Muna; Khanna, Savita; Gnyawali, Urmila; Bergdall, Valerie K; Sen, Chandan K

2011-11-01

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers. 2011 by the Wound Healing Society.

Inhibition of inflammatory and proliferative responses of human keratinocytes exposed to the sesquiterpene lactones dehydrocostuslactone and costunolide.

Directory of Open Access Journals (Sweden)

Claudia Scarponi

Full Text Available The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE and costunolide (CS, two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit

The effect of anthocyanins from red wine and blackberry on the integrity of a keratinocyte model using ECIS.

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Évora, Ana; de Freitas, Victor; Mateus, Nuno; Fernandes, Iva

2017-11-15

There is a growing market demand for the incorporation of plant-derived ingredients into new products for the cosmetic industry. Anthocyanins are polyphenols arising from plant secondary metabolism that have been shown to possess many bioactive properties such as free radical scavenging, antimicrobial, and chemopreventive activities. In this work, the biological activities of red wine and blackberry anthocyanins were assessed by developing a new keratinocyte barrier model using the HaCat cell line and a microelectrode-based biosensor device, referred to as Electric Cell-Substrate Impedance Sensing (ECIS). Cells were seeded at the optimal cellular density of 1.6 × 10 6 cells per mL and the half-time was calculated to be 3.55 ± 0.67 hours. The compounds' cytotoxicity was assessed and anthocyanin pigments showed no cytotoxicity towards keratinocyte cells. Wound healing assays were also performed using ECIS and it was observed that the tested pigments enhanced the healing rate of keratinocyte cells by reducing the healing time more than 50%. Cyanidin-3-glucoside presented the best results recovering 50% of the injured area in 1.48(±0.15) hours, followed by the blackberry anthocyanins (2.01 ± 0.18 hours), malvidin-3-glucoside (2.03 ± 0.09 hours) and red wine anthocyanins (2.36 ± 0.76 hours). All presented significant differences from the control 4.91(±1.11) hours.

Skin wound healing in different aged Xenopus laevis.

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Bertolotti, Evelina; Malagoli, Davide; Franchini, Antonella

2013-08-01

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8- and 15-month-old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re-epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti-inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti-tumor necrosis factor (TNF)-α, iNOS, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti-α-SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar-like pattern. The quantitative PCR analysis demonstrated an up-regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar-like tissue formation. Copyright © 2013 Wiley Periodicals, Inc.

A multi-centre clinical evaluation of reactive oxygen topical wound gel in 114 wounds.

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Dryden, M; Dickinson, A; Brooks, J; Hudgell, L; Saeed, K; Cutting, K F

2016-03-01

This article reports the outcomes of the use of Surgihoney RO (SHRO), topical wound dressing in a multi-centre, international setting. The aims were to explore the clinical effects of SHRO, including a reduction in bacterial load and biofilm and improvement in healing in a variety of challenging non-healing and clinically infected wounds. This was a non-comparative evaluation, where both acute and chronic wounds with established delayed healing were treated with the dressing. Clinicians prospectively recorded wound improvement or deterioration, level of wound exudate, presence of pain, and presence of slough and necrosis. Analysis of this data provided information on clinical performance of the dressing. Semi-quantitative culture to assess bacterial bioburden was performed where possible. We recruited 104 patients, mean age 61 years old, with 114 wounds. The mean duration of wounds before treatment was 3.7 months and the mean duration of treatment was 25.7 days. During treatment 24 wounds (21%) healed and the remaining 90 (79%) wounds improved following application of the dressing. No deterioration in any wound was observed. A reduction in patient pain, level of wound exudate and in devitalised tissue were consistently reported. These positive improvements in wound progress were reflected in the wound cultures that showed a reduction in bacterial load in 39 out of the 40 swabs taken. There were two adverse events recorded: a stinging sensation following application of the dressing was experienced by 2 patients, and 2 elderly patients died of causes unrelated to the dressing or to the chronic wound. These patients' wounds and their response to SHRO have been included in the analysis. SHRO was well tolerated and shows great promise as an effective potent topical antimicrobial in the healing of challenging wounds. Matthew Dryden has become a shareholder in Matoke Holdings, the manufacturer of Surgihoney RO, since the completion of this study. Keith Cutting is a

Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

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Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

2014-07-01

Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.

Toll like receptors gene expression of human keratinocytes cultured of severe burn injury.

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Cornick, Sarita Mac; Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Cezillo, Marcus V B; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

To evaluate the expression profile of genes related to Toll Like Receptors (TLR) pathways of human Primary Epidermal keratinocytes of patients with severe burns. After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific TLR pathways PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 21% of these genes were differentially expressed, of which 100% were repressed or hyporegulated. Among these, the following genes (fold decrease): HSPA1A (-58), HRAS (-36), MAP2K3 (-23), TOLLIP (-23), RELA (-18), FOS (-16), and TLR1 (-6.0). This study contributes to the understanding of the molecular mechanisms related to TLR pathways and underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

DNA Methylation Dynamics Regulate the Formation of a Regenerative Wound Epithelium during Axolotl Limb Regeneration.

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Cristian Aguilar

Full Text Available The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. We have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. We report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability.

5alpha-dihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization.

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Gilliver, S C; Ruckshanthi, J P D; Hardman, M J; Zeef, L A H; Ashcroft, G S

2009-01-01

The ongoing search for explanations as to why elderly males heal acute skin wounds more slowly than do their female counterparts (and are more strongly disposed to conditions of chronic ulceration) has identified endogenous oestrogens and androgens as being respectively enhancers and inhibitors of repair. We previously demonstrated that blocking the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) limits its ability to impair healing, suggesting that DHT is a more potent inhibitor of repair than is testosterone. The present study aimed to delineate the central mechanisms by which androgens delay repair. Whilst the contractile properties of neither rat wounds in vivo nor fibroblast-impregnated collagenous discs in vitro appeared to be influenced by androgen manipulations, the global blockade of DHT biosynthesis markedly accelerated re-epithelialization of incisional and excisional wounds and reduced local expression of beta-catenin, a key inhibitor of repair. Moreover, DHT retarded the in vitro migration of epidermal keratinocytes following scratch wounding. By contrast, it failed to influence the migratory and proliferative properties of dermal fibroblasts, suggesting that its primary inhibitory effect is upon re-epithelialization. These novel findings may be of particular significance in the context of chronic ulceration, for which being male is a key risk factor.

Human tissue inhibitor of metalloproteinases-1 improved wound healing in diabetes through its anti-apoptotic effect.

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Lao, Guojuan; Ren, Meng; Wang, Xiaoyi; Zhang, Jinglu; Huang, Yanrui; Liu, Dan; Luo, Hengcong; Yang, Chuan; Yan, Li

2017-09-08

Impaired wound healing accompanies severe cell apoptosis in diabetic patients. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was known to have effects on promoting growth and anti-apoptosis for cells. We aimed to determine the actual levels of TIMP-1 and cell apoptosis in: (i) the biopsies of diabetic and non-diabetic foot tissue and (ii) the human fibroblasts with or without treatments of advanced glycation end-products (AGEs). Next, we aimed to determine the improved levels of cell apoptosis and wound healing after the treatments of either active protein of TIMP-1 or in vivo expression of gene therapy vector-mediated TIMP-1 in both the human fibroblasts and the animal model of diabetic rats. The levels of TIMP-1 were significantly reduced in diabetic skin tissues and in AGEs-treated fibroblasts. Both AGEs-treated cells were effectively protected from apoptosis by active protein of TIMP-1 at appropriate dose level. So did the induced in vivo TIMP-1 expression after gene delivery. Similar effects were also found on the significant improvement of impaired wound healing in diabetic rats. We concluded that TIMP-1 improved wound healing through its anti-apoptotic effect. Treatments with either active protein TIMP-1 or TIMP-1 gene therapy delivered in local wound sites may be used as a strategy for accelerating diabetic wound healing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Topical application of dressing with amino acids improves cutaneous wound healing in aged rats.

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Corsetti, Giovanni; D'Antona, Giuseppe; Dioguardi, Francesco Saverio; Rezzani, Rita

2010-09-01

The principal goal in treating surgical and non-surgical wounds, in particular for aged skin, is the need for rapid closure of the lesion. Cutaneous wound healing processes involve four phases including an inflammatory response with the induction of pro-inflammatory cytokines. If inflammation develops in response to bacterial infection, it can create a problem for wound closure. Reduced inflammation accelerates wound closure with subsequent increased fibroblast function and collagen synthesis. On the contrary, prolonged chronic inflammation results in very limited wound healing. Using histological and immunohistochemical techniques, we investigated the effects of a new wound dressing called Vulnamin that contains four essential amino acids for collagen and elastin synthesis plus sodium ialuronate (Na-Ial), compared with Na-Ial alone, in closure of experimental cutaneous wounds of aged rats. Our results showed that the application of Vulnamin dressings modulated the inflammatory response with a reduction in the number of inflammatory cells and inducible nitric oxide synthase (iNOS) immunolocalisation, while increasing endothelial nitric oxide synthase (eNOS) and transforming growth factor-beta1 (TGF-beta1) immunolocalisation. Furthermore, the dressing increased the distribution density of fibroblasts and aided the synthesis of thin collagen fibers resulting in a reduction in healing time. The nutritive approach using this new wound dressing can provide an efficacious and safe strategy to accelerate wound healing in elderly subjects, simplifying therapeutic procedures and leading to an improved quality of life. 2009 Elsevier GmbH. All rights reserved.

Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing.

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Maquerlot, François; Galiacy, Stephane; Malo, Michel; Guignabert, Christophe; Lawrence, Daniel A; d'Ortho, Maria-Pia; Barlovatz-Meimon, Georgia

2006-11-01

Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.

Improving outcomes following penetrating colon wounds: application of a clinical pathway.

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Miller, Preston R; Fabian, Timothy C; Croce, Martin A; Magnotti, Louis J; Elizabeth Pritchard, F; Minard, Gayle; Stewart, Ronald M

2002-06-01

managed in the CP group (27% vs. 19%). Abscess rate was lower in the CP group (27% vs. 37%), as was suture line leak rate (7% vs. 14%). Colon related mortality in the CP group was 5% as compared with 12% in the PS group. The clinical pathway for destructive colon wound management has improved outcomes as measured by anastomotic leak rates and colon related mortality. The data demonstrated the need for colostomy in the face of shock and comorbidities. Institution of this pathway results in colostomy for only 7% of all colon wounds.

Serial cultivation of human scalp hair follicle keratinocytes.

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Weterings, P J; Roelofs, H M; Vermorken, A J; Bloemendal, H

1983-01-01

A method is described for the serial cultivation of adult human hair follicle keratinocytes. Plucked scalp hair follicles, placed on bovine eye lens capsules as a growth substrate, give rise to quickly expanding colonies within a few days. After trypsinization, the cells are replated with irradiated 3T3 cells as 'feeders'. Using this combination of techniques the keratinocytes can be subcultured up to four times. In this way about 10(7) keratinocytes can be generated from one single hair follicle. Moreover, the technique enables cryogenic storage of the cells, allowing for instance, convenient transportation. Subcultured hair follicle keratinocytes can be plated on glass coverslips. This allows immunofluorescence studies. The keratin cytoskeletons visualized using an antiserum against human keratin.

Managing painful chronic wounds: the Wound Pain Management Model

DEFF Research Database (Denmark)

Price, Patricia; Fogh, Karsten; Glynn, Chris

2007-01-01

of the pain experience: location, duration, intensity, quality, onset and impact on activities of daily living. Holistic management must be based on a safe and effective mix of psychosocial approaches together with local and systemic pain management. It is no longer acceptable to ignore or inadequately...... to the wound should be handled as one of the main priorities in chronic wound management together with addressing the cause. Management of pain in chronic wounds depends on proper assessment, reporting and documenting patient experiences of pain. Assessment should be based on six critical dimensions...... document persistent wound pain and not to develop a treatment and monitoring strategy to improve the lives of persons with chronic wounds. Unless wound pain is optimally managed, patient suffering and costs to health care systems will increase. Udgivelsesdato: 2007-Apr...

Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

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Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

2015-01-01

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. PMID:25589789

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms, HaCaT keratinocytes and affects skin deposition.

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Holmes, Amy M; Lim, Julian; Studier, Hauke; Roberts, Michael S

2016-12-01

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC 50 ranges from 31.25 to 125 μg/mL depending on the bacterial species) and HaCaT keratinocytes (IC 50 78.65 μg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC 50 of 7.85 μg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.

Copper Metal-Organic Framework Nanoparticles Stabilized with Folic Acid Improve Wound Healing in Diabetes.

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Xiao, Jisheng; Zhu, Yunxiao; Huddleston, Samantha; Li, Peng; Xiao, Baixue; Farha, Omar K; Ameer, Guillermo A

2018-02-27

The successful treatment of chronic nonhealing wounds requires strategies that promote angiogenesis, collagen deposition, and re-epithelialization of the wound. Copper ions have been reported to stimulate angiogenesis; however, several applications of copper salts or oxides to the wound bed are required, leading to variable outcomes and raising toxicity concerns. We hypothesized that copper-based metal-organic framework nanoparticles (Cu-MOF NPs), referred to as HKUST-1, which are rapidly degraded in protein solutions, can be modified to slowly release Cu 2+ , resulting in reduced toxicity and improved wound healing rates. Folic acid was added during HKUST-1 synthesis to generate folic-acid-modified HKUST-1 (F-HKUST-1). The effect of folic acid incorporation on NP stability, size, hydrophobicity, surface area, and copper ion release profile was measured. In addition, cytotoxicity and in vitro cell migration processes due to F-HKUST-1 and HKUST-1 were evaluated. Wound closure rates were assessed using the splinted excisional dermal wound model in diabetic mice. The incorporation of folic acid into HKUST-1 enabled the slow release of copper ions, which reduced cytotoxicity and enhanced cell migration in vitro. In vivo, F-HKUST-1 induced angiogenesis, promoted collagen deposition and re-epithelialization, and increased wound closure rates. These results demonstrate that folic acid incorporation into HKUST-1 NPs is a simple, safe, and promising approach to control Cu 2+ release, thus enabling the direct application of Cu-MOF NPs to wounds.

Identification of p63+ keratinocyte progenitor cells in circulation and their matrix-directed differentiation to epithelial cells.

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Nair, Renjith P; Krishnan, Lissy K

2013-04-11

In the event of chronic diabetes or burn wounds, accomplishing skin regeneration is a major concern. Autologous skin grafting is the most effective remedy, but the tissue harvest may create more nonhealing wounds. Currently available skin substitutes have a limited clinical outcome because of immune reactions arising from the xenobiotic scaffold or allogenous cells. Autologous stem cells that can be collected without an additional injury may be a viable option for skin-tissue engineering. Presence of a low number of keratinocyte progenitor cells (KPCs) within the peripheral blood mononuclear cell (PBMNC) population has been indicated. Identification, isolation, expansion, and differentiation of KPCs is necessary before they are considered for skin regeneration, which is the focus of this study. Culture of isolated human PBMNCs on a cell-specific matrix was carried out to induce differentiation of KPCs. Flow cytometry and reverse transcriptase polymerase chain reaction were done for epithelial stem cell marker p63 and lineage markers cytokeratin 5 and cytokeratin 14, to track differentiation. Proliferation was confirmed by quantifying the proliferating cell nuclear antigen-expressing cells. Immunostaining with epithelial cell markers, involucrin and filaggrin, was carried out to establish terminal differentiation. Microscopic analysis confirmed growth and survival of KPCs on the dermal fibroblast monolayer and on a transplantable fibrin sheet. We demonstrated that KPCs are p63(+) and CD34-. The specifically designed composition of the extracellular matrix was found to support selective adhesion, proliferation, and differentiation of p63(+) KPCs. The PBMNC culture for 12 days under controlled conditions resulted in a homogenous population that expressed cytokeratins, and >90% of the cells were found to proliferate. Subculture for 5 days resulted in expression of filaggrin and involucrin, suggesting terminal differentiation. Transfer of matrix-selected KPCs to a

The role of secreted heat shock protein-90 (Hsp90) in wound healing - how could it shape future therapeutics?

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Guo, Jiacong; Chang, Cheng; Li, Wei

2017-08-01

Defects in tissue repair or wound healing pose a clinical, economic and social problem worldwide. Despite decades of studies, there have been few effective therapeutic treatments. Areas covered: We discuss the possible reasons for why growth factor therapy did not succeed. We point out the lack of human disorder-relevant animal models as another blockade for therapeutic development. We summarize the recent discovery of secreted heat shock protein-90 (Hsp90) as a novel wound healing agent. Expert commentary: Wound healing is a highly complex and multistep process that requires participations of many cell types, extracellular matrices and soluble molecules to work together in a spatial and temporal fashion within the wound microenvironment. The time that wounds remain open directly correlates with the clinical mortality associated with wounds. This time urgency makes the healing process impossible to regenerate back to the unwounded stage, rather forces it to take many shortcuts in order to protect life. Therefore, for therapeutic purpose, it is crucial to identify so-called 'driver genes' for the life-saving phase of wound closure. Keratinocyte-secreted Hsp90α was discovered in 2007 and has shown the promise by overcoming several key hurdles that have blocked the effectiveness of growth factors during wound healing.

Innate and adaptive immunity gene expression of human keratinocytes cultured of severe burn injury.

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Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Lanziani, Larissa Elias; Ferreira, Lydia Masako; Gragnani, Alfredo

2014-01-01

Evaluate the expression profile of genes related to Innate and Adaptive Immune System (IAIS) of human Primary Epidermal keratinocytes (hPEKP) of patients with severe burns. After obtaining viable fragments of skin with and without burning, culture hKEP was initiated by the enzymatic method using Dispase (Sigma-Aldrich). These cells were treated with Trizol(r) (Life Technologies) for extraction of total RNA. This was quantified and analyzed for purity for obtaining cDNA for the analysis of gene expression using specific IAIS PCR Arrays plates (SA Biosciences). After the analysis of gene expression we found that 63% of these genes were differentially expressed, of which 77% were repressed and 23% were hyper-regulated. Among these, the following genes (fold increase or decrease): IL8 (41), IL6 (32), TNF (-92), HLA-E (-86), LYS (-74), CCR6 (- 73), CD86 (-41) and HLA-A (-35). This study contributes to the understanding of the molecular mechanisms underlying wound infection caused by the burn. Furthermore, it may provide new strategies to restore normal expression of these genes and thereby change the healing process and improve clinical outcome.

Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time

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G. Elliott

1992-01-01

Full Text Available Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG E than confluent ones. As interleukin-1 (IL-1 stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1α (100 U/ml stimulated PGE2 synthesis by proliferating (7 days in culture but not differentiating (14 days in culture keratinocytes. Similar effects were observed using tumour necrosis factor-α. Both arachidonic acid (AA and the calcium ionophore A23187 stimulated PGE2 synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.

Distorted leukocyte migration, angiogenesis, wound repair and metastasis in Tspan8 and Tspan8/CD151 double knockout mice indicate complementary activities of Tspan8 and CD51.

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Zhao, Kun; Erb, Ulrike; Hackert, Thilo; Zöller, Margot; Yue, Shijing

2018-02-01

The tetraspanin Tspan8 supports via associated integrins and proteases tumor progression and angiogenesis. To shed light on its activities in non-transformed cells, we generated a Tspan8 knockout (ko) mouse, comparing leukocyte migration, angiogenesis, wound healing and tumor growth with wild type, CD151ko and Tspan8/CD151ko (dbko) mice. CD151ko mice were included as CD151 activities resemble that of Tspan8, and dbko mice to exclude mutual substitution. Tspan8ko and dbko mice show no pathological phenotype. However, delayed type hypersensitivity reactions are mitigated in Tspan8ko mice, angiogenesis is severely impaired in Tspan8ko, CD151ko and dbko mice, with Tspan8 mostly affecting lymphangiogenesis. Distinct contributions of CD151 and Tspan8 to skin wound healing rely on preferentially CD151 anchoring basal keratinocytes and Tspan8 promoting motility. Proliferation of wounded skin keratinocytes is not affected. Metastasis formation of a melanoma and a Tspan8-expressing pancreatic cancer line was impaired in Tspan8ko and dbko mice, pointing towards a contribution of host Tspan8 to tumor progression. In line with the importance of tetraspanins in exosome-mediated intercellular communication, defects became mitigated by Tspan8/CD151-competent serum exosomes, which offers a most promising therapeutic option for chronic wounds and arteriosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifunctional activities of KSLW synthetic antimicrobial decapeptide: Implications for wound healing

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Williams, Richard Leroy

Wound healing is a complex process leading to the maintenance of skin integrity. Stress is known to increase susceptibility to bacterial infection, alter proinflammatory cytokine expression, and delay wound closure. Recently, antimicrobial peptides have generated interest due to their prokaryotic selectivity, decreased microbial resistance and multifunctional roles in wound healing, including fibroblast stimulation, keratinocyte migration and leukocyte migration. The objective of this dissertation project was to evaluate the effect of a synthetic antimicrobial decapeptide (KSLW) on bacterial clearance inflammation, and wound closure during stress-impaired healing. SKH-1 mice were randomly assigned to either control or restraint-stressed (RST) groups. Punch biopsy wounds (3.5 mm in diameter) were created bilaterally on the dorsal skin. Wounds were injected with 50 microL of empty carriers or KSLW prepared in Pluronic-F68, phospholipid micelles, or saline. Bacterial assays of harvested wounds were conducted on BHI agar. Wound closure was determined by photoplanimetry. Cytokine and growth factor mRNA expression was assessed with real-time RT-PCR. Human neutrophil migration assays and checkerboard analyses were performed using Transweli plates, and counting on hemacytometer. Oxidative burst activity was measured by spectrophotometric analysis of 2,7-dichlorofluorescein oxidation. KSLW-treatment resulted in significant reductions in bacterial load among RST mice, with no difference from control after 24h. The effect was sustained 5 days post-wounding, in RST mice treated with KSLW-F68. Temporal analysis of gene induction revealed reversals of stress-induced altered expression of growth factors, proinflammatory cytokines, and chemokines essential for favorable wound healing, at various time points. KSLW-treatment in RST mice demonstrated faster wound closure throughout the stress period. KSLW, at micromolar concentrations, demonstrated a significant effect on neutrophil

The Reparative Abilities of Menstrual Stem Cells Modulate the Wound Matrix Signals and Improve Cutaneous Regeneration

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Jimena Cuenca

2018-05-01

Full Text Available Considerable advances have been made toward understanding the cellular and molecular mechanism of wound healing, however, treatments for chronic wounds remain elusive. Emerging concepts utilizing mesenchymal stem cells (MSCs from umbilical cord, adipose tissue and bone marrow have shown therapeutical advantages for wound healing. Based on this positive outcome, efforts to determine the optimal sources for MSCs are required in order to improve their migratory, angiogenic, immunomodulatory, and reparative abilities. An alternative source suitable for repetitive, non-invasive collection of MSCs is from the menstrual fluid (MenSCs, displaying a major practical advantage over other sources. This study aims to compare the biological functions and the transcriptomic pattern of MenSCs with umbilical cord MSCs in conditions resembling the wound microenvironment. Consequently, we correlate the specific gene expression signature from MenSCs with changes of the wound matrix signals in vivo. The direct comparison revealed a superior clonogenic and migratory potential of MenSCs as well as a beneficial effect of their secretome on human dermal fibroblast migration in vitro. Furthermore, MenSCs showed increased immunomodulatory properties, inhibiting T-cell proliferation in co-culture. We further, investigated the expression of selected genes involved in wound repair (growth factors, cytokines, chemokines, AMPs, MMPs and found considerably higher expression levels in MenSCs (ANGPT1 1.5-fold; PDGFA 1.8-fold; PDGFB 791-fold; MMP3 21.6-fold; ELN 13.4-fold; and MMP10 9.2-fold. This difference became more pronounced under a pro-inflammatory stimulation, resembling wound bed conditions. Locally applied in a murine excisional wound splinting model, MenSCs showed a significantly improved wound closure after 14 days, as well as enhanced neovascularization, compared to the untreated group. Interestingly, analysis of excised wound tissue revealed a significantly higher

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

International Nuclear Information System (INIS)

Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia

2007-01-01

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10

AMPK regulation of the growth of cultured human keratinocytes

International Nuclear Information System (INIS)

Saha, Asish K.; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

2006-01-01

AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). At concentrations of 10 -4 and 10 -3 M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10 -6 M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D 3 (10 -7 and 10 -6 M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p 3 is AMPK-independent

Nerve signaling regulates basal keratinocyte proliferation in the blastema apical epithelial cap in the axolotl (Ambystoma mexicanum).

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Satoh, Akira; Bryant, Susan V; Gardiner, David M

2012-06-15

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration. Copyright © 2012 Elsevier Inc. All rights reserved.

Decorin gene expression and its regulation in human keratinocytes

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Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico); Kuri-Harcuch, Walid, E-mail: walidkuri@gmail.com [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico)

2011-07-22

Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

Differentiation of human scalp hair follicle keratinocytes in culture.

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Weterings, P J; Verhagen, H; Wirtz, P; Vermorken, A J

1984-01-01

The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.

Y chromosome and vimentin used to trace the fate of allogeneic keratinocytes delivered to the wound by the recombined human/pig skin

Czech Academy of Sciences Publication Activity Database

Pokorná, Eva; Brož, L.; Veselý, Pavel; Matoušková, Eva

2001-01-01

Roč. 47, č. 4 (2001), s. 128-134 ISSN 0015-5500 R&D Projects: GA MZd IZ4368; GA MZd NK6126 Keywords : allogeneic keratinocytes * xenodermis * Y-chromosome FISH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.519, year: 2001

Chemical peeling by SA-PEG remodels photo-damaged skin: suppressing p53 expression and normalizing keratinocyte differentiation.

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Dainichi, Teruki; Amano, Satoshi; Matsunaga, Yukiko; Iriyama, Shunsuke; Hirao, Tetsuji; Hariya, Takeshi; Hibino, Toshihiko; Katagiri, Chika; Takahashi, Motoji; Ueda, Setsuko; Furue, Masutaka

2006-02-01

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

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Marchal Joëlle

2005-10-01

Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

Establishment of primary keratinocyte culture from horse tissue biopsates

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Jernej OGOREVC

2015-12-01

Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

Development of the mechanical properties of engineered skin substitutes after grafting to full-thickness wounds.

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Sander, Edward A; Lynch, Kaari A; Boyce, Steven T

2014-05-01

Engineered skin substitutes (ESSs) have been reported to close full-thickness burn wounds but are subject to loss from mechanical shear due to their deficiencies in tensile strength and elasticity. Hypothetically, if the mechanical properties of ESS matched those of native skin, losses due to shear or fracture could be reduced. To consider modifications of the composition of ESS to improve homology with native skin, biomechanical analyses of the current composition of ESS were performed. ESSs consist of a degradable biopolymer scaffold of type I collagen and chondroitin-sulfate (CGS) that is populated sequentially with cultured human dermal fibroblasts (hF) and epidermal keratinocytes (hK). In the current study, the hydrated biopolymer scaffold (CGS), the scaffold populated with hF dermal skin substitute (DSS), or the complete ESS were evaluated mechanically for linear stiffness (N/mm), ultimate tensile load at failure (N), maximum extension at failure (mm), and energy absorbed up to the point of failure (N-mm). These biomechanical end points were also used to evaluate ESS at six weeks after grafting to full-thickness skin wounds in athymic mice and compared to murine autograft or excised murine skin. The data showed statistically significant differences (p clinical morbidity from graft loss.

The reduction in inflammation and impairment in wound healing by using strontium chloride hexahydrate.

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Berksoy Hayta, Sibel; Durmuş, Kasim; Altuntaş, Emine Elif; Yildiz, Esin; Hisarciklıo, Mehmet; Akyol, Melih

2018-03-01

Numerous growth factors, cytokine, mitogen and chemotactic factors are involved in wound healing. Even though inflammation is important for the stimulation of proliferative phase, excessive inflammation also causes impairment in wound healing. Strontium salts suppress keratinocyte-induced TNF-alpha and interleukin-1 and interleukin-6 in in vitro cultures. This study was conducted to determine the effects of administration of topical strontium chloride hexahydrate on wound healing through TNF-alpha and TGF-beta in surgical wound healing model of in-vivo rat skin. Twenty-four rats were used in the study. After approximately 2 cm cutaneous-subcutaneous incision was horizontally carried out on the mid-neckline of the rats, the incision was again closed using 2.0 vicryl. The rats were assigned into three groups including eight rats in each group. Placebo emollient ointment and also the ointments, which were containing 5% and 10% strontium chloride hexahydrate and were prepared at the same base with placebo ointment, were administered to the groups by a blind executor twice a day for a week. At the end of seventh day, the rats were sacrificed and cutaneous and subcutaneous tissue of their wound site was resected for histopathological examination. Scoring of histopathological wound healing and scoring of tissue TNF-alpha and TGF-beta level with immunohistochemical staining were performed. The groups, to which both 5% and 10% strontium chloride hexahydrate was administered, had lower immunohistochemical TNF-alpha levels and histopathological wound scores compared to controls, which was statistically significant (p < 0.05). Strontium chloride hexahydrate can lead to impairment in wound healing by suppressing inflammation through TNF-alpha.

Simultaneous Delivery of Highly Diverse Bioactive Compounds from Blend Electrospun Fibers for Skin Wound Healing.

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Peh, Priscilla; Lim, Natalie Sheng Jie; Blocki, Anna; Chee, Stella Min Ling; Park, Heyjin Chris; Liao, Susan; Chan, Casey; Raghunath, Michael

2015-07-15

Blend emulsion electrospinning is widely perceived to destroy the bioactivity of proteins, and a blend emulsion of water-soluble and nonsoluble molecules is believed to be thermodynamically unstable to electrospin smoothly. Here we demonstrate a method to retain the bioactivity of disparate fragile biomolecules when electrospun. Using bovine serum albumin as a carrier protein; water-soluble vitamin C, fat soluble vitamin D3, steroid hormone hydrocortisone, peptide hormone insulin, thyroid hormone triiodothyronine (T3), and peptide epidermal growth factor (EGF) were simultaneously blend-spun into PLGA-collagen nanofibers. Upon release, vitamin C maintained the ability to facilitate Type I collagen secretion by fibroblasts, EGF stimulated skin fibroblast proliferation, and insulin potentiated adipogenic differentiation. Transgenic cell reporter assays confirmed the bioactivity of vitamin D3, T3, and hydrocortisone. These factors concertedly increased keratinocyte and fibroblast proliferation while maintaining keratinocyte basal state. This method presents an elegant solution to simultaneously deliver disparate bioactive biomolecules for wound healing applications.

Improved wound management by regulated negative pressure-assisted wound therapy and regulated, oxygen- enriched negative pressure-assisted wound therapy through basic science research and clinical assessment

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Moris Topaz

2012-01-01

Full Text Available Regulated negative pressure-assisted wound therapy (RNPT should be regarded as a state-of-the-art technology in wound treatment and the most important physical, nonpharmaceutical, platform technology developed and applied for wound healing in the last two decades. RNPT systems maintain the treated wound′s environment as a semi-closed, semi-isolated system applying external physical stimulations to the wound, leading to biological and biochemical effects, with the potential to substantially influence wound-host interactions, and when properly applied may enhance wound healing. RNPT is a simple, safe, and affordable tool that can be utilized in a wide range of acute and chronic conditions, with reduced need for complicated surgical procedures, and antibiotic treatment. This technology has been shown to be effective and safe, saving limbs and lives on a global scale. Regulated, oxygen-enriched negative pressure-assisted wound therapy (RO-NPT is an innovative technology, whereby supplemental oxygen is concurrently administered with RNPT for their synergistic effect on treatment and prophylaxis of anaerobic wound infection and promotion of wound healing. Understanding the basic science, modes of operation and the associated risks of these technologies through their fundamental clinical mechanisms is the main objective of this review.

Chemosensory Information Processing between Keratinocytes and Trigeminal Neurons

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Sondersorg, Anna Christina; Busse, Daniela; Kyereme, Jessica; Rothermel, Markus; Neufang, Gitta; Gisselmann, Günter; Hatt, Hanns; Conrad, Heike

2014-01-01

Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2′,4′-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling. PMID:24790106

Teaching wound care to family medicine residents on a wound care service

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Little SH

2013-08-01

Full Text Available Sahoko H Little,1,2 Sunil S Menawat,1,3 Michael Worzniak,1 Michael D Fetters2 1Oakwood Annapolis Family Medicine Residency, Wayne, Michigan, USA; 2University of Michigan, Department of Family Medicine, Ann Arbor, Michigan, USA; 3Ghent Family Medicine Residency, Eastern Virginia Medical School, Norfolk, Virginia, USA Abstract: Primary care physicians often care for patients with chronic wounds, and they can best serve patients if they have knowledge and proficient skills in chronic wound care, including sharp debridement. The Oakwood Annapolis Family Medicine Residency in Michigan, USA developed a Wound Care Service, incorporating wound care training during the surgical rotation. Effectiveness of the wound care training was evaluated through pre- and posttesting of residents, to assess changes in knowledge and comfort in treating chronic wounds. The results demonstrate significant improvement in residents’ knowledge and comfort in wound care. This innovation demonstrates the feasibility of educating residents in chronic wound care through hands-on experience. Keywords: wound care education, primary care, residency education, surgery rotation, curriculum development

Trends in Surgical Wound Healing

DEFF Research Database (Denmark)

Gottrup, F.

2008-01-01

The understanding of acute and chronic wound pathophysiology has progressed considerably over the past decades. Unfortunately, improvement in clinical practice has not followed suit, although new trends and developments have improved the outcome of wound treatment in many ways. This review focuse...

3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

International Nuclear Information System (INIS)

Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

2014-01-01

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase–Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals

3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

Energy Technology Data Exchange (ETDEWEB)

Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-ichi [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Kotani, Eiji [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan); Hirano, Tomoko [Venture Laboratory, Kyoto Institute of Technology, Kyoto (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa (Japan); Matsumoto, Goichi [Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, Yokohama (Japan); Mori, Hajime, E-mail: hmori@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan)

2014-09-01

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase–Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals.

Wound-Healing Potential of Cultured Epidermal Sheets Is Unaltered after Lyophilization: A Preclinical Study in Comparison to Cryopreserved CES

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H. Jang

2013-01-01

Full Text Available Lyophilized Cultured Epidermal Sheets (L-CES have been reported to be as effective as the cryopreserved CES (F-CES in treating skin ulcers. However, unlike F-CES, no preclinical study assessing wound-healing effects has been conducted for L-CES. The present study was set out to investigate the microstructure, cytokine profile, and wound-healing effects of L-CES in comparison to those of F-CES. Keratinocytes were cultured to prepare CES, followed by cryopreservation at −70°C and lyophilization. Under microscopic observation, intact cells with apparent intracellular junctions were observed in L-CES. The L-CES, like fresh CES, consisted of three to four well-maintained epidermal layers, as shown by the expression of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES and F-CES, and both CES were comparable to fresh CES. TGF-α, EGF, VEGF, IL-1α, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson’s trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

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Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Effect of Nanodiamond and Nanoplatinum Liquid, DPV576, on Human Primary Keratinocytes.

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Ghoneum, Mamdooh H; Katano, Hideki; Agrawal, Sudhanshu; Ganguly, Sreerupa; Agrawal, Anshu

2017-01-01

Nanofabrics are now being used in a wide range of products that come into direct contact with skin, including carpet, clothing, and medical fabrics. In the current study, we examined the effect of a dispersed aqueous mixture of nanodiamond (ND) and nanoplatinum (NP) (DPV576) on human primary keratinocytes with respect to transient receptor potential vanilloid (TRPV) channel expression, secretion of cytokines and production of nerve growth factor (NGF). Keratinocytes were treated with DPV576 at concentrations of 1:10 and 1:100 dilutions for 24 hours in vitro, and their activation of was determined by production of cytokines TNF-α, IL-1β, and prostaglandin (PGE2), and by production of NGF. Inhibitor experiments were carried out by incubating keratinocytes with the TRPV4-selective antagonist HC-067047. TRPV receptor expression (TRPV1, TRPV3 and TRPV4) on keratinocytes as well as changes in Ca2+ potential were examined by flow cytometry. DPV576 treatment of keratinocytes resulted in the following effects: (1) stimulation of keratinocytes as indicated by the significant secretion of cytokines IL-1β, TNF-α, and PGE2, an effect noted only at higher concentration (1:10); (2) significant decrease in the expression of TRPV4 on keratinocytes with a spike in the calcium flux, but no change in the expression of TRPV1 and TRPV3; (3) induction of cytokine secretion independent of TRPV4, as the addition of TRPV4 inhibitor had no significant effect on the cytokine production from keratinocytes; (4) induction of NGF secretion by keratinocytes. These results demonstrate that DPV576 activates keratinocytes via multiple signaling pathways which may reduce stress associated with inflammation, pain, and circadian rhythms. ND/NP-coated fabrics that target the modulation of local inflammation, pain, and circadian rhythms could potentially be of benefit to humans.

Lower limb revascularisation preceding surgical wound coverage - an interdisciplinary algorithm for chronic wound closure.

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Aust, M C; Spies, M; Guggenheim, M; Gohritz, A; Kall, S; Rosenthal, H; Pichlmaier, M; Oehlert, G; Vogt, P M

2008-08-01

Chronic wounds may occur or persist due to arterial insufficiency. Despite the high prevalence of arterial occlusive disease, a search of the literature has yielded a paucity of data on the benefit of interventions to recanalise lower extremity arteries prior to surgical closure of chronic wounds. To investigate the correlation of simple clinical examinations and apparative diagnostics for the detection of arterial occlusive disease of the lower extremity in patients with chronic wounds, and to evaluate the benefit of vascular procedures to optimise wound perfusion before surgical closure. During a 6-year period, 150 patients with chronic lower extremity wounds (no healing for more than 30 days) were included into this prospective study. All patients underwent palpation of foot pulses, Doppler sonography and measurement of occlusive pressures. Positive clinical findings were re-evaluated by angiography. All patients with peripheral extremity vessel occlusions underwent vascular interventions (percutaneous transluminal angioplasty with stenting, open thrombectomy or vascular bypass surgery) prior to surgical wound closure. In all 34 patients (21%) with missing foot pulses, suspicious Doppler signals or pathological occlusive pressure measurements, the clinical diagnosis of arterial occlusion was confirmed by angiography. An arterial pathology had previously been diagnosed in merely two of those patients. Nineteen patients underwent percutaneous transluminal angioplasty and 21 stents were implanted; in 10 cases, open thrombectomy or vascular bypasses were performed. In all 34 patients, sufficient peripheral recanalisation and improved wound perfusion were successfully achieved. For definitive wound closure, microsurgical tissue transplantation was performed in 15 patients. Angiography was performed prior to surgery. In 11 patients, regional or local flaps were used. Six patients received split skin grafting only; two wounds healed conservatively following vascular

Possible role of epidermal keratinocytes in the construction of acupuncture meridians.

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Denda, Mitsuhiro; Tsutsumi, Moe

2014-04-01

Acupuncture meridians consist of a network of acupuncture points on the skin, stimulation of which is well established to have a variety of physiological effects. We have previously demonstrated that epidermal keratinocytes contain multiple sensory systems for temperature, mechanical stimuli, electric potentials and other stimuli. These sensory systems generate changes in the calcium-ion concentration in the epidermis, so epidermal keratinocytes can generate spatially-localized electro-physiological patterns in the skin. We have previously demonstrated signaling between epidermal keratinocytes and peripheral nerve systems. Therefore, stimuli sensed by epidermal keratinocytes might be transferred to the unmyelinated nerve fibers that are known to exist in the epidermis and, thence, to the spinal cord and brain. We propose that epidermal keratinocytes form an information-gathering network in the skin and that this network plays a key role in whole-body homeostasis in response to the changing environment. We also hypothesize that this network corresponds to the acupuncture meridians. As supporting examples, we present some striking calcium propagation patterns observed in cultured human keratinocytes after adenosine-triphosphate (ATP) stimulation. These results support the ideas that keratinocytes can generate spatially-restricted signaling patterns after environmental stimulation and that the cultures might be in-vitro models of meridians as an information-gathering network in skin. Copyright © 2014. Published by Elsevier B.V.

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo.

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Wölfle, Ute; Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M

2017-08-22

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

[Errors in wound management].

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Filipović, Marinko; Novinscak, Tomislav

2014-10-01

Chronic ulcers have adverse effects on the patient quality of life and productivity, thus posing financial burden upon the healthcare system. Chronic wound healing is a complex process resulting from the interaction of the patient general health status, wound related factors, medical personnel skill and competence, and therapy related products. In clinical practice, considerable improvement has been made in the treatment of chronic wounds, which is evident in the reduced rate of the severe forms of chronic wounds in outpatient clinics. However, in spite of all the modern approaches, efforts invested by medical personnel and agents available for wound care, numerous problems are still encountered in daily practice. Most frequently, the problems arise from inappropriate education, of young personnel in particular, absence of multidisciplinary approach, and inadequate communication among the personnel directly involved in wound treatment. To perceive them more clearly, the potential problems or complications in the management of chronic wounds can be classified into the following groups: problems mostly related to the use of wound coverage and other etiology related specificities of wound treatment; problems related to incompatibility of the agents used in wound treatment; and problems arising from failure to ensure aseptic and antiseptic performance conditions.

Development and evaluation of a new composite Laserskin graft.

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Lam, P K; Chan, E S; To, E W; Lau, C H; Yen, S C; King, W W

1999-11-01

Tremendous effort has been made to improve the graft take rate of cultured epidermal autograph. The purpose of this study is to develop and evaluate a new composite Laserskin graft (CLSG) as a human skin substitute for wound resurfacing. The seeding efficacy of cultured keratinocytes on plain Laserskin was compared with the 3T3 cell-seeded Laserskin and allogenic fibroblast-populated Laserskin. Three different types of CLSG, 2 cm in diameter each, were prepared and tested in rats. Type A CLSG consisted of proliferative allogenic rat fibroblasts on both sides of the Laserskin with autologous keratinocytes also on the upper side. Fibroblasts and keratinocytes were seeded only on the upper side of the Laserskin in type B CLSG. Keratinocytes alone were seeded on plain Laserskin in type C CLSG. Type B CLSG consisting of autologous keratinocytes and autologous dermal fibroblasts was tested on five selected wounds (5x5 cm each) of a patient with full-thickness burn. In another burn patient, type B CLSG consisting of autologous keratinocytes and allogenic dermal fibroblasts was grafted onto three wounds (5x5 cm each). The seeding efficacy of human keratinocytes on plain Laserskin increased from 75% to 95% when proliferative allogenic fibroblasts were grown as a feeder layer on the Laserskin. The seeding efficacy of rat keratinocytes increased from 36% to 88% in the presence of a proliferative allogenic fibroblast feeder layer, whereas human/rat keratinocytes had respective seeding efficacy of 98%/91% on Laserskin preseeded with mitomycin C-treated 3T3 cells. Skin biopsies of grafted type A CLSG on day 14 after grafting showed complete epithelialization without severe inflammation in 16 of 20 (80%) grafted surgical wounds in rats. There were eight (40%) and seven (35%) "takes" of the CLSG in types B and C, respectively. The infection rate in type B CLSG was two (10%). There was one (5%) infection in types A and C. The respective take rates on the two patients grafted with

Factors Affecting Wound Healing

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Guo, S.; DiPietro, L.A.

2010-01-01

Wound healing, as a normal biological process in the human body, is achieved through four precisely and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. Many factors can interfere with one or more phases of this process, thus causing improper or impaired wound healing. This article reviews the recent literature on the most significant factors that affect cutaneous wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include oxygenation, infection, age and sex hormones, stress, diabetes, obesity, medications, alcoholism, smoking, and nutrition. A better understanding of the influence of these factors on repair may lead to therapeutics that improve wound healing and resolve impaired wounds. PMID:20139336

Wound Healing in Older Adults.

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Gould, Lisa J; Fulton, Ana Tuya

2016-02-01

Impaired wound healing in the elderly represents a major clinical problem that is growing as our population ages. Wound healing is affected by age and by co-morbid conditions, particularly diabetes and obesity. This is particularly important in Rhode Island as the state has a very high percentage of vulnerable older adults. A multi- disciplinary approach that incorporates the skills of a comprehensive wound center with specialized nursing, geriatric medicine and palliative care will facilitate rapid wound healing, reduce costs and improve outcomes for our older adults that suffer from 'problem wounds'.

Effect of platelet lysate on human cells involved in different phases of wound healing.

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Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

2013-01-01

Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pplatelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin β4.

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Niu, Gengming; Ye, Taiyang; Qin, Liuliang; Bourbon, Pierre M; Chang, Cheng; Zhao, Shengqiang; Li, Yan; Zhou, Lei; Cui, Pengfei; Rabinovitz, Issac; Mercurio, Arthur M; Zhao, Dezheng; Zeng, Huiyan

2015-01-01

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin β4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin β4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin β4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing. © FASEB.

The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.

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Pellegrini, G; Ranno, R; Stracuzzi, G; Bondanza, S; Guerra, L; Zambruno, G; Micali, G; De Luca, M

1999-09-27

Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be life-saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are proposed, a careful evaluation of epidermal stem cell preservation is essential for the clinical performance of this life-saving technology. Fibrin was chosen as a potential substrate for keratinocyte cultivation. Stem cells were monitored by clonal analysis using the culture system originally described by Rheinwald and Green as a reference. Massive full-thickness burns were treated with the composite allodermis/cultured autograft technique. We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and long-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on massive full-thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

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Takikawa, Megumi; Ishihara, Masayuki; Takabayashi, Yuki; Sumi, Yuki; Takikawa, Makoto; Yoshida, Ryuichi; Nakamura, Shingo; Hattori, Hidemi; Yanagibayashi, Satoshi; Yamamoto, Naoto; Kiyosawa, Tomoharu

2015-04-13

The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

Débridement and Autologous Lipotransfer for Chronic Ulceration of the Diabetic Foot and Lower Limb Improves Wound Healing.

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Stasch, Tilman; Hoehne, Julius; Huynh, Tuan; De Baerdemaeker, Randy; Grandel, Siegfried; Herold, Christian

2015-12-01

The application of autologous lipotransfer (fat grafting, lipofilling) in reconstructive surgery is steadily becoming more popular as evidence of the regenerative and reparative effects of fat becomes better known. The authors investigated the use of autologous lipotransfer for treatment of chronic diabetic and other foot and lower limb ulcers. Twenty-six patients with nonhealing wounds were treated with surgical débridement and autologous lipotransfer (using the débridement and autologous lipotransfer method). The mean age of the wounds before intervention was 16.7 months. Wound size after débridement averaged 5.1 ± 2.6 cm2. On average, 7.1 ± 3.3 cc of lipoaspirate was transferred into the wound area. Twenty-two of 25 wounds (88 percent) healed completely within a mean of 68.0 ± 33.0 days. A reduction of wound size by 50 percent was achieved after an average of 4 weeks. In one patient with an ulcer within particularly scarred tissues on the lower limb, a repeated session of lipotransfer led to complete wound healing after another 4 weeks. The authors describe a simple and useful technique to improve wound healing in diabetic feet and chronic lower limb ulcers with a background of peripheral vascular disease, where other interventional options to achieve wound healing have failed.

Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

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Ju-Suk Nam

2014-03-01

Full Text Available Wingless-type (Wnt signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα. Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.

Re-appraisal of keratinocytes' role in vitiligo pathogenesis

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Ola Ahmed Bakry

2018-01-01

Full Text Available Background: Vitiligo is a common pigmentary disorder. Studies on its pathogenesis extensively investigated melanocytes' abnormalities and few studies searched for keratinocytes' role in disease development. Liver X receptor-α (LXR-α is a member of nuclear hormone receptors that acts as a transcription factor. Its target genes are the main regulators of melanocyte functions. Aim: The aim of this study is to investigate keratinocytes' role in vitiligo pathogenesis through immunohistochemical expression of LXR-α in lesional, perilesional, and distant nonlesional vitiligo skin. Materials and Methods: This case–control study was carried out on 44 participants. These included 24 patients with vitiligo and 20 age- and sex-matched normal individuals as a control group. Biopsies, from cases, were taken from lesional, perilesional, and distant nonlesional areas. Evaluation was done using immunohistochemical technique. Results: Keratinocyte LXR-α expression was upregulated in the lesional and perilesional skin (follicular and interfollicular epidermis compared with control skin (P<0.001 for all. There was significant association between higher histoscore (H-score in lesional epidermis (P<0.001 and in hair follicle (P=0.001 and the presence of angiogenesis. There was significant association between higher H-score in lesional epidermis and suprabasal vacuolization (P=0.02. No significant association was found between H-score or expression percentage and clinical data of selected cases. Conclusion: LXR-α upregulation is associated with keratinocyte damage in vitiligo lesional skin that leads to decreased keratinocyte-derived mediators and growth factors supporting the growth and/or melanization of surrounding melanocytes. Therefore, melanocyte function and survival are affected.

Differential Activation of Human Keratinocytes by Leishmania Species Causing Localized or Disseminated Disease.

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Scorza, Breanna M; Wacker, Mark A; Messingham, Kelly; Kim, Peter; Klingelhutz, Aloysius; Fairley, Janet; Wilson, Mary E

2017-10-01

All Leishmania species parasites are introduced into mammalian skin through a sand fly bite, but different species cause distinct clinical outcomes. Mouse studies suggest that early responses are critical determinants of subsequent adaptive immunity in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant cell in the epidermis. We hypothesized that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection. Incubation of primary or immortalized human keratinocytes with Leishmania infantum or Leishmania major, which cause visceral or cutaneous leishmaniasis, respectively, elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of proinflammatory genes for IL-6, IL-8, tumor necrosis factor, and IL-1B, whereas keratinocytes exposed to several L. major isolates did not. Furthermore, keratinocyte-monocyte co-incubation studies across a 4 µM semipermeable membrane suggested that L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication (P Leishmania species that may affect the course of disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Podoplanin expression in peritumoral keratinocytes predicts aggressive behavior in extramammary Paget's disease.

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Cho, Zaigen; Konishi, Eiichi; Kanemaru, Mai; Isohisa, Taro; Arita, Takahiro; Kawai, Minako; Tsutsumi, Miho; Mizutani, Hiromi; Takenaka, Hideya; Ozawa, Toshiyuki; Tsuruta, Daisuke; Katoh, Norito; Asai, Jun

2017-07-01

Recent studies have demonstrated podoplanin expression in several tumors, which has been associated with lymph node metastasis and poor prognosis. Podoplanin expression in peritumoral cells such as cancer-associated fibroblasts also correlates with tumor progression in several cancers. However, podoplanin expression and its association with extramammary Paget's disease (EMPD) remain unclear. In this study, we examined whether the presence of podoplanin expression in tumor cells or peritumoral basal keratinocytes correlated with aggressive behavior in patients with EMPD and investigated the mechanisms of podoplanin-mediated tumor invasion in this disorder. Skin samples of 37 patients with EMPD were investigated by immunohistochemical analysis. The functions of podoplanin in keratinocytes were examined in vitro by RT-PCR and with invadopodia gelatin-degradation assays using HaCaT cells. Podoplanin was not identified in tumor cells in all cases. Podoplanin expression in peritumoral basal keratinocytes was observed in 25 patients (67.6%). In in situ EMPD, 50% of cases (9 in 18) exhibited podoplanin-positive keratinocytes, whereas 84.2% (16 in 19) demonstrated positive staining in invasive EMPD (P<0.05). Podoplanin expression in peritumoral keratinocytes was also associated with tumor thickness (P<0.005). By immunohistochemical analysis, podoplanin-positive peritumoral keratinocytes were found to be negative for E-cadherin, one of the major adhesion molecules of keratinocytes, which might contribute to tumor invasion into the dermis through a crack in the basal cell layer induced by down-regulation of cell adhesion therein. We further found that podoplanin-positive keratinocytes exhibited invadopodia, which are thought to function in the migration of cancer cells through tissue barriers, indicating that podoplanin-positive peritumoral basal keratinocytes might assist tumor invasion by degrading the extracellular matrix. The presence of podoplanin expression in

Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects

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Dai, Tianhong; Tanaka, Masamitsu; Huang, Ying-Ying; Hamblin, Michael R

2011-01-01

Since its discovery approximately 200 years ago, chitosan, as a cationic natural polymer, has been widely used as a topical dressing in wound management owing to its hemostatic, stimulation of healing, antimicrobial, nontoxic, biocompatible and biodegradable properties. This article covers the antimicrobial and wound-healing effects of chitosan, as well as its derivatives and complexes, and its use as a vehicle to deliver biopharmaceuticals, antimicrobials and growth factors into tissue. Studies covering applications of chitosan in wounds and burns can be classified into in vitro, animal and clinical studies. Chitosan preparations are classified into native chitosan, chitosan formulations, complexes and derivatives with other substances. Chitosan can be used to prevent or treat wound and burn infections not only because of its intrinsic antimicrobial properties, but also by virtue of its ability to deliver extrinsic antimicrobial agents to wounds and burns. It can also be used as a slow-release drug-delivery vehicle for growth factors to improve wound healing. The large number of publications in this area suggests that chitosan will continue to be an important agent in the management of wounds and burns. PMID:21810057

Saliva and wound healing.

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Brand, Henk S; Ligtenberg, Antoon J M; Veerman, Enno C I

2014-01-01

Oral wounds heal faster and with less scar formation than skin wounds. One of the key factors involved is saliva, which promotes wound healing in several ways. Saliva creates a humid environment, thus improving the survival and functioning of inflammatory cells that are crucial for wound healing. In addition, saliva contains several proteins which play a role in the different stages of wound healing. Saliva contains substantial amounts of tissue factor, which dramatically accelerates blood clotting. Subsequently, epidermal growth factor in saliva promotes the proliferation of epithelial cells. Secretory leucocyte protease inhibitor inhibits the tissue-degrading activity of enzymes like elastase and trypsin. Absence of this protease inhibitor delays oral wound healing. Salivary histatins in vitro promote wound closure by enhancing cell spreading and cell migration, but do not stimulate cell proliferation. A synthetic cyclic variant of histatin exhibits a 1,000-fold higher activity than linear histatin, which makes this cyclic variant a promising agent for the development of a new wound healing medication. Conclusively, recognition of the many roles salivary proteins play in wound healing makes saliva a promising source for the development of new drugs involved in tissue regeneration.

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

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McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice

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Geiger, Adolf, E-mail: ageiger@dreirosen-pharma.com; Walker, Audrey, E-mail: awalker@dreirosen-pharma.com; Nissen, Erwin, E-mail: enissen@dreirosen-pharma.com

2015-11-13

Diabetic ulcers represent a substantial societal and healthcare burden worldwide and scarcely respond to current treatment strategies. This study was addressed to evaluate the therapeutic potential of exosomes secreted by human circulating fibrocytes, a population of mesenchymal progenitors involved in normal wound healing via paracrine signaling. The exosomes released from cells sequentially stimulated with platelet-derived growth factor-BB and transforming growth factor-β1, in the presence of fibroblast growth factor 2, did not show potential immunogenicity. These exosomes exhibited in-vitro proangiogenic properties, activated diabetic dermal fibroblasts, induced the migration and proliferation of diabetic keratinocytes, and accelerated wound closure in diabetic mice in vivo. Important components of the exosomal cargo were heat shock protein-90α, total and activated signal transducer and activator of transcription 3, proangiogenic (miR-126, miR-130a, miR-132) and anti-inflammatory (miR124a, miR-125b) microRNAs, and a microRNA regulating collagen deposition (miR-21). This proof-of-concept study demonstrates the feasibility of the use of fibrocytes-derived exosomes for the treatment of diabetic ulcers. - Highlights: • Fibrocytes have shown potent wound healing properties in vitro and in vivo. • Their clinical use is precluded by low numbers and antigen-presenting function. • We isolated exosomes with no immunogenicity potential from human fibrocytes. • Their cargo included microRNAs and proteins that are known healing promoters. • They accelerated wound closure in diabetic mice in a dose-dependent manner.

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.

Science.gov (United States)

Mohamadzadeh, M; Müller, M; Hultsch, T; Enk, A; Saloga, J; Knop, J

1994-12-01

To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

Biomaterials and Nanotherapeutics for Enhancing Skin Wound Healing

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Das, Subhamoy; Baker, Aaron B.

2016-01-01

Wound healing is an intricate process that requires complex coordination between many cell types and an appropriate extracellular microenvironment. Chronic wounds often suffer from high protease activity, persistent infection, excess inflammation, and hypoxia. While there has been intense investigation to find new methods to improve cutaneous wound care, the management of chronic wounds, burns, and skin wound infection remain challenging clinical problems. Ideally, advanced wound dressings can provide enhanced healing and bridge the gaps in the healing processes that prevent chronic wounds from healing. These technologies have great potential for improving outcomes in patients with poorly healing wounds but face significant barriers in addressing the heterogeneity and clinical complexity of chronic or severe wounds. Active wound dressings aim to enhance the natural healing process and work to counter many aspects that plague poorly healing wounds, including excessive inflammation, ischemia, scarring, and wound infection. This review paper discusses recent advances in the development of biomaterials and nanoparticle therapeutics to enhance wound healing. In particular, this review focuses on the novel cutaneous wound treatments that have undergone significant preclinical development or are currently used in clinical practice. PMID:27843895

Biomaterials and Nanotherapeutics for Enhancing Skin Wound Healing

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Subhamoy Das

2016-10-01

Full Text Available Wound healing is an intricate process that requires complex coordination between many cells and an appropriate extracellular microenvironment. Chronic wounds often suffer from high protease activity, persistent infection, excess inflammation, and hypoxia. While there has been intense investigation to find new methods to improve cutaneous wound care; the management of chronic wounds, burns, and skin wound infection remain challenging clinical problems. Ideally, advanced wound dressings can provide enhanced healing and bridge the gaps in the healing processes that prevent chronic wounds from healing. These technologies have great potential for improving outcomes in patients with poorly healing wounds but face significant barriers in addressing the heterogeneity and clinical complexity of chronic or severe wounds. Active wound dressings aim to enhance the natural healing process and work to counter many aspects that plague poorly healing wounds including excessive inflammation, ischemia, scarring and wound infection. This review paper discusses recent advances in the development of biomaterials and nanoparticle therapeutics to enhance wound healing. In particular, this review focuses on the novel cutaneous wound treatments that have undergone significant preclinical development or currently used in clinical practice.

Apitherapeutics and phage-loaded nanofibers as wound dressings with enhanced wound healing and antibacterial activity.

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Sarhan, Wessam A; Azzazy, Hassan Me

2017-09-01

Develop green wound dressings which exhibit enhanced wound-healing ability and potent antibacterial effects. Honey, polyvinyl alcohol, chitosan nanofibers were electrospun and loaded with bee venom, propolis and/or bacteriophage against the multidrug-resistant Pseudomonas aeruginosa and examined for their antibacterial, wound-healing ability and cytotoxicity. Among different formulations of nanofibers, honey, polyvinyl alcohol, chitosan-bee venom/bacteriophage exhibited the most potent antibacterial activity against all tested bacterial strains (Gram-positive and -negative strains) and achieved nearly complete killing of multidrug-resistant P. aeruginosa. In vivo testing revealed enhanced wound-healing results and cytotoxicity testing proved improved biocompatibility. The developed biocompatible nanofibers represent competitive wound-healing dressings with potent antibacterial and wound-healing activity.

Keratinocytes propagated in serum-free, feeder-free culture conditions fail to form stratified epidermis in a reconstituted skin model.

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Rebecca Lamb

Full Text Available Primary human epidermal stem cells isolated from skin tissues and subsequently expanded in tissue culture are used for human therapeutic use to reconstitute skin on patients and to generate artificial skin in culture for academic and commercial research. Classically, epidermal cells, known as keratinocytes, required fibroblast feeder support and serum-containing media for serial propagation. In alignment with global efforts to remove potential animal contaminants, many serum-free, feeder-free culture methods have been developed that support derivation and growth of these cells in 2-dimensional culture. Here we show that keratinocytes grown continually in serum-free and feeder-free conditions were unable to form into a stratified, mature epidermis in a skin equivalent model. This is not due to loss of cell potential as keratinocytes propagated in serum-free, feeder-free conditions retain their ability to form stratified epidermis when re-introduced to classic serum-containing media. Extracellular calcium supplementation failed to improve epidermis development. In contrast, the addition of serum to commercial, growth media developed for serum-free expansion of keratinocytes facilitated 3-dimensional stratification in our skin equivalent model. Moreover, the addition of heat-inactivated serum improved the epidermis structure and thickness, suggesting that serum contains factors that both aid and inhibit stratification.

Collagen-based wound dressings for the treatment of diabetes-related foot ulcers: a systematic review

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Holmes C

2013-01-01

Full Text Available Crystal Holmes,1 James S Wrobel,1 Mark P MacEachern,2 Blaise R Boles31Department of Internal Medicine, University of Michigan Medical School, 2A Alfred Taubman Health Sciences Library, University of Michigan, 3Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USABackground: Diabetic foot ulcers are a major source of morbidity, limb loss, and mortality. A prolonged inflammatory response, extracellular matrix degradation irregularities, and increased bacteria presence have all been hypothesized as major contributing factors in the delayed healing of diabetic wounds. Collagen components such as fibroblast and keratinocytes are fundamental to the process of wound healing and skin formation. Wound dressings that contain collagen products create a biological scaffold matrix that supports the regulation of extracellular components and promotes wound healing.Methods: A systematic review of studies reporting collagen wound dressings used in the treatment of Diabetic foot ulcers was conducted. Comprehensive searches were run in Ovid MEDLINE, PubMed, EMBASE, and ISI Web of Science to capture citations pertaining to the use of collagen wound dressings in the treatment of diabetic foot ulcers. The searches were limited to human studies reported in English.Results: Using our search strategy, 26 papers were discussed, and included 13 randomized designs, twelve prospective cohorts, and one retrospective cohort, representing 2386 patients with diabetic foot ulcers. Our design was not a formal meta-analysis. In those studies where complete epithelialization, 58% of collagen-treated wounds completely healed (weighted mean 67%. Only 23% of studies reported control group healing with 29% healing (weighted mean 11% described for controls.Conclusion: Collagen- based wound dressings can be an effective tool in the healing of diabetic foot wounds. The current studies show an overall increase in healing rates despite

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

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Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

2013-02-01

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

A Clinicoepidemiological Profile of Chronic Wounds in Wound Healing Department in Shanghai.

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Sun, Xiaofang; Ni, Pengwen; Wu, Minjie; Huang, Yao; Ye, Junna; Xie, Ting

2017-03-01

The aim of the study was to update the clinical database of chronic wounds in order to derive an evidence based understanding of the condition and hence to guide future clinical management in China. A total of 241 patients from January 1, 2011 to April 30, 2016 with chronic wounds of more than 2 weeks' duration were studied in wound healing department in Shanghai. Results revealed that among all the patients the mean age was 52.5 ± 20.2 years (range 2-92 years). The mean initial area of wounds was 30.3 ± 63.0 cm 2 (range 0.25-468 cm 2 ). The mean duration of wounds was 68.5 ± 175.2 months (range 0.5-840 months). The previously reported causes of chronic wounds were traumatic or surgical wounds (n = 82, 34.0%), followed by pressure ulcers (n = 59, 24.5%). To study the effects of age, patients were divided into 2 groups: less than 60 years (wounds etiology between the 2 age groups was analyzed, and there was significant statistical difference ( P wounds, chi-square test was used. There were significant differences in the factor of wound infection. ( P = .035, 95% CI = 0.031-0.038) Regarding therapies, 72.6% (n = 175) of the patients were treated with negative pressure wound therapy. Among all the patients, 29.9% (n = 72) of them were completely healed when discharged while 62.7% (n = 150) of them improved. The mean treatment cost was 12055.4 ± 9206.3 Chinese Yuan (range 891-63626 Chinese Yuan). In conclusion, traumatic or surgical wounds have recently become the leading cause of chronic wounds in Shanghai, China. Etiology of the 2 age groups was different. Infection could significantly influence the wound outcome.

The Wound CARE Instrument: the process for developing standards for wound management education and programming.

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Orsted, Heather L; Woodbury, M Gail; Stevenson, Kimberly

2012-06-01

This article describes the collaborative process undertaken by the Canadian Association for Enterostomal Therapy and the Canadian Association of Wound Care in an effort to improve the quality of wound prevention and management education and programming. The end result of this process is the Wound CARE Instrument which promotes an interprofessional, collaborative appraisal process to support the development, adoption or adaption of wound management educational events and programs. © 2011 The Authors. © 2011 Blackwell Publishing Ltd and Medicalhelplines.com Inc.

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

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Salerno, Simona, E-mail: s.salerno@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Messina, Antonietta [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); Giordano, Francesca [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende, (CS) (Italy); Bader, Augustinus [Biomedical-Biotechnological Center, BBZ, University of Leipzig, D-04103 Leipzig (Germany); Drioli, Enrico [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy); WCU Energy Engineering Department, Hanyang University, Seoul (Korea, Republic of); De Bartolo, Loredana, E-mail: l.debartolo@itm.cnr.it [Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87036, Rende (CS) (Italy)

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.

Optimization and integration of nanosilver on polycaprolactone nanofibrous mesh for bacterial inhibition and wound healing in vitro and in vivo

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Liu M

2017-09-01

proliferating cell nuclear antigen (PCNA in keratinocytes and longer length of neo-formed epidermis, PCL/NS accelerated wound healing by promoting re-epithelialization via enhancing keratinocyte proliferation in infectious wounds. Keywords: polycaprolactone nanofibrous mesh, mussel inspired, nanosilver, anti-infection activity, wound healing, re-epithelialization

Kinetics of growth and differentiation of cultured human epidermal keratinocytes

International Nuclear Information System (INIS)

Albers, K.M.

1985-01-01

A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with 14 C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the 14 C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of 14 C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle

Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

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Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

2016-10-01

In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. Copyright © 2016 Elsevier B.V. All rights reserved.

Cultivation of human dermal fibroblasts and epidermal keratinocytes on keratin-coated silica bead substrates.

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Tan, Bee Yi; Nguyen, Luong T H; Kim, Hyo-Sop; Kim, Jae-Ho; Ng, Kee Woei

2017-10-01

Human hair keratin is promising as a bioactive material platform for various biomedical applications. To explore its versatility further, human hair keratin was coated onto monolayers of silica beads to produce film-like substrates. This combination was hypothesized to provide a synergistic effect in improving the biochemical properties of the resultant composite. Atomic force microscopy analysis showed uniform coatings of keratin on the silica beads with a slight increase in the resulting surface roughness. Keratin-coated silica beads had higher surface energy and relatively lower negative charge than those of bare silica beads. To investigate cell response, human dermal fibroblasts (HDFs), and human epidermal keratinocytes (HEKs) were cultured on the substrates over 4 days. Results showed that keratin coatings significantly enhanced the metabolic activity of HDFs and encouraged cell spreading but did not exert any significant effects on HEKs. HDF expression of collagen I was significantly more intense on the keratin-coated compared to the bare silica substrates. Furthermore, HDF secretion of various cytokines suggested that keratin coatings triggered active cell responses related to wound healing. Collectively, our study demonstrated that human hair keratin-coated silica bead monolayers have the potential to modulate HDF behavior in culture and may be exploited further. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2789-2798, 2017. © 2017 Wiley Periodicals, Inc.

Photodynamic toxicity of hematoporphyrin derivatives to human keratinocytes in culture.

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Kappus, H; Reinhold, C; Artuc, M

Human keratinocytes in culture were able to take up hematoporphyrin derivatives (HPDs) used during photodynamic chemotherapy of tumors. In the absence of light, HPDs showed no cytotoxic effects to keratinocytes. However, after irradiation with visible light, HPDs induced immediate cytotoxicity as measured by the neutral red uptake assay. On the other hand, cell attachment as measured by protein estimation was not affected. When the cells treated with HPDs and irradiated with light were cultured for a further 72 h, they partially lost their ability to attach to the collagen surface. Most of the cells remaining attached after 72 h were no longer viable following treatment with HPDs and light. All parameters measured depended on the intracellular concentration of HPDs used (7-50 ng/10(5) cells) and the time of irradiation (0-30 min). These results suggest that human keratinocytes are a good model to study cytotoxic effects of photodynamically active drugs. Further, keratinocytes were unable to recover after damage caused by HPDs and light.

Noncultured keratinocyte/melanocyte cosuspension: effect on reepithelialization and repigmentation--a randomized, placebo-controlled study.

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Back, Christopher; Dearman, Bronwyn; Li, Amy; Neild, Tim; Greenwood, John E

2009-01-01

Randomized controlled trials in the literature investigating the efficacy of noncultured keratinocyte/melanocyte suspensions are scarce; however, the advocates of such techniques press the value of their application based largely on case studies and anecdote. Caucasian patients with burn hypopigmentation seldom request cosmetic revision making worthwhile clinical trials difficult so that informal case treatments with new therapies generate anecdotal results. A randomized, placebo-controlled trial was carried out to evaluate whether cosuspensions of noncultured skin cells are capable of (1) decreasing the time to reepithelialization and (2) reestablishing pigmentation in vitiligo leukoderma following epidermal/superficial dermal ablation (in the knowledge that a positive result would make the technique likely to be successful in burn hypopigmentation). Vitiligo is common and is socially more debilitating such that suitable trial subjects for new therapies from this pool are more forthcoming. This study demonstrated that suspensions of noncultured keratinocytes and melanocytes do not decrease the time to epithelialization of superficial partial thickness wounds compared with controls. It also suggested that the achievement, quality, and duration of any pigmentation were unpredictable and largely disappointing. Some pigmentation was recorded in placebo-treated areas indicating an effect of the method of epidermal ablation in these patients. These findings have mandated a complete review of the use of these techniques in burn care at the Royal Adelaide Hospital; they have been omitted from surgical protocols where the aim of use was to speed reepithelialization. Their infrequent use in burns hypopigmentation will continue contingent on the successful repigmentation of a test patch.

Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

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Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

2014-04-01

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

Rapid preparation of a noncultured skin cell suspension that promotes wound healing.

Science.gov (United States)

Yoon, Cheonjae; Lee, Jungsuk; Jeong, Hyosun; Lee, Sungjun; Sohn, Taesik; Chung, Sungphil

2017-06-01

Autologous skin cell suspensions have been used for wound healing in patients with burns and against normal pigmentation in vitiligo. To separate cells and the extracellular matrix from skin tissue, most researchers use enzymatic digestion. Therefore, this process is difficult to perform during a routine surgical procedure. We aimed to prepare a suspension of noncultured autologous skin cells (NCSCs) using a tissue homogenizer as a new method instead of harsh biochemical reagents. The potential clinical applicability of NCSCs was analyzed using a nude-rat model of burn healing. After optimization of the homogenizer settings, cell viability ranged from 52 to 89%. Scanning electron microscopy showed evidence of keratinocyte-like cell morphology, and several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the NCSCs. The rat model revealed that NCSCs accelerated skin regeneration. NCSCs could be generated using a tissue homogenizer for enhancement of wound healing in vivo. In the NCSC group of wounds, on day 7 of epithelialization, granulation was observed, whereas on day 14, there was a significant increase in skin adnexa regeneration as compared to the control group (PBS treatment; p study suggests that the proposed process is rapid and does not require the use of biochemical agents. Thus, we recommend a combination of surgical treatment with the new therapy for a burn as an effective method.

An aligned porous electrospun fibrous membrane with controlled drug delivery - An efficient strategy to accelerate diabetic wound healing with improved angiogenesis.

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Ren, Xiaozhi; Han, Yiming; Wang, Jie; Jiang, Yuqi; Yi, Zhengfang; Xu, He; Ke, Qinfei

2018-04-01

A chronic wound in diabetic patients is usually characterized by poor angiogenesis and delayed wound closure. The exploration of efficient strategy to significantly improve angiogenesis in the diabetic wound bed and thereby accelerate wound healing is still a significant challenge. Herein, we reported a kind of aligned porous poly (l-lactic acid) (PlLA) electrospun fibrous membranes containing dimethyloxalylglycine (DMOG)-loaded mesoporous silica nanoparticles (DS) for diabetic wound healing. The PlLA electrospun fibers aligned in a single direction and there were ellipse-shaped nano-pores in situ generated onto the surface of fibers, while the DS were well distributed in the fibers and the DMOG as well as Si ion could be controlled released from the nanopores on the fibers. The in vitro results revealed that the aligned porous composite membranes (DS-PL) could stimulate the proliferation, migration and angiogenesis-related gene expression of human umbilical vein endothelial cells (HUVECs) compared with the pure PlLA membranes. The in vivo study further demonstrated that the prepared DS-PL membranes significantly improved neo-vascularization, re-epithelialization and collagen formation as well as inhibited inflammatory reaction in the diabetic wound bed, which eventually stimulated the healing of the diabetic wound. Collectively, these results suggest that the combination of hierarchical structures (nanopores on the aligned fibers) with the controllable released DMOG drugs as well as Si ions from the membranes, which could create a synergetic effect on the rapid stimulation of angiogenesis in the diabetic wound bed, is a potential novel therapeutic strategy for highly efficient diabetic wound healing. A chronic wound in diabetic patients is usually characterized by the poor angiogenesis and the delayed wound closure. The main innovation of this study is to design a new kind of skin tissue engineered scaffold, aligned porous poly (l-lactic acid) (PlLA) electrospun

Wound Disruption Following Colorectal Operations.

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Moghadamyeghaneh, Zhobin; Hanna, Mark H; Carmichael, Joseph C; Mills, Steven; Pigazzi, Alessio; Nguyen, Ninh T; Stamos, Michael J

2015-12-01

Postoperative wound disruption is associated with high morbidity and mortality. We sought to identify the risk factors and outcomes of wound disruption following colorectal resection. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) database was used to examine the clinical data of patients who underwent colorectal resection from 2005 to 2013. Multivariate regression analysis was performed to identify risk factors of wound disruption. We sampled a total of 164,297 patients who underwent colorectal resection. Of these, 2073 (1.3 %) had wound disruption. Patients with wound disruption had significantly higher mortality (5.1 vs. 1.9 %, AOR: 1.46, P = 0.01). The highest risk of wound disruption was seen in patients with wound infection (4.8 vs. 0.9 %, AOR: 4.11, P disruption such as chronic steroid use (AOR: 1.71, P disruption compared to open surgery (AOR: 0.61, P disruption occurs in 1.3 % of colorectal resections, and it correlates with mortality of patients. Wound infection is the strongest predictor of wound disruption. Chronic steroid use, obesity, severe COPD, prolonged operation, non-elective admission, and serum albumin level are strongly associated with wound disruption. Utilization of the laparoscopic approach may decrease the risk of wound disruption when possible.

Stereotyped distribution of proliferating keratinocytes in disorders affecting the epidermis

International Nuclear Information System (INIS)

Pierard-Franchimont, C.; Pierard, G.E.

1989-01-01

We used the technique of autoradiography after incorporation of tritiated thymidine ( 3 H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of 3 H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of 3 H-TdR-labeled keratinocytes in the various layers of the epidermis

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing.

Science.gov (United States)

di Martino, O; Tito, A; De Lucia, A; Cimmino, A; Cicotti, F; Apone, F; Colucci, G; Calabrò, V

2017-01-01

Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus , family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE). We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Hibiscus syriacus Extract from an Established Cell Culture Stimulates Skin Wound Healing

Directory of Open Access Journals (Sweden)

O. di Martino

2017-01-01

Full Text Available Higher plants are the source of a wide array of bioactive compounds that support skin integrity and health. Hibiscus syriacus, family Malvaceae, is a plant of Chinese origin known for its antipyretic, anthelmintic, and antifungal properties. The aim of this study was to assess the healing and hydration properties of H. syriacus ethanolic extract (HSEE. We established a cell culture from Hibiscus syriacus leaves and obtained an ethanol soluble extract from cultured cells. The properties of the extract were tested by gene expression and functional analyses on human fibroblast, keratinocytes, and skin explants. HSEE treatment increased the healing potential of fibroblasts and keratinocytes. Specifically, HSEE significantly stimulated fibronectin and collagen synthesis by 16 and 60%, respectively, while fibroblasts contractility was enhanced by 30%. These results were confirmed on skin explants, where HSEE accelerated the wound healing activity in terms of epithelium formation and fibronectin production. Moreover, HSEE increased the expression of genes involved in skin hydration and homeostasis. Specifically, aquaporin 3 and filaggrin genes were enhanced by 20 and 58%, respectively. Our data show that HSEE contains compounds capable of stimulating expression of biomarkers relevant to skin regeneration and hydration thereby counteracting molecular pathways leading to skin damage and aging.

Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

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Mehic, Denis; Bakiri, Latifa; Ghannadan, Minoo; Wagner, Erwin F; Tschachler, Erwin

2005-01-01

Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

Effects of Human Mesenchymal Stem Cells Coculture on Calcium-Induced Differentiation of Normal Human Keratinocytes.

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Sah, Shyam Kishor; Kim, Hae Young; Lee, Ji Hae; Lee, Seong-Wook; Kim, Hyung-Sik; Kim, Yeon-Soo; Kang, Kyung-Sun; Kim, Tae-Yoon

2017-06-01

The influence of mesenchymal stem cells (MSCs) on keratinocytes in altered microenvironments is poorly understood. Here, we cocultured umbilical cord blood-derived MSCs with normal human epidermal keratinocytes to evaluate their paracrine effect in the presence of high extracellular calcium (Ca 2+ ) concentration. High Ca 2+ environment to keratinocytes can disrupt normal skin barrier function due to abnormal/premature differentiation of keratinocytes. Surprisingly, we found that MSCs suppress both proliferation and differentiation of keratinocytes under a high Ca 2+ environment in transforming growth factors β1 (TGFβ1)-dependent manner. Furthermore, we determined that MSCs can regulate the mitogen-activated protein kinases, phosphatidylinositol 3-kinase/protein kinase B, and protein kinase C pathways in Ca 2+ -induced differentiated keratinocytes. Knockdown of TGFβ1 from MSCs results in decreased suppression of differentiation with significantly increased proliferation of keratinocytes compared with control MSCs. MSCs-derived TGFβ1 further induced growth inhibition of keratinocyte in high extracellular Ca 2+ environment as analyzed by a decrease in DNA synthesis, accumulation of phosphorylated retinoblastoma protein, cdc2, and increased mRNA level of p21, and independent of TGFβ1/SMAD pathway. Taken together, we found that MSCs-derived TGFβ1 is a critical regulator of keratinocyte function, and involves multiple proximal signaling cascades. Stem Cells 2017;35:1592-1602. © 2017 AlphaMed Press.

Keratinocytes at the uppermost layer of epidermis might act as sensors of atmospheric pressure change.

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Denda, Mitsuhiro

2016-01-01

It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.

Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

Science.gov (United States)

Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

2016-01-01

Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

The incidence and multiplicity rates of keratinocyte cancers in Australia.

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Pandeya, Nirmala; Olsen, Catherine M; Whiteman, David C

2017-10-16

To assess the incidence and multiplicity of keratinocyte cancers (basal cell carcinoma [BCC] and squamous cell carcinoma [SCC]) excised in Australia, and to examine variations by age, sex, state, and prior skin cancer history. Analysis of individual-level Medicare data for keratinocyte cancer treatments (identified by eight specific MBS item codes) during 2011-2014. Histological data from the QSkin prospective cohort study were analysed to estimate BCC and SCC incidence. A 10% systematic random sample of all people registered with Medicare during 1997-2014. People aged at least 20 years in 2011 who made at least one claim for any MBS medical service during 2011-2014 (1 704 193 individuals). Age-standardised incidence rates (ASRs) and standardised incidence ratios (SIRs). The person-based incidence of keratinocyte cancer excisions in Australia was 1531 per 100 000 person-years; incidence increased with age, and was higher for men than women (SIR, 1.43; 95% CI, 1.42-1.45). Lesion-based incidence was 3154 per 100 000 person-years. The estimated ASRs for BCC and SCC were 770 per 100 000 and 270 per 100 000 person-years respectively. During 2011-2014, 3.9% of Australians had one keratinocyte cancer excised, 2.7% had more than one excised; 74% of skin cancers were excised from patients who had two or more lesions removed. Multiplicity was strongly correlated with age; most male patients over 70 were treated for multiple lesions. Keratinocyte cancer incidence was eight times as high among people with a prior history of excisions as among those without. The incidence and multiplicity of keratinocyte cancer in Australia are very high, causing a large disease burden that has not previously been quantified.

Shining a Light on Black Holes in Keratinocytes.

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Bowman, Shanna L; Marks, Michael S

2018-03-01

The mechanisms by which melanins are transferred from melanocytes and stored within keratinocytes to generate skin pigmentation are hotly debated. Correia et al. and Hurbain et al. provide evidence that melanin cores of melanosomes are secreted from melanocytes and taken up and stored within non-degradative membranous organelles in keratinocytes in the basal epidermis of human skin. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation.

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van Pelt, F N; Haring, R M; Weterings, P J

1991-01-01

Micronucleus formation in cultured human keratinocytes was studied after exposure to benzo[a]pyrene, cyclophosphamide and 12-O-tetradecanoylphorbol-13-acetate without the addition of an exogenous metabolizing system. The first two agents need bioactivation by specific isoenzymes of cytochrome P-450 to form genotoxic intermediates. Benzo[a]pyrene induced the micronucleus formation in both uninduced and Aroclor 1254-pretreated cultures. Clastogenic effects of cyclophosphamide were observed only in Aroclor 1254-pretreated cells. The tumour promotor 12-O-tetradecanoylphorbol-13-acetate did not affect the frequency of micronuclei in human keratinocytes. The data indicate that cultured human keratinocytes can be used to study the tissue-specific response to genotoxic agents as well as interindividual variation in biotransformation capacity.

A Cooperative Copper Metal-Organic Framework-Hydrogel System Improves Wound Healing in Diabetes.

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Xiao, Jisheng; Chen, Siyu; Yi, Ji; Zhang, Hao; Ameer, Guillermo A

2017-01-05

Chronic non-healing wounds remain a major clinical challenge that would benefit from the development of advanced, regenerative dressings that promote wound closure within a clinically relevant time frame. The use of copper ions has shown promise in wound healing applications possibly by promoting angiogenesis. However, reported treatments that use copper ions require multiple applications of copper salts or oxides to the wound bed, exposing the patient to potentially toxic levels of copper ions and resulting in variable outcomes. Herein we set out to assess whether copper metal organic framework nanoparticles (HKUST-1 NPs) embedded within an antioxidant thermoresponsive citrate-based hydrogel would decrease copper ion toxicity and accelerate wound healing in diabetic mice. HKUST-1 and poly-(polyethyleneglycol citrate-co- N -isopropylacrylamide) (PPCN) were synthesized and characterized. HKUST-1 NP stability in a protein solution with and without embedding them in PPCN hydrogel was determined. Copper ion release, cytotoxicity, apoptosis, and in vitro migration processes were measured. Wound closure rates and wound blood perfusion were assessed in vivo using the splinted excisional dermal wound diabetic mouse model. HKUST-1 NP disintegrated in protein solution while HKUST-1 NPs embedded in PPCN (H-HKUST-1) were protected from degradation and copper ions were slowly released. Cytotoxicity and apoptosis due to copper ion release were significantly reduced while dermal cell migration in vitro and wound closure rates in vivo were significantly enhanced. In vivo , H-HKUST-1 induced angiogenesis, collagen deposition, and re-epithelialization during wound healing in diabetic mice. These results suggest that a cooperatively stabilized, copper ion-releasing H-HKUST-1 hydrogel is a promising innovative dressing for the treatment of chronic wounds.

Attachment and growth of human keratinocytes in a serum-free environment.

Science.gov (United States)

Gilchrest, B A; Calhoun, J K; Maciag, T

1982-08-01

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes.

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Sayedyahossein, Samar; Nini, Lylia; Irvine, Timothy S; Dagnino, Lina

2012-10-01

Phagocytic melanosome uptake by epidermal keratinocytes is a central protective mechanism against damage induced by ultraviolet radiation. Phagocytosis requires formation of pseudopodia via actin cytoskeleton rearrangements. Integrin-linked kinase (ILK) is an important modulator of actin cytoskeletal dynamics. We have examined the role of ILK in regulation of phagocytosis, using epidermal keratinocytes isolated from mice with epidermis-restricted Ilk gene inactivation. ILK-deficient cells exhibited severely impaired capacity to engulf fluorescent microspheres in response to stimulation of the keratinocyte growth factor (KGF) receptor or the protease-activated receptor-2. KGF induced ERK phosphorylation in ILK-expressing and ILK-deficient cells, suggesting that ILK is not essential for KGF receptor signaling. In contrast, KGF promoted activation of Rac1 and formation of pseudopodia in ILK-expressing, but not in ILK-deficient cells. Rac1-deficient keratinocytes also showed substantially impaired phagocytic ability, underlining the importance of ILK-dependent Rac1 function for particle engulfment. Finally, cross-modulation of KGF receptors by integrins may be another important element, as integrin β1-deficient keratinocytes also fail to show significant phagocytosis in response to KGF. Thus, we have identified a novel signaling pathway essential for phagocytosis in keratinocytes, which involves ILK-dependent activation of Rac1 in response to KGF, resulting in the formation of pseudopodia and particle uptake.

Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

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Zheng, Ruijin [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Environmental Health Science, New York Medical College, Valhalla, NY (United States); Mishin, Vladimir; Black, Adrienne T. [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Shakarjian, Michael P. [Environmental Health Science, New York Medical College, Valhalla, NY (United States); Kong, Ah-Ng Tony; Laskin, Debra L. [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Environmental and Occupational Medicine, Rutgers University-Robert Wood Johnson Medical School, Piscataway, NJ (United States)

2014-03-01

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

Wound healing in plastic surgery: does age matter? An American College of Surgeons National Surgical Quality Improvement Program study.

Science.gov (United States)

Karamanos, Efstathios; Osgood, Geoff; Siddiqui, Aamir; Rubinfeld, Ilan

2015-03-01

Increasing age has traditionally been associated with impairment in wound healing after operative interventions. This is based mostly on hearsay and anecdotal information. This idea fits with the authors’ understanding of biology in older organisms. This dictum has not been rigorously tested in clinical practice. The American College of Surgeons National Surgical Quality Improvement Program database was retrospectively queried for all patients undergoing plastic surgery from 2005 to 2010. Variables extracted included basic demographics, comorbidities, previous steroid and tobacco use, wound classification at the end of the surgery, and development of postoperative surgical-site infections. Multivariate analyses were used to investigate the impact of aging in wound dehiscence. A total of 25,967 patients were identified. Overall, the incidence of wound dehiscence was 0.75 percent (n = 196). When patients younger than 30 years were compared to older patient groups, no difference in the probability of developing wound dehiscence was noted. Specifically, the groups of patients aged 61 to 70 years and older than 70 years did not have statistically significant wound healing deficiencies [adjusted OR, 0.63 (95 percent CI, 0.11 to 3.63), adjusted p = 0.609; 2.79 (0.55 to 14.18), adjusted p = 0.217, for 61 to 70 years and older than 70 years, respectively]. Factors independently associated with wound dehiscence included postoperative abscess development, paraplegia, quadriplegia, steroid and tobacco use, deep surgical-site infection development, increasing body mass index, and wound classification at the end of surgery. In patients undergoing plastic surgery, wound dehiscence is a rare complication (0.75 percent). Aging is not associated with an increased incidence of wound dehiscence. Risk, III.

Human keratinocytes restrict chikungunya virus replication at a post-fusion step

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Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

2015-02-15

Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

Human keratinocytes restrict chikungunya virus replication at a post-fusion step

International Nuclear Information System (INIS)

Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe

2015-01-01

Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Science.gov (United States)

Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

2013-01-01

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

Science.gov (United States)

Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

2014-08-01

Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

Science.gov (United States)

Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

2015-08-02

Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence

Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces.

Science.gov (United States)

Dorkhan, Marjan; Yücel-Lindberg, Tülay; Hall, Jan; Svensäter, Gunnel; Davies, Julia R

2014-06-21

A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated. The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium. SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells. Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to

Porous CS based membranes with improved antimicrobial properties for the treatment of infected wound in veterinary applications

International Nuclear Information System (INIS)

Tonda-Turo, C.; Ruini, F.; Argentati, M.; Di Girolamo, N.; Robino, P.; Nebbia, P.; Ciardelli, G.

2016-01-01

Recently, much attention has been given to the use of innovative solution for the treatment of infected wounds in animals. Current applied treatments are often un-effective leading to infection propagation and animal death. Novel engineered membranes based on chitosan (CS) can be prepared to combine local antimicrobial effect, high flexibility and easy manipulation. In this work, CS crosslinked porous membranes with improved antimicrobial properties were prepared via freeze-drying technique to promote wound healing and to reduce the bacterial proliferation in infected injuries. Silver nanoparticles (AgNPs) and gentamicin sulfate (GS) were incorporated into the CS matrices to impart antibacterial properties on a wild range of strains. CS based porous membranes were tested for their physicochemical, thermal, mechanical as well as swelling and degradation behavior at physiological condition. Additionally, GS release profile was investigated, showing a moderate burst effect in the first days followed by a decreasing release rate which it was maintained for at least 56 days. Moreover, porous membranes loaded with GS or AgNPs showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. The bacterial strains used in this work were collected in chelonians after carapace injuries to better mimic the environment after trauma. - Highlights: • Innovative scaffolds for wound healing in veterinary applications • Novel engineered membranes based on chitosan with improved antibacterial properties • Highly flexible and versatile membranes for infected wounds

Porous CS based membranes with improved antimicrobial properties for the treatment of infected wound in veterinary applications

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Tonda-Turo, C., E-mail: chiara.tondaturo@polito.it [Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Turin (Italy); Ruini, F. [Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Turin (Italy); Argentati, M. [Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Turin (Italy); Clinic for Exotic Animals, CVS, Via Sandro Giovannini 53, 00137 Rome (Italy); Di Girolamo, N. [Clinic for Exotic Animals, CVS, Via Sandro Giovannini 53, 00137 Rome (Italy); Robino, P.; Nebbia, P. [Department of Veterinary Sciences, University of Turin, Largo Braccini 2, 10095 Grugliasco, Turin (Italy); Ciardelli, G. [Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129, Turin (Italy)

2016-03-01

Recently, much attention has been given to the use of innovative solution for the treatment of infected wounds in animals. Current applied treatments are often un-effective leading to infection propagation and animal death. Novel engineered membranes based on chitosan (CS) can be prepared to combine local antimicrobial effect, high flexibility and easy manipulation. In this work, CS crosslinked porous membranes with improved antimicrobial properties were prepared via freeze-drying technique to promote wound healing and to reduce the bacterial proliferation in infected injuries. Silver nanoparticles (AgNPs) and gentamicin sulfate (GS) were incorporated into the CS matrices to impart antibacterial properties on a wild range of strains. CS based porous membranes were tested for their physicochemical, thermal, mechanical as well as swelling and degradation behavior at physiological condition. Additionally, GS release profile was investigated, showing a moderate burst effect in the first days followed by a decreasing release rate which it was maintained for at least 56 days. Moreover, porous membranes loaded with GS or AgNPs showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. The bacterial strains used in this work were collected in chelonians after carapace injuries to better mimic the environment after trauma. - Highlights: • Innovative scaffolds for wound healing in veterinary applications • Novel engineered membranes based on chitosan with improved antibacterial properties • Highly flexible and versatile membranes for infected wounds.

Liposome Entrapment of Bacteriophages Improves Wound Healing in a Diabetic Mouse MRSA Infection

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Sanjay Chhibber

2018-03-01

Full Text Available Diabetic populations are more prone to developing wound infections which results in poor and delayed wound healing. Infection with drug resistant organisms further worsen the situation, driving searches for alternative treatment approaches such as phage therapy. Major drawback of phage therapy, however, is low phage persistence in situ, suggesting further refinement of the approach. In the present work we address this issue by employing liposomes as delivery vehicles. A liposome entrapped phage cocktail was evaluated for its ability to resolve a Staphylococcus aureus-induced diabetic excission wound infection. Two characterized S. aureus specific lytic phages, MR-5 and MR-10 alone, in combination (cocktail, or entrapped in liposomes (versus as free phages were assesed for their therapeutic efficacy in resolving diabetic wound infection. Mice treated with free phage cocktail showed significant reduction in wound bioburden, greater wound contraction and faster tissue healing than with free monophage therapy. However, to further enhance the availability of viable phages the encapsulation of phage cocktail in the liposomes was done. Results of in vitro stability studies and in vivo phage titer determination, suggests that liposomal entrapment of phage cocktail can lead to better phage persistence at the wound site. A 2 log increase in phage titre, however, was observed at the wound site with liposome entrapped as compared to the free phage cocktail, and this was associaed with increased rates of infection resolution and wound healing. Entrapment of phage cocktails within liposomes thus could represent an attractive approach for treatment of bacterial infections, not responding to antibiotis as increased phage persistence in vitro and in vivo at the wound site was observed.

Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

International Nuclear Information System (INIS)

Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T.

1991-01-01

Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes

Bilayered nanofibrous 3D hierarchy as skin rudiment by emulsion electrospinning for burn wound management.

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Pal, Pallabi; Dadhich, Prabhash; Srivas, Pavan Kumar; Das, Bodhisatwa; Maulik, Dhrubajyoti; Dhara, Santanu

2017-08-22

Mimicking skin extracellular matrix hierarchy, the present work aims to develop a bilayer skin graft comprising a porous cotton-wool-like 3D layer with membranous structure of PCL-chitosan nanofibers. Emulsion electrospinning with differential stirring periods of PCL-chitosan emulsion results in development of a bilayer 3D structure with varied morphology. The electrospun membrane has fiber diameter ∼274 nm and pore size ∼1.16 μm while fluffy 3D layer has fiber diameter ∼1.62 μm and pore size ∼62 μm. The 3D layer was further coated with collagen I isolated from Cirrhinus cirrhosus fish scales to improve biofunctionality. Surface coating with collagen I resulted in bundling the fibers together, thereby increasing their average diameter to 2.80 μm and decreasing pore size to ∼45 μm. The architecture and composition of the scaffold promotes efficient cellular activity where interconnected porosity with ECM resembling collagen I coating assists cellular adhesion, infiltration, and proliferation from initial days of fibroblast seeding, while keratinocytes migrate on the surface only without infiltrating in the membranous nanofiber layer. Anatomy of the scaffold arising due to variation in pore size distribution at different layers thereby facilitates compartmentalization and prevents initial cellular transmigration. The scaffold also assists in extracellular matrix protein synthesis and keratinocyte stratification in vitro. Further, the scaffold effectively integrates and attaches with third-degree burn wound margins created in rat models and accelerates healing in comparison to standard Tegaderm dressing™. The bilayer scaffold is thus a promising, readily available, cost-effective, off-the-shelf matrix as a skin substitute.

A Cooperative Copper Metal-Organic Framework-Hydrogel System Improves Wound Healing in Diabetes

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Xiao, Jisheng; Chen, Siyu; Yi, Ji; Zhang, Hao; Ameer, Guillermo A.

2016-01-01

Chronic non-healing wounds remain a major clinical challenge that would benefit from the development of advanced, regenerative dressings that promote wound closure within a clinically relevant time frame. The use of copper ions has shown promise in wound healing applications possibly by promoting angiogenesis. However, reported treatments that use copper ions require multiple applications of copper salts or oxides to the wound bed, exposing the patient to potentially toxic levels of copper io...

The protective effects of piceatannol from passion fruit (Passiflora edulis) seeds in UVB-irradiated keratinocytes.

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Maruki-Uchida, Hiroko; Kurita, Ikuko; Sugiyama, Kenkichi; Sai, Masahiko; Maeda, Kazuhisa; Ito, Tatsuhiko

2013-01-01

The use of naturally occurring botanicals with substantial antioxidant activity to prevent photoageing is receiving increasing attention. We have previously identified piceatannol and scirpusin B, which is a dimer of piceatannol, as strong antioxidants that are present in passion fruit (Passiflora edulis) seeds. In the present study, the effects of passion fruit seed extract, piceatannol, and scirpusin B on human keratinocytes were investigated. The passion fruit seed extract and piceatannol upregulated the glutathione (GSH) levels in keratinocytes in a dose-dependent manner, indicating that piceatannol is an active component of the passion fruit seed extract in keratinocytes. The pretreatment with piceatannol also suppressed the UVB-induced generation of reactive oxygen species (ROS) in the keratinocytes. In addition, the transfer of the medium from the UVB-irradiated keratinocytes to non-irradiated fibroblasts enhanced matrix-metalloproteinase (MMP)-1 activity, and this MMP-1 induction was reduced when the keratinocytes were pretreated with piceatannol. These results suggest that piceatannol attenuates the UVB-induced activity of MMP-1 along with a reduction of ROS generation in keratinocytes. Thus, piceatannol and passion fruit seed extract containing high amounts of piceatannol are potential anti-photoageing cosmetic ingredients.

How Does Chronic Cigarette Smoke Exposure Affect Human Skin? A Global Proteomics Study in Primary Human Keratinocytes.

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Rajagopalan, Pavithra; Nanjappa, Vishalakshi; Raja, Remya; Jain, Ankit P; Mangalaparthi, Kiran K; Sathe, Gajanan J; Babu, Niraj; Patel, Krishna; Cavusoglu, Nükhet; Soeur, Jeremie; Pandey, Akhilesh; Roy, Nita; Breton, Lionel; Chatterjee, Aditi; Misra, Namita; Gowda, Harsha

2016-11-01

Cigarette smoking has been associated with multiple negative effects on human skin. Long-term physiological effects of cigarette smoke are through chronic and not acute exposure. Molecular alterations due to chronic exposure to cigarette smoke remain unclear. Primary human skin keratinocytes chronically exposed to cigarette smoke condensate (CSC) showed a decreased wound-healing capacity with an increased expression of NRF2 and MMP9. Using quantitative proteomics, we identified 4728 proteins, of which 105 proteins were overexpressed (≥2-fold) and 41 proteins were downregulated (≤2-fold) in primary skin keratinocytes chronically exposed to CSC. We observed an alteration in the expression of several proteins involved in maintenance of epithelial barrier integrity, including keratin 80 (5.3 fold, p value 2.5 × 10 -7 ), cystatin A (3.6-fold, p value 3.2 × 10 -3 ), and periplakin (2.4-fold, p value 1.2 × 10 -8 ). Increased expression of proteins associated with skin hydration, including caspase 14 (2.2-fold, p value 4.7 × 10 -2 ) and filaggrin (3.6-fold, p value 5.4 × 10 -7 ), was also observed. In addition, we report differential expression of several proteins, including adipogenesis regulatory factor (2.5-fold, p value 1.3 × 10 -3 ) and histone H1.0 (2.5-fold, p value 6.3 × 10 -3 ) that have not been reported earlier. Bioinformatics analyses demonstrated that proteins differentially expressed in response to CSC are largely related to oxidative stress, maintenance of skin integrity, and anti-inflammatory responses. Importantly, treatment with vitamin E, a widely used antioxidant, could partially rescue adverse effects of CSC exposure in primary skin keratinocytes. The utility of antioxidant-based new dermatological formulations in delaying or preventing skin aging and oxidative damages caused by chronic cigarette smoke exposure warrants further clinical investigations and multi-omics research.

The Future of Data-Driven Wound Care.

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Woods, Jon S; Saxena, Mayur; Nagamine, Tasha; Howell, Raelina S; Criscitelli, Theresa; Gorenstein, Scott; M Gillette, Brian

2018-04-01

Care for patients with chronic wounds can be complex, and the chances of poor outcomes are high if wound care is not optimized through evidence-based protocols. Tracking and managing every variable and comorbidity in patients with wounds is difficult despite the increasing use of wound-specific electronic medical records. Harnessing the power of big data analytics to help nurses and physicians provide optimized care based on the care provided to millions of patients can result in better outcomes. Numerous applications of machine learning toward workflow improvements, inpatient monitoring, outpatient communication, and hospital operations can improve overall efficiency and efficacy of care delivery in and out of the hospital, while reducing adverse events and complications. This article provides an overview of the application of big data analytics and machine learning in health care, highlights important recent advances, and discusses how these technologies may revolutionize advanced wound care. © AORN, Inc, 2018.

Does treatment of split-thickness skin grafts with negative-pressure wound therapy improve tissue markers of wound healing in a porcine experimental model?

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Ward, Christopher; Ciraulo, David; Coulter, Michael; Desjardins, Steven; Liaw, Lucy; Peterson, Sarah

2012-08-01

Negative-pressure wound therapy (NPWT) has been used for to treat wounds for more than 15 years and, more recently, has been used to secure split-thickness skin grafts. There are some data to support this use of NPWT, but the actual mechanism by which NPWT speeds healing or improves skin graft take is not entirely known. The purpose of this project was to assess whether NPWT improved angiogenesis, wound healing, or graft survival when compared with traditional bolster dressings securing split-thickness skin grafts in a porcine model. We performed two split-thickness skin grafts on each of eight 30 kg Yorkshire pigs. We took graft biopsies on postoperative days 2, 4, 6, 8, and 10 and submitted the samples for immunohistochemical staining, as well as standard hematoxylin and eosin staining. We measured the degree of vascular ingrowth via immunohistochemical staining for von Willenbrand's factor to better identify blood vessel epithelium. We determined the mean cross-sectional area of blood vessels present for each representative specimen, and then compared the bolster and NPWT samples. We also assessed each graft for incorporation and survival at postoperative day 10. Our analysis of the data revealed that there was no statistically significant difference in the degree of vascular ingrowth as measured by mean cross-sectional capillary area (p = 0.23). We did not note any difference in graft survival or apparent incorporation on a macroscopic level, although standard hematoxylin and eosin staining indicated that microscopically, there seemed to be better subjective graft incorporation in the NPWT samples and a nonsignificant trend toward improved graft survival in the NPWT group. We were unable to demonstrate a significant difference in vessel ingrowth when comparing NPWT and traditional bolster methods for split-thickness skin graft fixation. More studies are needed to elucidate the manner by which NPWT exerts its effects and the true clinical magnitude of these

Negative pressure wound therapy for the treatment of infected wounds with exposed knee joint after patellar fracture.

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Lee, Sang Yang; Niikura, Takahiro; Miwa, Masahiko; Sakai, Yoshitada; Oe, Keisuke; Fukazawa, Takahiro; Kawakami, Yohei; Kurosaka, Masahiro

2011-06-14

Treatment of soft tissue defects with exposed bones and joints, resulting from trauma, infection, and surgical complications, represents a major challenge. The introduction of negative pressure wound therapy has changed many wound management practices. Negative pressure wound therapy has recently been used in the orthopedic field for management of traumatic or open wounds with exposed bone, nerve, tendon, and orthopedic implants. This article describes a case of a patient with a large soft tissue defect and exposed knee joint, in which negative pressure wound therapy markedly improved wound healing. A 50-year-old man presented with an ulceration of his left knee with exposed joint, caused by severe wound infections after open reduction and internal fixation of a patellar fracture. After 20 days of negative pressure wound therapy, a granulated wound bed covered the exposed bones and joint.To our knowledge, this is the first report of negative pressure wound therapy used in a patient with a large soft tissue defect with exposed knee joint. Despite the chronic wound secondary to infection, healing was achieved through the use of the negative pressure wound therapy, thus promoting granulation tissue formation and closing the joint. We suggest negative pressure wound therapy as an alternative option for patients with lower limb wounds containing exposed bones and joints when free flap transfer is contraindicated. Our result added to the growing evidence that negative pressure wound therapy is a useful adjunctive treatment for open wounds around the knee joint. Copyright 2011, SLACK Incorporated.

The peanut lectin-binding glycoproteins of human epidermal keratinocytes

International Nuclear Information System (INIS)

Morrison, A.I.; Keeble, S.; Watt, F.M.

1988-01-01

The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

Use of a risk assessment method to improve the safety of negative pressure wound therapy.

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Lelong, Anne-Sophie; Martelli, Nicolas; Bonan, Brigitte; Prognon, Patrice; Pineau, Judith

2014-06-01

To conduct a risk analysis of the negative pressure wound therapy (NPWT) care process and to improve the safety of NPWT, a working group of nurses, hospital pharmacists, physicians and hospital managers performed a risk analysis for the process of NPWT care. The failure modes, effects and criticality analysis (FMECA) method was used for this analysis. Failure modes and their consequences were defined and classified as a function of their criticality to identify priority actions for improvement. By contrast to classical FMECA, the criticality index (CI) of each consequence was calculated by multiplying occurrence, severity and detection scores. We identified 13 failure modes, leading to 20 different consequences. The CI of consequences was initially 712, falling to 357 after corrective measures were implemented. The major improvements proposed included the establishment of 6-monthly training cycles for nurses, physicians and surgeons and the introduction of computerised prescription for NPWT. The FMECA method also made it possible to prioritise actions as a function of the criticality ranking of consequences and was easily understood and used by the working group. This study is, to our knowledge, the first to use the FMECA method to improve the safety of NPWT. © 2012 The Authors. International Wound Journal © 2012 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Effect of platelet lysate on human cells involved in different phases of wound healing.

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Maria Chiara Barsotti

Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Stem Cells and Engineered Scaffolds for Regenerative Wound Healing

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Biraja C. Dash

2018-03-01

Full Text Available The normal wound healing process involves a well-organized cascade of biological pathways and any failure in this process leads to wounds becoming chronic. Non-healing wounds are a burden on healthcare systems and set to increase with aging population and growing incidences of obesity and diabetes. Stem cell-based therapies have the potential to heal chronic wounds but have so far seen little success in the clinic. Current research has been focused on using polymeric biomaterial systems that can act as a niche for these stem cells to improve their survival and paracrine activity that would eventually promote wound healing. Furthermore, different modification strategies have been developed to improve stem cell survival and differentiation, ultimately promoting regenerative wound healing. This review focuses on advanced polymeric scaffolds that have been used to deliver stem cells and have been tested for their efficiency in preclinical animal models of wounds.

Stem Cells and Engineered Scaffolds for Regenerative Wound Healing.

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Dash, Biraja C; Xu, Zhenzhen; Lin, Lawrence; Koo, Andrew; Ndon, Sifon; Berthiaume, Francois; Dardik, Alan; Hsia, Henry

2018-03-09

The normal wound healing process involves a well-organized cascade of biological pathways and any failure in this process leads to wounds becoming chronic. Non-healing wounds are a burden on healthcare systems and set to increase with aging population and growing incidences of obesity and diabetes. Stem cell-based therapies have the potential to heal chronic wounds but have so far seen little success in the clinic. Current research has been focused on using polymeric biomaterial systems that can act as a niche for these stem cells to improve their survival and paracrine activity that would eventually promote wound healing. Furthermore, different modification strategies have been developed to improve stem cell survival and differentiation, ultimately promoting regenerative wound healing. This review focuses on advanced polymeric scaffolds that have been used to deliver stem cells and have been tested for their efficiency in preclinical animal models of wounds.

Aldefluor protocol to sort keratinocytes stem cells from skin

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Noronha, Samuel Marcos Ribeiro; Gragnani, Alfredo; Pereira, Thiago Antônio Calado; Correa, Silvana Aparecida Alves; Bonucci, Jessica; Ferreira, Lydia Masako

2017-01-01

Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 c...

Growth Hormone-Releasing Peptide 6 Enhances the Healing Process and Improves the Esthetic Outcome of the Wounds

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Yssel Mendoza Marí

2016-01-01

Full Text Available In addition to its cytoprotective effects, growth hormone-releasing peptide 6 (GHRP-6 proved to reduce liver fibrotic induration. CD36 as one of the GHRP-6 receptors appears abundantly represented in cutaneous wounds granulation tissue. The healing response in a scenario of CD36 agonistic stimulation had not been previously investigated. Excisional full-thickness wounds (6 mmØ were created in the dorsum of Wistar rats and topically treated twice a day for 5 days. The universal model of rabbit’s ears hypertrophic scars was implemented and the animals were treated daily for 30 days. Treatments for both species were based on a CMC jelly composition containing GHRP-6 400 μg/mL. Wounds response characterization included closure dynamic, RT-PCR transcriptional profile, histology, and histomorphometric procedures. The rats experiment indicated that GHRP-6 pharmacodynamics involves attenuation of immunoinflammatory mediators, their effector cells, and the reduction of the expression of fibrotic cytokines. Importantly, in the hypertrophic scars rabbit’s model, GHRP-6 intervention dramatically reduced the onset of exuberant scars by activating PPARγ and reducing the expression of fibrogenic cytokines. GHRP-6 showed no effect on the reversion of consolidated lesions. This evidence supports the notion that CD36 is an active and pharmacologically approachable receptor to attenuate wound inflammation and accelerate its closure so as to improve wound esthetic.

Phototherapy improves healing of cutaneous wounds in nourished and undernourished Wistar rats.

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Pinheiro, Antonio Luiz Barbosa; Meireles, Gyselle Cynthia Silva; de Barros Vieira, Alessandro Leonardo; Almeida, Darcy; Carvalho, Carolina Montagn; dos Santos, Jean Nunes

2004-01-01

A wound represents the interruption of the continuity of tissue that is followed by damage or cellular death. Wound healing occurs due to a competitive mechanism between the synthesis and lysis of collagen. Any factor that increases collagen lysis or reduces its synthesis may result in changes in the healing process, i.e., nutritional deficiencies. Phototherapies have been suggested as an effective method to improve wound healing. This study evaluated, histologically, the differences in the healing of cutaneous wounds in nourished and undernourished rats following laser therapy or illumination by polarized light. Fifty nourished or undernourished Wistar rats had a standardized wound created on the dorsum and were divided into 6 subgroups: Group 1--Control (Standard diet; n=5); Group 2--Control (DBR; n=5); Group 3--Standard diet + laser therapy (lambda635nm; 20J/cm2, n=5; or 40J/cm2, n=5); Group 4--Standard diet + Bioptron (lambda400-2000nm; 20J/cm2, n=5; or 40 J/cm2, n=5); Group 5--DBR + laser therapy (lambda635nm; 20J/cm2, n=5; or 40J/cm2, n=5); Group 6--DBR + Bioptron (lambda400-2000nm; 20J/cm2, n=5; or 40 J/cm2, n=5). The first application of the treatment was carried out immediately after surgery and repeated every 24 h during 7 days. Specimens were routinely processed (wax, cut and stained with H&E and Picrosirius stain) and analyzed under light microscopy. Analysis included re-epithelization, inflammatory infiltrate, and fibroblastic proliferation. Picrosirius stained slides were used to perform descriptive analysis of the collagen fibers. The results showed the best results for nourished and undernourished groups treated with polarized light at a dose of 20J/cm2 and the undernourished groups irradiated with the laser light. It is concluded that the nutritional status influenced the progression of the healing process as well as the quality of the healed tissue and that the use of both modalities of phototherapy resulted in a positive biomodulatory effect in

New techniques for wound debridement.

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Madhok, Brijesh M; Vowden, Kathryn; Vowden, Peter

2013-06-01

Debridement is a crucial component of wound management. Traditionally, several types of wound debridement techniques have been used in clinical practice such as autolytic, enzymatic, biodebridement, mechanical, conservative sharp and surgical. Various factors determine the method of choice for debridement for a particular wound such as suitability to the patient, the type of wound, its anatomical location and the extent of debridement required. Recently developed products are beginning to challenge traditional techniques that are currently used in wound bed preparation. The purpose of this review was to critically evaluate the current evidence behind the use of these newer techniques in clinical practice. There is some evidence to suggest that low frequency ultrasound therapy may improve healing rates in patients with venous ulcers and diabetic foot ulcers. Hydrosurgery debridement is quick and precise, but the current evidence is limited and further studies are underway. Debridement using a monofilament polyester fibre pad and plasma-mediated bipolar radiofrequency ablation are both very new techniques. The initial evidence is limited, and further studies are warranted to confirm their role in management of chronic wounds. © 2013 The Authors. International Wound Journal © 2013 John Wiley & Sons Ltd and Medicalhelplines.com Inc.

The use of negative pressure wound therapy in the treatment of infected wounds. Case studies

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Daniel de Alcântara Jones

Full Text Available ABSTRACT OBJECTIVE: To evaluate the results and benefits obtained from the topical use of negative pressure wound therapy (NPWT in patients with infected wounds. METHODS: This was a retrospective study of 20 patients (17 males and three females, mean age 42 years with infected wounds treated using NPWT. The infected wounds were caused by trauma. The treatment system used was VAC.(r (Vacuum Assisted Closure, KCI, San Antonio, United States applied to the wound in continuous mode from 100 to 125 mmHg. The parameters related to the wounds (location, number of VAC changes, the size of the defects in the soft parts, and the evolution of the state of the wound, length of hospital stay, length of intravenous antibiotic therapy, and complications related to the use of this therapy were evaluated. RESULTS: The mean length of the hospital stay, use of NPWT, and antibacterial therapy were 41 days, 22.5 days, and 20 days respectively. The use of the VAC led to a mean reduction of 29% in the wound area (95.65-68.1 cm2; p < 0.05. Only one patient did not show any improvement in the final appearance of the wound with complete eradication of the infection. No complication directly caused by NPWT was observed. CONCLUSION: NPWT stimulates infection-free scar tissue formation in a short time, and is a quick and comfortable alternative to conventional infected wounds treatment methods.

Effects of topical topiramate in wound healing in mice.

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Jara, Carlos Poblete; Bóbbo, Vanessa Cristina Dias; Carraro, Rodrigo Scarpari; de Araujo, Thiago Matos Ferreira; Lima, Maria H M; Velloso, Licio A; Araújo, Eliana P

2018-02-23

Recent studies have indicated that systemic topiramate can induce an improvement on the aesthetic appearance of skin scars. Here, we evaluated topical topiramate as an agent to improve wound healing in C57/BL6 mice. Mice were inflicted with a 6.0 mm punch to create two wounds in the skin of the dorsal region. Thereafter, mice were randomly assigned to either vehicle or topical topiramate (20 µl of 2% cream) once a day for 14 days, beginning on the same day as wound generation. We analyzed the wound samples over real-time PCR, Western blotting, and microscopy. There was no effect of the topiramate treatment on the time for complete reepithelization of the wound. However, on microscopic analysis, topiramate treatment resulted in increased granulation tissue, thicker epidermal repair, and improved deposition of type I collagen fibers. During wound healing, there were increased expressions of anti-inflammatory markers, such as IL-10, TGF-β1, and reduced expression of the active form of JNK. In addition, topiramate treatment increased the expression of active forms of two intermediaries in the insulin-signaling pathway, IRS-1 and Akt. Finally, at the end of the wound-healing process, topiramate treatment resulted in increased expression of SOX-2, a transcription factor that is essential to maintain cell self-renewal of undifferentiated embryonic stem cells. We conclude that topical topiramate can improve the overall quality of wound healing in the healthy skin of mice. This improvement is accompanied by reduced expression of markers involved in inflammation and increased expression of proteins of the insulin-signaling pathway.

The Effects of Antifungal Azoles on Inflammatory Cytokine Production in Human Keratinocytes

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K Zomorodian

2008-04-01

Full Text Available ABSTRACT: Introduction & Objective: Azoles drugs are being used successfully in treatment of fungal infections. Recently, immunosuppressive effects of some of these agents have been reported. Keratinocytes, as the major cells of the skin, have an important role in innate immunity against pathogenic agents. Considering the scanty of information about the effects of azoles on immune responces, this study was conducted to assess the expression and secretion of inflammatory cytokines in keratinocytes following treatment with azole drugs. Materials & Methods: This is an exprimental study conducted in in molecular biology division in Tehran University of Medical Sciences and Immunodermatology Department in Vienna Medical University. Primery keratinocytes were cultured and treated with different concentrations of fluconazole, itraconazole, ketoconazole and griseofulvin. Secreted IL1, IL6 and TNF-α by keratinocytes in culture supernatant were measured by quantitative enzyme immunoassay technique. Moreover, expression of the genes encoding IL1 and IL8 was evaluated by Real Time-PCR. Results: Treatment of keratinocytes with different concentrations of fluconazole and low concentration of ketoconazole resulted in decrease in IL1 secretion, but Itraconazole and griseofulvin did not show such an effect at the same concentrations. In addition, none of the examined drugs had an effect on secretion level of IL6 and TNF-α. Quantitative analysis of IL1 and IL8 encoding genes revealed that transcription on these genes might be suppressed following treatment with fluconazole or ketoconazole. Conclusion: Fluconazole and ketoconazole might modulate the expression and secretion of IL1 and IL8 and affect the direction of immune responses induced by keratinocytes

Lactobacillus rhamnosus GG Inhibits the Toxic Effects of Staphylococcus aureus on Epidermal Keratinocytes

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Mohammedsaeed, Walaa; McBain, Andrew J.; Cruickshank, Sheena M.

2014-01-01

Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 108 CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. PMID:25015889

Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

International Nuclear Information System (INIS)

Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

2016-01-01

The effects and the underlying mechanisms of hydrogen sulfide (H 2 S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H 2 S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H 2 S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H 2 S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H 2 S promotes keratinocyte proliferation and differentiation. • The effects of H 2 S on proliferation and differentiation is modulated by autophagy. • Exogenous H 2 S has no effect on keratinocyte apoptosis.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

Science.gov (United States)

Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

Significant Differences in Nurses’ Knowledge of Basic Wound Management

DEFF Research Database (Denmark)

Zarchi, Kian; Latif, Seemab; Haugaard, Vibeke B

2014-01-01

Wounds represent a growing healthcare problem due to an aging population. Nurses play a key role in wound management and their theoretical understanding of basic wound management may be expected to influence the quality of wound therapy fundamentally. In this study, we evaluated the level...... of knowledge of wound management in 136 Danish nurses working in 3 different settings: advanced wound care clinics, home care and general hospital departments. We found that hospital nurses had less theoretical knowledge than home care nurses and nurses working at advanced wound care clinics. We also found...... of wound management in Denmark and suggests how improvements might be achieved....

MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

International Nuclear Information System (INIS)

Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming

2016-01-01

MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

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Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming, E-mail: lizm_1001@sina.com

2016-02-26

MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

Pimecrolimus enhances TLR2/6-induced expression of antimicrobial peptides in keratinocytes.

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Büchau, Amanda S; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Gallo, Richard L

2008-11-01

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

Directory of Open Access Journals (Sweden)

L. V. Altukhova

2015-09-01

Full Text Available Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160×103 fibroblasts and (130–140×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in

AKT delays the early-activated apoptotic pathway in UVB-irradiated keratinocytes via BAD translocation.

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Claerhout, Sofie; Decraene, David; Van Laethem, An; Van Kelst, Sofie; Agostinis, Patrizia; Garmyn, Marjan

2007-02-01

Upon irradiation with a high dose of UVB, keratinocytes undergo apoptosis as a protective mechanism. In previous work, we demonstrated the existence of an early-activated UVB-induced apoptotic pathway in growth factor-depleted human keratinocytes, which can be substantially delayed by the exclusive supplementation of IGF-1. We now show that in human keratinocytes, IGF-1 inhibits the onset of UVB-triggered apoptosis through a transcriptional independent, AKT-mediated mechanism, involving BAD serine 136 phosphorylation. Our results show that the early UVB-induced apoptosis in growth factor-depleted human keratinocytes is exclusively triggered through the mitochondrial pathway. It is accompanied by BAX translocation, cytochrome c release, and procaspase-9 cleavage, but not by procaspase-8 or BID cleavage. In human keratinocytes, IGF-1 supplementation inhibits these events in a transcription-independent manner. Both IGF-1 supplementation and the transduction of a membrane-targeted form of AKT result in a shift of the BH3-only protein BAD from the mitochondria to the cytoplasm, paralleled by an increase of AKT-specific Ser136 phospho-BAD bound to 14-3-3zeta protein. These data indicate that AKT-induced BAD phosphorylation and its subsequent cytoplasmic sequestration by 14-3-3zeta is a major mechanism responsible for the postponement of UVB-induced apoptosis in human keratinocytes.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

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Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Preparation and characterization of novel antibacterial castor oil-based polyurethane membranes for wound dressing application.

Science.gov (United States)

Yari, Abbas; Yeganeh, Hamid; Bakhshi, Hadi; Gharibi, Reza

2014-01-01

Preparation of novel antibacterial and cytocompatible polyurethane membranes as occlusive dressing, which can provide moist and sterile environment over mild exudative wounds is considered in this work. In this regard, an epoxy-terminated polyurethane (EPU) prepolymer based on castor oil and glycidyltriethylammonium chloride (GTEAC) as a reactive bactericidal agent were synthesized. Polyurethane membranes were prepared through cocuring of EPU and different content of GTEAC with 1,4-butane diamine. The physical and mechanical properties, as well as cytocompatibility and antibacterial performance of prepared membranes were studied. Depending on their chemical formulations, the equilibrium water absorption and water vapor transmission rate values of the membranes were in ranges of 3-85% and 53-154g m(-2) day(-1), respectively. Therefore, these transparent membranes can maintain for a long period the moist environment over the wounds with low exudates. Detailed cytotoxicity analysis of samples against mouse L929 fibroblast and MCA-3D keratinocyte cells showed good level of cytocompatibility of membranes after purification via extraction of residual unreacted GTEAC moieties. The antibacterial activity of the membranes against Escherichia coli and Staphylococcus aureus bacteria was also studied. The membrane containing 50% GTEAC exhibited an effective antibacterial activity, while showed acceptable cytocompatibility and therefore, can be applied as an antibacterial occlusive wound dressing. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

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Xie, Xin [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Dai, Hui [Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang Province (China); Zhuang, Binyu [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Chai, Li; Xie, Yanguang [Institute of Dermatology of Heilongjiang Province, Harbin, 150001, Heilongjiang Province (China); Li, Yuzhen, E-mail: liyuzhen@medmail.com.cn [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China)

2016-04-08

The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

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Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan); Akagawa, Mitsugu [Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531 (Japan); Tsuji-Naito, Kentaro, E-mail: knaito@dhc.co.jp [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan)

2016-07-08

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes

International Nuclear Information System (INIS)

Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Woo, So-Youn

2017-01-01

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF. - Highlights: • Psoriasis-like skin inflammation increase dermal mast cells. • Keratinocyte produce stem cell factor in psoriasis-like skin inflammation. • Keratinocyte promote mast cell proliferation by stem cell factor dependent manner

Hydroxypropyltrimethyl Ammonium Chloride Chitosan Functionalized-PLGA Electrospun Fibrous Membranes as Antibacterial Wound Dressing: In Vitro and In Vivo Evaluation

Directory of Open Access Journals (Sweden)

Shengbing Yang

2017-12-01

Full Text Available A novel poly(lactic-co-glycolic acid (PLGA-hydroxypropyltrimethyl ammonium chloride chitosan (HACC composite nanofiber wound dressing was prepared through electrospinning and the entrapment-graft technique as an antibacterial dressing for cutaneous wound healing. HACC with 30% degrees of substitution (DS was immobilized onto the surface of PLGA membranes via the reaction between carboxyl groups in PLGA after alkali treatment and the reactive groups (–NH2 in HACC molecules. The naked PLGA and chitosan graft PLGA (PLGA-CS membranes served as controls. The surface immobilization was characterized by scanning electron microscopy (SEM, atomic force microscopy (AFM, Fourier transform infrared (FTIR, thermogravimetric analysis (TGA and energy dispersive X-ray spectrometry (EDX. The morphology studies showed that the membranes remain uniform after the immobilization process. The effects of the surface modification by HACC and CS on the biological properties of the membranes were also investigated. Compared with PLGA and PLGA-CS, PLGA-HACC exhibited more effective antibacterial activity towards both Gram-positive (S. aureus and Gram-negative (P. aeruginosa bacteria. The newly developed fibrous membranes were evaluated in vitro for their cytotoxicity using human dermal fibroblasts (HDFs and human keratinocytes (HaCaTs and in vivo using a wound healing mice model. It was revealed that PLGA-HACC fibrous membranes exhibited favorable cytocompatibility and significantly stimulated adhesion, spreading and proliferation of HDFs and HaCaTs. PLGA-HACC exhibited excellent wound healing efficacy, which was confirmed using a full thickness excision wound model in S. aureus-infected mice. The experimental results in this work suggest that PLGA-HACC is a strong candidate for use as a therapeutic biomaterial in the treatment of infected wounds.

A Cross-sectional, Descriptive, Quality Improvement Project to Assess Undergraduate Nursing Students' Clinical Exposure to Patients With Wounds in an Introductory Nursing Course.

Science.gov (United States)

Pieper, Barbara; Keves-Foster, Mary Kathryn; Ashare, JoAnn; Zugcic, Mary; Albdour, Maha; Albdour, Dalia

2016-04-01

Because nurses frequently participate in decisions related to wound care, learning about wounds and their care during undergraduate education is critical. A cross-sectional, descriptive, quality improvement project was conducted in an introductory baccalaureate nursing course to identify: 1) the types of patients with wounds assigned to beginning students, 2) patient wound care procedures and dressings, and 3) student level of participation in wound care. Data were collected from the weekly notes recorded about students' (N = 49) patient care experiences in 3 acute care hospitals for 9 clinical days during 1 semester. Data were recorded on a paper-and-pencil form by instructors at the end of the clinical day and included type of wound, wound irrigation, dressing, technique of care, and student's participation. Descriptive statistics were used to examine the frequency and distribution of the wound characteristics and care assessed. Of the 284 patients assigned to students, 75 (26.4%) had a wound. The most common wound was a surgical incision (49, 65%) and was closed (36, 73.5%). Twenty-six (26) patients had a pressure ulcer, most commonly Stage II. The most common dressing was dry gauze (29). Damp gauze was used on 18 wounds. Wound irrigation was recorded for 24 wound protocols and performed with a bulb syringe or by pouring the solution from a container. Generally, nonsterile wound care was performed. Twenty-five (25) students performed wound care with the instructor, 16 watched the care performed by another clinician, and 10 participated with another nurse in the wound care. For 22 patients, the wound care was neither observed nor performed because either it was not time for the dressing to be changed or it was only to be changed by a medical team. From these data, it was concluded beginning nursing students had some, but limited, clinical experience with patients with wounds. Students' wound care experiences need further examination, especially across multiple

Matrix- and plasma-derived peptides promote tissue-specific injury responses and wound healing in diabetic swine.

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Sheets, Anthony R; Massey, Conner J; Cronk, Stephen M; Iafrati, Mark D; Herman, Ira M

2016-07-02

Non-healing wounds are a major global health concern and account for the majority of non-traumatic limb amputations worldwide. However, compared to standard care practices, few advanced therapeutics effectively resolve these injuries stemming from cardiovascular disease, aging, and diabetes-related vasculopathies. While matrix turnover is disrupted in these injuries, debriding enzymes may promote healing by releasing matrix fragments that induce cell migration, proliferation, and morphogenesis, and plasma products may also stimulate these processes. Thus, we created matrix- and plasma-derived peptides, Comb1 and UN3, which induce cellular injury responses in vitro, and accelerate healing in rodent models of non-healing wounds. However, the effects of these peptides in non-healing wounds in diabetes are not known. Here, we interrogated whether these peptides stimulate healing in a diabetic porcine model highly reminiscent of human healing impairments in type 1 and type 2-diabetes. After 3-6 weeks of streptozotocin-induced diabetes, full-thickness wounds were surgically created on the backs of adult female Yorkshire swine under general anesthesia. Comb1 and UN3 peptides or sterile saline (negative control) were administered to wounds daily for 3-7 days. Following sacrifice, wound tissues were harvested, and quantitative histological and immunohistochemical analyses were performed for wound closure, angiogenesis and granulation tissue deposition, along with quantitative molecular analyses of factors critical for angiogenesis, epithelialization, and dermal matrix remodeling. Comb1 and UN3 significantly increase re-epithelialization and angiogenesis in diabetic porcine wounds, compared to saline-treated controls. Additionally, fluorescein-conjugated Comb1 labels keratinocytes, fibroblasts, and vascular endothelial cells in porcine wounds, and Far western blotting reveals these cell populations express multiple fluorescein-Comb1-interacting proteins in vitro. Further

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization.

Science.gov (United States)

Haynes, S L; Shuttleworth, C A; Kielty, C M

1997-07-01

Fibrillin-containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal-epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal-epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal-epithelial junction is not well defined. We have used fresh tissues, freshly isolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin-1 and fibrillin-2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co-ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.

Wound Healing Effects of Rose Placenta in a Mouse Model of Full-Thickness Wounds

Directory of Open Access Journals (Sweden)

Yang Woo Kim

2015-11-01

Full Text Available BackgroundRosa damascena, a type of herb, has been used for wound healing in Eastern folk medicine. The goal of this study was to evaluate the effectiveness of rose placenta from R. damascena in a full-thickness wound model in mice.MethodsSixty six-week-old C57BL/6N mice were used. Full-thickness wounds were made with an 8-mm diameter punch. Two wounds were made on each side of the back, and wounds were assigned randomly to the control and experimental groups. Rose placenta (250 µg was injected in the experimental group, and normal saline was injected in the control group. Wound sizes were measured with digital photography, and specimens were harvested. Immunohistochemical staining was performed to assess the expression of epidermal growth factor (EGF, vascular endothelial growth factor (VEGF, transforming growth factor-β1 (TGF-β1, and CD31. Vessel density was measured. Quantitative analysis using an enzyme-linked immunosorbent assay (ELISA for EGF was performed. All evaluations were performed on postoperative days 0, 2, 4, 7, and 10. Statistical analyses were performed using the paired t-test.Results On days 4, 7, and 10, the wounds treated with rose placenta were significantly smaller. On day 2, VEGF and EGF expression increased in the experimental group. On days 7 and 10, TGF-β1 expression decreased in the experimental group. On day 10, vessel density increased in the experimental group. The increase in EGF on day 2 was confirmed with ELISA.ConclusionsRose placenta was found to be associated with improved wound healing in a mouse full-thickness wound model via increased EGF release. Rose placenta may potentially be a novel drug candidate for enhancing wound healing.

Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

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Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

2016-02-01

A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

An mHealth App for Decision-Making Support in Wound Dressing Selection (WounDS): Protocol for a User-Centered Feasibility Study.

Science.gov (United States)

Jordan, Scott; McSwiggan, Jane; Parker, Joanne; Halas, Gayle A; Friesen, Marcia

2018-04-24

include the potential to improve healthcare providers' competency in wound management and to improve wound healing through better alignment with evidence-based best practices in wound dressing selection, consistency in care from primary to community care, and subsequent downstream impacts in quality of life for patients. ©Scott Jordan, Jane McSwiggan, Joanne Parker, Gayle A. Halas, Marcia Friesen. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 24.04.2018.

A guide to wound managment in palliative care.

Science.gov (United States)

Naylor, Wayne A

2005-11-01

Wound management in palliative patients is often a very challenging area of care. There are many unique issues that can combine to produce complicated wound management scenarios, including the types of wounds and wound symptoms most commonly affecting palliative care patients, as well as the presence of concurrent disease and associated treatment. Problems exist with the availability of suitable dressings and balancing life expectancy with the goals of wound care. A significant, and possibly under-recognized, issue is the emotional and social distress experienced by these patients, which can be directly attributed to their wound. These problems must all be recognized and addressed in order to manage wounds effectively in this patient population. This article aims to explore these issues and offer advice on the management of wound-related symptoms, with the ultimate goal of improving patients' quality of life.

Analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes

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Yoshinori Sugiyama

2014-01-01

Full Text Available Aquaporin 9 (AQP9 is a member of the aquaglyceroporin family that transports glycerol, urea and other small solutes as well as water. Compared to the expression and function in epidermal keratinocytes of AQP3, another aquaglyceroporin, our knowledge of epidermal AQP9 remains elusive. In this study, we investigated the expression of AQP9 in the human epidermis and cultured keratinocytes. Immunofluorescence studies revealed that AQP9 expression is highly restricted to the stratum granulosum of the human epidermis, where occludin is also expressed at the tight junctions. Interestingly, the AQP3 staining decreased sharply below the cell layers in which AQP9 is expressed. In cultured normal human epidermal keratinocytes (NHEK, knock-down of AQP9 expression in the differentiated cells induced by RNA interference reduced glycerol uptake, which was not as pronounced as was the case with AQP3 knock-down cells. In contrast, similar reduction of urea uptake was detected in AQP9 and AQP3 knock-down cells. These findings suggested that AQP9 expression in NHEK facilitates at least the transport of glycerol and urea. Finally, we analyzed the effect of retinoic acid (RA, a potent stimulator of keratinocyte proliferation, on AQP3 and AQP9 mRNA expression in differentiated NHEK. Stimulation with RA at 1 μM for 24 h augmented AQP3 expression and down-regulated AQP9 expression. Collectively, these results indicate that AQP9 expression in epidermal keratinocytes is regulated in a different manner from that of AQP3.

Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

International Nuclear Information System (INIS)

Verrando, P.; Ortonne, J.P.

1985-01-01

Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders

The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

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Li Qi-Jing

2002-06-01

Full Text Available Abstract Background During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF, is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules. Results Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration. Conclusions These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

Development of a tissue-engineered human oral mucosa equivalent based on an acellular allogeneic dermal matrix: a preliminary report of clinical application to burn wounds.

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Iida, Takuya; Takami, Yoshihiro; Yamaguchi, Ryo; Shimazaki, Shuji; Harii, Kiyonori

2005-01-01

Tissue-engineered skin equivalents composed of epidermal and dermal components have been widely investigated for coverage of full-thickness skin defects. We developed a tissue-engineered oral mucosa equivalent based on an acellular allogeneic dermal matrix and investigated its characteristics. We also tried and assessed its preliminary clinical application. Human oral mucosal keratinocytes were separated from a piece of oral mucosa and cultured in a chemically-defined medium. The keratinocytes were seeded on to the acellular allogeneic dermal matrix and cultured. Histologically, the mucosa equivalent had a well-stratified epithelial layer. Immunohistochemical study showed that it was similar to normal oral mucosa. We applied this equivalent in one case with an extensive burn wound. The equivalent was transplanted three weeks after the harvest of the patient's oral mucosa and about 30% of the graft finally survived. We conclude that this new oral mucosa equivalent could become a therapeutic option for the treatment of extensive burns.

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

International Nuclear Information System (INIS)

Tong, P.; Mayes, D.; Wheeler, L.

1986-01-01

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca ++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca ++ medium (0.09 mM) as measured by incorporation of [ 3 H] thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC 50 of about 10 μM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 μM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number of control at 10 μM. These results are of importance since they suggest Ca ++ may influence the effect of retinoids on keratinocytes

GRHL3 binding and enhancers rearrange as epidermal keratinocytes transition between functional states.

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Rachel Herndon Klein

2017-04-01

Full Text Available Transcription factor binding, chromatin modifications and large scale chromatin re-organization underlie progressive, irreversible cell lineage commitments and differentiation. We know little, however, about chromatin changes as cells enter transient, reversible states such as migration. Here we demonstrate that when human progenitor keratinocytes either differentiate or migrate they form complements of typical enhancers and super-enhancers that are unique for each state. Unique super-enhancers for each cellular state link to gene expression that confers functions associated with the respective cell state. These super-enhancers are also enriched for skin disease sequence variants. GRHL3, a transcription factor that promotes both differentiation and migration, binds preferentially to super-enhancers in differentiating keratinocytes, while during migration, it binds preferentially to promoters along with REST, repressing the expression of migration inhibitors. Key epidermal differentiation transcription factor genes, including GRHL3, are located within super-enhancers, and many of these transcription factors in turn bind to and regulate super-enhancers. Furthermore, GRHL3 represses the formation of a number of progenitor and non-keratinocyte super-enhancers in differentiating keratinocytes. Hence, chromatin relocates GRHL3 binding and enhancers to regulate both the irreversible commitment of progenitor keratinocytes to differentiation and their reversible transition to migration.

News in wound healing and management

DEFF Research Database (Denmark)

Gottrup, Finn; Jørgensen, Bo; Karlsmark, Tonny

2009-01-01

-TNFalpha) and Lactobacillus plantarum cultures have also been successfully used in hard to heal, atypical wounds. Knowledge on influencing factors as smoking and biofilm on the healing process has also been improved. Smoking results in delayed healing and increased risk of postoperative infection, whereas the role of biofilm...... is still at an exploratory level. Organizing models for optimal wound management are constantly being developed and refined. SUMMARY: Recent knowledge on the importance of new dressing materials containing active substances, new treatments for atypical wounds, influencing factors on the healing process...

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

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Santos, Jorge M; Camões, Sérgio P; Filipe, Elysse; Cipriano, Madalena; Barcia, Rita N; Filipe, Mariana; Teixeira, Mariana; Simões, Sandra; Gaspar, Manuela; Mosqueira, Diogo; Nascimento, Diana S; Pinto-do-Ó, Perpétua; Cruz, Pedro; Cruz, Helder; Castro, Matilde; Miranda, Joana P

2015-05-09

The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM

Hypoperfusion and Wound Healing: Another Dimension of Wound Assessment.

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Smollock, Wendy; Montenegro, Paul; Czenis, Amy; He, Yuan

2018-02-01

To examine the correlation between mean arterial pressure (MAP) and wound healing indices and describe an analytical process that can be used accurately and prospectively when evaluating all types of skin ulcerations. A correlational study in a long-term-care facility.Participants (N = 230) were adults residing in a long-term-care facility with an average age of 77.8 years (range, 35-105). Assessment through both an index of wound healing and wound surface area. Signs of wound healing included a reduction of surface area and surface necrosis and increased granulation or epithelialization. Aggregate analyses for all wound locations revealed a positive correlation between the MAP and index of wound healing (r = 0.86, n = 501, P wound healing was noted for all wound locations in this data set when MAP values were 80 mm Hg or less (r = 0.95, n = 141, P wounds and MAP of less than 80 mm Hg yielded a very strong positive correlation. The data indicated that as perfusion decreased, wounds within the sample population declined (r = 0.93, n = 102, P wound healing or worsened wounds. A predictability of wounds stalling or declining related to the MAP was observed, regardless of topical treatment or standard-of-care interventions. Therefore, the data also suggest that remediating states of low perfusion should take precedence in making treatment decisions.

Selenoproteins are essential for proper keratinocyte function and skin development.

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Aniruddha Sengupta

2010-08-01

Full Text Available Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec. Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14 expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

Wound care centers

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Pressure ulcer - wound care center; Decubitus ulcer - wound care center; Diabetic ulcer - wound care center; Surgical wound - wound ... Common types of non-healing wounds include: Pressure sores Surgical ... flow, or swollen legs Certain wounds may not heal well due to: ...

Formulation of Novel Layered Sodium Carboxymethylcellulose Film Wound Dressings with Ibuprofen for Alleviating Wound Pain

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Lenka Vinklárková

2015-01-01

Full Text Available Effective assessment and management of wound pain can facilitate both improvements in healing rates and overall quality of life. From a pharmacological perspective, topical application of nonsteroidal anti-inflammatory drugs in the form of film wound dressings may be a good choice. Thus, the aim of this work was to develop novel layered film wound dressings containing ibuprofen based on partially substituted fibrous sodium carboxymethylcellulose (nonwoven textile Hcel NaT. To this end, an innovative solvent casting method using a sequential coating technique has been applied. The concentration of ibuprofen which was incorporated as an acetone solution or as a suspension in a sodium carboxymethylcellulose dispersion was 0.5 mg/cm2 and 1.0 mg/cm2 of film. Results showed that developed films had adequate mechanical and swelling properties and an advantageous acidic surface pH for wound application. An in vitro drug release study implied that layered films retained the drug for a longer period of time and thus could minimize the frequency of changing the dressing. Films with suspended ibuprofen demonstrated higher drug content uniformity and superior in vitro drug release characteristics in comparison with ibuprofen incorporation as an acetone solution. Prepared films could be potential wound dressings for the effective treatment of wound pain in low exuding wounds.

Formulation of Novel Layered Sodium Carboxymethylcellulose Film Wound Dressings with Ibuprofen for Alleviating Wound Pain

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Vinklárková, Lenka; Vetchý, David; Bernatonienė, Jurga

2015-01-01

Effective assessment and management of wound pain can facilitate both improvements in healing rates and overall quality of life. From a pharmacological perspective, topical application of nonsteroidal anti-inflammatory drugs in the form of film wound dressings may be a good choice. Thus, the aim of this work was to develop novel layered film wound dressings containing ibuprofen based on partially substituted fibrous sodium carboxymethylcellulose (nonwoven textile Hcel NaT). To this end, an innovative solvent casting method using a sequential coating technique has been applied. The concentration of ibuprofen which was incorporated as an acetone solution or as a suspension in a sodium carboxymethylcellulose dispersion was 0.5 mg/cm2 and 1.0 mg/cm2 of film. Results showed that developed films had adequate mechanical and swelling properties and an advantageous acidic surface pH for wound application. An in vitro drug release study implied that layered films retained the drug for a longer period of time and thus could minimize the frequency of changing the dressing. Films with suspended ibuprofen demonstrated higher drug content uniformity and superior in vitro drug release characteristics in comparison with ibuprofen incorporation as an acetone solution. Prepared films could be potential wound dressings for the effective treatment of wound pain in low exuding wounds. PMID:26090454

Prolonged C1 Inhibitor Administration Improves Local Healing of Burn Wounds and Reduces Myocardial Inflammation in a Rat Burn Wound Model

NARCIS (Netherlands)

Begieneman, Mark P. V.; Kubat, Bela; Ulrich, Magda M. W.; Hahn, Nynke E.; Stumpf-Stolker, Yvette; Tempelaars, Miranda; Middelkoop, Esther; Zeerleder, Sacha; Wouters, Diana; van Ham, Marieke S.; Niessen, Hans W. M.; Krijnen, Paul A. J.

2012-01-01

In a previous study, the authors found persistent presence of acute inflammation markers such as C-reactive protein and complement factors locally in burn wounds. This persistence of acute inflammation may not only delay local burn wound healing but also have a systemic effect, for instance on the

Galectin-7 overexpression is associated with the apoptotic process in UVB-induced sunburn keratinocytes

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Bernerd, Francoise; Sarasin, Alain; Magnaldo, Thierry

1999-01-01

Galectin-7 is a β-galactoside binding protein specifically expressed in stratified epithelia and notably in epidermis, but barely detectable in epidermal tumors and absent from squamous carcinoma cell lines. Galectin-7 gene is an early transcriptional target of the tumor suppressor protein P53 [Polyak, K., Xia, Y., Zweier, J., Kinzler, K. & Vogelstein, B. (1997) Nature (London) 389, 300–305]. Because p53 transcriptional activity is increased by genotoxic stresses we have examined the possible effects of ultraviolet radiations (UVB) on galectin-7 expression in epidermal keratinocytes. The amounts of galectin-7 mRNA and protein are increased rapidly after UVB irradiation of epidermal keratinocytes. The increase of galectin-7 is parallel to P53 stabilization. UVB irradiation of skin reconstructed in vitro and of human skin ex vivo demonstrates that galectin-7 overexpression is associated with sunburn/apoptotic keratinocytes. Transfection of a galectin-7 expression vector results in a significant increase in terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive keratinocytes. The present findings demonstrate a keratinocyte-specific protein involved in the UV-induced apoptosis, an essential process in the maintenance of epidermal homeostasis. PMID:10500176

In vitro tests and ethnopharmacological investigations: wound healing as an example.

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Houghton, P J; Hylands, P J; Mensah, A Y; Hensel, A; Deters, A M

2005-08-22

In vitro tests are now widely employed in ethnopharmacological research because of ethical reasons and their usefulness in bioactive-guided fractionation and determination of active compounds. For many disease conditions, a variety of in vitro tests can now be employed as the biochemical mechanisms underlying disease and healing processes are understood. Approaches to the in vitro investigations of wound healing processes are exemplified by studies on extracts of Buddleja species and three Ghanaian species Spathodea campanulata, Commelina diffusa and Secamone afzelii. Most studies have been carried out on Buddleja officinalis or Buddleja globosa. The extracts have been shown to have anti-inflammatory and antioxidant properties due to flavonoids, triterpenoids, diterpenoids and caffeic acid derivatives. There appears to a slight effect on proliferation of fibroblasts at lower concentrations, but this was not significant, and higher concentrations appeared to be cytotoxic. Novel findings are the ability of Buddleja globosa leaf extracts to induce differentiation in keratinocytes and to alter the profile of proteins produced by cultured fibroblasts. Extracts also had some effect on lattice contraction. The three Ghanaian species examined show a mixture of antimicrobial and antioxidant activities. The evolution over recent years of tests for wound healing, from in vivo tests to cell-based systems and chemical reactions and on to investigations into effects on secondary messengers and protein expression, is described.

Topical Erythropoietin Treatment Accelerates the Healing of Cutaneous Burn Wounds in Diabetic Pigs Through an Aquaporin-3-Dependent Mechanism.

Science.gov (United States)

Hamed, Saher; Ullmann, Yehuda; Egozi, Dana; Keren, Aviad; Daod, Essam; Anis, Omer; Kabha, Hoda; Belokopytov, Mark; Ashkar, Manal; Shofti, Rona; Zaretsky, Asaph; Schlesinger, Michal; Teot, Luc; Liu, Paul Y

2017-08-01

We have previously reported that the topical application of erythropoietin (EPO) to cutaneous wounds in rats and mice with experimentally induced diabetes accelerates their healing by stimulating angiogenesis, reepithelialization, and collagen deposition, and by suppressing the inflammatory response and apoptosis. Aquaporins (AQPs) are integral membrane proteins whose function is to regulate intracellular fluid hemostasis by enabling the transport of water and glycerol. AQP3 is the AQP that is expressed in the skin where it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, we provide the results of an investigation that examined the contribution of AQP3 to the mechanism of EPO action on the healing of burn wounds in the skin of pigs with experimentally induced type 1 diabetes. We found that topical EPO treatment of the burns accelerated their healing through an AQP3-dependent mechanism that activates angiogenesis, triggers collagen and hyaluronic acid synthesis and the formation of the extracellular matrix (ECM), and stimulates reepithelialization by keratinocytes. We also found that incorporating fibronectin, a crucial constituent of the ECM, into the topical EPO-containing gel, can potentiate the accelerating action of EPO on the healing of the burn injury. © 2017 by the American Diabetes Association.

Ibuprofen loaded PLA nanofibrous scaffolds increase proliferation of human skin cells in vitro and promote healing of full thickness incision wounds in vivo.

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Mohiti-Asli, M; Saha, S; Murphy, S V; Gracz, H; Pourdeyhimi, B; Atala, A; Loboa, E G

2017-02-01

This article presents successful incorporation of ibuprofen in polylactic acid (PLA) nanofibers to create scaffolds for the treatment of both acute and chronic wounds. Nanofibrous PLA scaffolds containing 10, 20, or 30 wt % ibuprofen were created and ibuprofen release profiles quantified. In vitro cytotoxicity to human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF) of the three scaffolds with varying ibuprofen concentrations were evaluated and compared to pure PLA nanofibrous scaffolds. Thereafter, scaffolds loaded with ibuprofen at the concentration that promoted human skin cell viability and proliferation (20 wt %) were evaluated in vivo in nude mice using a full thickness skin incision model to determine the ability of these scaffolds to promote skin regeneration and/or assist with scarless healing. Both acellular and HEK and HDF cell-seeded 20 wt % ibuprofen loaded nanofibrous bandages reduced wound contraction compared with wounds treated with Tegaderm™ and sterile gauze. Newly regenerated skin on wounds treated with cell-seeded 20 wt % ibuprofen bandages exhibited significantly greater blood vessel formation relative to acellular ibuprofen bandages. We have found that degradable anti-inflammatory scaffolds containing 20 wt % ibuprofen promote human skin cell viability and proliferation in vitro, reduce wound contraction in vivo, and when seeded with skin cells, also enhance new blood vessel formation. The approaches and results reported here hold promise for multiple skin tissue engineering and wound healing applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 327-339, 2017. © 2015 Wiley Periodicals, Inc.

Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

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Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

2015-02-01

Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. © FASEB.

Differential Apoptosis in Mucosal and Dermal Wound Healing

Science.gov (United States)

Johnson, Ariel; Francis, Marybeth; DiPietro, Luisa Ann

2014-01-01

Objectives: Dermal and mucosal healing are mechanistically similar. However, scarring and closure rates are dramatically improved in mucosal healing, possibly due to differences in apoptosis. Apoptosis, nature's preprogrammed form of cell death, occurs via two major pathways, extrinsic and intrinsic, which intersect at caspase3 (Casp3) cleavage and activation. The purpose of this experiment was to identify the predominant pathways of apoptosis in mucosal and dermal wound healing. Approach: Wounds (1 mm biopsy punch) were made in the dorsal skin (n=3) or tongue (n=3) of female Balb/C mice aged 6 weeks. Wounds were harvested at 6 h, 24 h, day 3 (D3), D5, D7, and D10. RNA was isolated and analyzed using real time reverse transcriptase–polymerase chain reaction. Expression levels for genes in the intrinsic and extrinsic apoptotic pathways were compared in dermal and mucosal wounds. Results: Compared to mucosal healing, dermal wounds exhibited significantly higher expression of Casp3 (at D5; phealing compared to skin. Conclusion: Expression patterns of key regulators of apoptosis in wound healing indicate that apoptosis occurs predominantly through the intrinsic pathway in the healing mucosa, but predominantly through the extrinsic pathway in the healing skin. The identification of differences in the apoptotic pathways in skin and mucosal wounds may allow the development of therapeutics to improve skin healing. PMID:25493209

Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

Science.gov (United States)

Srivastava, Ankit; Ståhle, Mona; Pivarcsi, Andor; Sonkoly, Enikö

2018-05-08

Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

Application of Three - dimensional Wound Analyzer in the Small Wound Area Measurement during the Process of Wound Healing.

Science.gov (United States)

Sheng, Jiajun; Li, Haihang; Jin, Jian; Liu, Tong; Ma, Bing; Liu, Gongcheng; Zhu, Shihui

2018-02-20

The objective of this study was to determinate the reliability of 3-dimensional wound analyzer (3-DWMD) in the wound area measurement for animal small area in the process of wound healing. Seven Sprague-Dawley rats were used to establish the skin defect model. And the wound area and time consumption were measured on days 0, 5, 10, 15 using 3-DWMD, investigators, and planimetry method. The measurement results using 3-DWMD and investigators were analyzed comparative with that using planimetry method separately. A total 46 wounds, including 32 irregular wounds and regular 14 wounds, were measured. No matter calculating the irregular wounds or the regular wounds, there was no significant difference between 3-DWMD group and planimetry group in measuring wound area (P > 0.05). However, a statistically significant difference was found in time-consuming for measuring wound area between 3-DWMD group and planimetry group (P area, and its measurement results were consistent with planimetry method. Therefore, such measuring equipment has clinical reference value for measuring precision area of the wound in the process of wound healing.

Gene expression studies on human keratinocytes transduced with human growth hormone gene for a possible utilization in gene therapy

International Nuclear Information System (INIS)

Mathor, Monica Beatriz.

1994-01-01

Taking advantage of the recent progress in the DNA-recombinant techniques and of the potentiality of normal human keratinocytes primary culture to reconstitute the epidermis, it was decided to genetically transform these keratinocytes to produce human growth hormone under controllable conditions that would be used in gene therapy at this hormone deficient patients. The first step to achieve this goal was to standardize infection of keratinocytes with retrovirus producer cells containing a construct which included the gene of bacterial b-galactosidase. The best result was obtained cultivating the keratinocytes for 3 days in a 2:1 mixture of retrovirus producer cells and 3T3-J2 fibroblasts irradiated with 60 Gy, and splitting these infected keratinocytes on 3T3-J2 fibroblasts feeder layer. Another preliminary experiment was to infect normal human keratinocytes with interleukin-6 gene (hIL-6) that, in pathologic conditions, could be reproduced by keratinocytes and secreted to the blood stream. Thus, we verify that infected keratinocytes secrete an average amount of 500 ng/10 6 cell/day of cytokin during the in vitro life time, that certify the stable character of the injection. These keratinocytes, when grafted in mice, secrete hIL-6 to the blood stream reaching levels of 40 pg/ml of serum. After these preliminary experiments, we construct a retroviral vector with the human growth hormone gene (h GH) driven by human metallothionein promoter (h PMT), designated DChPMTGH. Normal human keratinocytes were infected with DChPMTGH producer cells, following previously standardized protocol, obtaining infected keratinocytes secreting to the culture media 340 ng h GH/10 6 cell/day without promoter activation. This is the highest level of h GH secreted in human keratinocytes primary culture described in literature. The h GH value increases approximately 10 times after activation with 100 μM Zn +2 for 8-12 hours. (author). 158 refs., 42 figs., 6 tabs

Death penalty for keratinocytes: apoptosis versus cornification.

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Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

2005-11-01

Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

The molecular biology in wound healing & non-healing wound.

Science.gov (United States)

Qing, Chun

2017-08-01

The development of molecular biology and other new biotechnologies helps us to recognize the wound healing and non-healing wound of skin in the past 30 years. This review mainly focuses on the molecular biology of many cytokines (including growth factors) and other molecular factors such as extracellular matrix (ECM) on wound healing. The molecular biology in cell movement such as epidermal cells in wound healing was also discussed. Moreover many common chronic wounds such as pressure ulcers, leg ulcers, diabetic foot wounds, venous stasis ulcers, etc. usually deteriorate into non-healing wounds. Therefore the molecular biology such as advanced glycation end products (AGEs) and other molecular factors in diabetes non-healing wounds were also reviewed. Copyright © 2017 Daping Hospital and the Research Institute of Surgery of the Third Military Medical University. Production and hosting by Elsevier B.V. All rights reserved.

VEGF, FGF-2 and TGFβ expression in the normal and regenerating epidermis of geckos: implications for epidermal homeostasis and wound healing in reptiles.

Science.gov (United States)

Subramaniam, Noeline; Petrik, James J; Vickaryous, Matthew K

2018-05-01

The skin is a bilayered organ that serves as a key barrier between an organism and its environment. In addition to protecting against microbial invasion, physical trauma and environmental damage, skin participates in maintaining homeostasis. Skin is also capable of spontaneous self-repair following injury. These functions are mediated by numerous pleiotrophic growth factors, including members of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor β (TGFβ) families. Although growth factor expression has been well documented in mammals, particularly during wound healing, for groups such as reptiles less is known. Here, we investigate the spatio-temporal pattern of expression of multiple growth factors in normal skin and following a full-thickness cutaneous injury in the representative lizard Eublepharis macularius, the leopard gecko. Unlike mammals, leopard geckos can heal cutaneous wounds without scarring. We demonstrate that before, during and after injury, keratinocytes of the epidermis express a diverse panel of growth factor ligands and receptors, including: VEGF, VEGFR1, VEGFR2, and phosphorylated VEGFR2; FGF-2 and FGFR1; and phosphorylated SMAD2, TGFβ1, and activin βA. Unexpectedly, only the tyrosine kinase receptors VEGFR1 and FGFR1 were dynamically expressed, and only during the earliest phases of re-epithelization; otherwise all the proteins of interest were constitutively present. We propose that the ubiquitous pattern of growth factor expression by keratinocytes is associated with various roles during tissue homeostasis, including protection against ultraviolet photodamage and coordinated body-wide skin shedding. © 2018 Anatomical Society.

Response of human epidermal keratinocytes to UV light

International Nuclear Information System (INIS)

Kartasova, A.A.

1987-01-01

This thesis presents a study on the response of human epidermal keratinocytes to UV light as well as to other agents like 4-NQO and TPA. The effects of ultraviolet (UV) light on the protein synthesis in cultured keratinocytes are presented in ch. III. The next chapter describes the construction of a cDNA library using mRNA isolated from UV irradiated kernatinocytes. This library was differentially screened with cDNA probes synthesized on mRNA from either UV irradiated or nonirradiated cells. Several groups of cDNA clones corresponding to transcripts whose level in the cytoplasm seem to be affected by exposure to UV light have been isolated and characterized by cross-hybridization, sequencing and Northern blot analysis. More detailed analysis of some of the cDNA clones is presented in the two chapters following ch. IV. The complete cDNA sequence of the proteinase inhibitor cystatin A and the modulation of its expression by UV light and the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in keratinocytes are described in ch. V. Two other groups of cDNA clones have been isolated which do not cross-hybridize with each other on Southern blots. However, the primary structures of the proteins deduced from the nucleotide sequences of these two groups of cDNA clones are very similar. 212 refs.; 33 figs.; 2 tabs

Delineating miRNA profile induced by chewing tobacco in oral keratinocytes

Directory of Open Access Journals (Sweden)

Mohd Younis Bhat

2017-10-01

Full Text Available The major established etiologic risk factor for oral cancer is tobacco (chewed, smoked and snuffed forms. Chewing form of tobacco is predominantly used in India making it the leading cause of oral cancer. Despite being one of the leading causes of oral cancer, the molecular alterations induced by chewing tobacco remains largely unclear. Carcinogenic effect of chewing tobacco is through chronic and not acute exposure. To understand the molecular alterations induced by chewing tobacco, we developed a cell line model where non-neoplastic oral keratinocytes were chronically exposed to chewing tobacco for a period of 6 months. This resulted in increased cellular proliferation and invasive ability of normal oral keratinocytes. Using this cellular model we studied the differential expression of miRNAs associated with chewing tobacco and the altered signaling pathways through which the aberrantly expressed miRNAs affect tumorigenesis. miRNA sequencing  was carried out using Illumina HiSeq 2500 platform  which resulted in the identification of 427 annotated miRNAs of which 10 were significantly dysregulated (≥ 4 fold; p-value ≤ 0.05 in tobacco exposed cells compared to untreated parental cells. To study the altered signaling in oral keratinocytes chronically exposed to chewing tobacco, we employed quantitative proteomics to characterize the dysregulated proteins. Integration of miRNA sequencing data with proteomic data resulted in identification of 36 proven protein targets which (≥1.5 fold; p-value ≤ 0.05 showed expression correlation with the 10 significantly dysregulated miRNAs. Pathway analysis of the dysregulated targets revealed enrichment of interferon signaling and mRNA processing related pathways in the chewing tobacco exposed cells. In addition, we also identified 6 novel miRNA in oral keratinocytes chronically exposed to chewing tobacco extract. Our study provides a framework to understand the oncogenic transformation induced by

Aging influences wound healing in patients with chronic lower extremity wounds treated in a specialized Wound Care Center.

Science.gov (United States)

Wicke, Corinna; Bachinger, Andreas; Coerper, Stephan; Beckert, Stefan; Witte, Maria B; Königsrainer, Alfred

2009-01-01

With the dramatic increase in the aging population, the study and care of wounds in the elderly have become priority topics for both researchers and clinicians. The effects of aging on wound healing in humans have remained controversial. The study was a 5-year epidemiological evaluation of standardized data collected regularly during patients' visits at a specialized Wound Care Center with the aim to determine the key factors influencing the healing of chronic lower extremity wounds. In this analysis of 1,158 chronic wounds, the frequency of wound closure was statistically significantly lower in older patients compared with younger patients. The share of closed wounds decreased by nearly 25% in the elderly patients (>or=70 years). The relationship between the patient's age and the proportion of wound closure was nonlinear. The effect of aging on the frequency of wound closure of chronic wounds became clinically apparent after age 60. The chronicity of the wounds was illustrated by their recurrent nature, their long duration, the presence of multiple wounds, and the frequency of concurrent infection. Comorbidity was documented by the coprevalence of up to three underlying diseases related to impaired wound healing. The present study clearly showed that aging affects chronic wound healing negatively.

Accessing wound-care information on the Internet: the implications for patients.

Science.gov (United States)

Bovill, E S; Hormbrey, E; Gillespie, P H; Banwell, P E

2001-02-01

The Internet and the World Wide Web have revolutionised communication and provide a unique forum for the exchange of information. It has been proposed that the Internet has given the public more access to medical information resources and improved patient education. This study assessed the impact of the Internet on the availability of information on wound care management. The search phrases 'wound care', 'wound healing' and 'wounds' were analysed using a powerful Metacrawler search engine (www.go2net.com). Web site access was classified according to the target audience (wound-care specialists, other health professionals, patients) and the author (societies, institutions or commercial companies). The largest proportion of web sites were commercially based (32%). Of the total number, 23% specifically targeted patients, mostly by advertising. Only 20% were aimed at wound specialists. Extensive surfing was required to obtain wound-care information, and objective information sites were under-represented. Regulated, easily accessible, objective information sites on wound-healing topics are needed for improved patient education and to balance the existing commercial bias.

miR-125b inhibits keratinocyte proliferation and promotes keratinocyte apoptosis in oral lichen planus by targeting MMP-2 expression through PI3K/Akt/mTOR pathway.

Science.gov (United States)

Wang, Jing; Luo, Hong; Xiao, Yan; Wang, Luyao

2016-05-01

Oral lichen planus (OLP) is a chronic inflammatory mucosal disease that involves the degeneration of keratinocytes. However, the etiology and mechanisms of OLP pathogenesis have not been fully elucidated. In this study, we used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to mimic a local OLP immune environment, and investigated the regulatory role of miR-125b in keratinocyte proliferation and apoptosis under OLP conditions. Immunohistochemical analysis and quantitative real-time PCR (qRT-PCR) assay showed that MMP-2 expression was up-regulated and miR-125b expression was down-regulated in both OLP mucosa tissues and LPS-incubated HaCaT cells. Western blot analysis indicated that miR-125b overexpression suppressed LPS-induced MMP-2 expression in HaCaT cells. Molecularly, our results confirmed that MMP-2 is a target gene of miR-125b in HaCaT cells. The effect of miR-125b on cell proliferation was revealed by CCK-8 assay, BrdU assay and cell cycle analysis, which illustrated that miR-125b overexpression impeded LPS-induced HaCaT cell proliferation. Flow cytometry analysis further demonstrated that miR-125b overexpression promoted HaCaT cell apoptosis. Moreover, these effects were involved in PI3K/Akt/mTOR activation, as miR-125b overexpression inhibited LPS-enhanced expression of p-Akt and p-mTOR proteins. Taken together, these data confirm that miR-125b might inhibit keratinocyte proliferation and promote keratinocyte apoptosis in OLP pathogenesis by targeting MMP-2 through PI3K/Akt/mTOR pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Changes in dermal matrix in the absence of Rac1 in keratinocytes

DEFF Research Database (Denmark)

Stanley, Alanna; Pedersen, Esben Ditlev Kølle; Brakebusch, Cord

2016-01-01

Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed....... As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction...... that this increase in the diameter of collagen fibrils due to inflammation may serve as pre-fibrotic marker enabling earlier determination of fibrosis and earlier treatment. This study has revealed previously unknown effects on the ECM due to the deletion of Rac1 in keratinocytes....

Effect of 1,24R-dihydroxyvitamin D3 on the growth of human keratinocytes.

LENUS (Irish Health Repository)

Matsumoto, K

1990-02-01

The effect of 1,24R-dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic analogue of a biologically active form of vitamin D3 (1,25-dihydroxyvitamin D3, 1,25(OH)2D3), on the growth of human keratinocytes cultured in serum-free medium was investigated. The growth of cultured normal human keratinocytes was inhibited by 65% by 10(-8)M 1,24R(OH)2D3 and by 90% by 10(-7)M 1,24(OH)2D3. It inhibited cell growth almost completely at 10(-6)M. The DNA synthesis of keratinocytes was also inhibited with 1,24R(OH)2D3 by 27% at 10(-8)M, 59% at 10(-7)M, and 92% at 10(-6)M. The inhibition of cell growth and DNA synthesis were more remarkable by 1,24R(OH)2D3 than by 1,25(OH)2D3. 1,24R(OH)2D3 also inhibited the growth of keratinocytes derived from patients with psoriasis vulgaris; the growth inhibitory effect was again more remarkable with 1,24R(OH)2D3 than with 1,25(OH)2D3. The viability and protein synthesis of keratinocytes were not affected by 1,24R(OH)2D3, suggesting that the growth inhibitory effect is due to its biological activity, not to cytotoxicity. The binding of [3H]-labeled 1,25(OH)2D3 to its receptor in the cytosolic fraction of cultured keratinocytes was competitively substituted by unlabeled 1,24R(OH)2D3 as well as 1,25(OH)2D3, suggesting that 1,24R(OH)2D3 binds to the 1,25(OH)2D3 receptor. It was found that the affinity of 1,24R(OH)2D3 for the receptor was slightly higher than that of 1,25(OH)2D3. These results demonstrate that 1,24R(OH)2D3 functions as a potent growth inhibitor in vitro in human keratinocytes from both normal and psoriatic epidermis, and it possesses a higher affinity for the 1,25(OH)2D3 receptor in cultured human keratinocytes. The difference in affinity of 1,24R(OH)2D3 for the 1,25(OH)2D3 receptor correlates with its greater inhibition of keratinocyte growth than 1,25(OH)2D3. 1,24R(OH)2D3 may be useful in the treatment of psoriasis.

Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide.

Science.gov (United States)

van Pelt, F N; Haring, R M; Overkamp, M J; Weterings, P J

1991-02-01

A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.

Combination of HIF-1α gene transfection and HIF-1-activated bone marrow-derived angiogenic cell infusion improves burn wound healing in aged mice.

Science.gov (United States)

Du, J; Liu, L; Lay, F; Wang, Q; Dou, C; Zhang, X; Hosseini, S M; Simon, A; Rees, D J; Ahmed, A K; Sebastian, R; Sarkar, K; Milner, S; Marti, G P; Semenza, G L; Harmon, J W

2013-11-01

Impaired burn wound healing in the elderly represents a major clinical problem. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In a murine burn wound model, aged mice had impaired wound healing associated with reduced levels of HIF-1. When gene therapy with HIF-1 alone did not correct these deficits, we explored the potential benefit of HIF-1 gene therapy combined with the intravenous infusion of bone marrow-derived angiogenic cells (BMDACs) cultured with dimethyloxalylglycine (DMOG). DMOG is known to reduce oxidative degradation of HIF-1. The mice treated with a plasmid DNA construct expressing a stabilized mutant form of HIF-1α (CA5-HIF-1α)+BMDACs had more rapid wound closure. By day 17, there were more mice with completely closed wounds in the treated group (χ(2), P=0.05). The dermal blood flow measured by laser Doppler showed significantly increased wound perfusion on day 11. Homing of BMDACs to the burn wound was dramatically enhanced by CA5-HIF-1α gene therapy. HIF-1α mRNA expression in the burn wound was increased after transfection with CA5-HIF-1α plasmid. Our findings offer insight into the pathophysiology of burns in the elderly and point to potential targets for developing new therapeutic strategies.

SDF-1 improves wound healing ability of glucocorticoid-treated adipose tissue-derived mesenchymal stem cells.

Science.gov (United States)

Kato, Toshiki; Khanh, Vuong Cat; Sato, Kazutoshi; Takeuchi, Kosuke; Carolina, Erica; Yamashita, Toshiharu; Sugaya, Hisashi; Yoshioka, Tomokazu; Mishima, Hajime; Ohneda, Osamu

2017-11-18

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

Directory of Open Access Journals (Sweden)

Mathilde Couteaudier

2015-03-01

Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

Flow cytometry of human primary epidermal and follicular keratinocytes.

Science.gov (United States)

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

Sustainability of keratinocyte gene transfer and cell survival in vivo.

Science.gov (United States)

Choate, K A; Khavari, P A

1997-05-20

The epidermis is an attractive site for therapeutic gene delivery because it is accessible and capable of delivering polypeptides to the systemic circulation. A number of difficulties, however, have emerged in attempts at cutaneous gene delivery, and central among these is an inability to sustain therapeutic gene production. We have examined two major potential contributing factors, viral vector stamina and involvement of long-lived epidermal progenitor cells. Human keratinocytes were either untreated or transduced with a retroviral vector for beta-galactosidase (beta-Gal) at > 99% efficiency and then grafted onto immunodeficient mice to regenerate human epidermis. Human epidermis was monitored in vivo after grafting for clinical and histologic appearance as well as for gene expression. Although integrated vector sequences persisted unchanged in engineered epidermis at 10 weeks post-grafting, retroviral long terminal repeat (LTR)-driven beta-Gal expression ceased in vivo after approximately 4 weeks. Endogenous cellular promoters, however, maintained consistently normal gene expression levels without evidence of time-dependent decline, as determined by immunostaining with species-specific antibodies for human involucrin, filaggrin, keratinocyte transglutaminase, keratin 10, type VII collagen, and Laminin 5 proteins out to week 14 post-grafting. Transduced human keratinocytes generated multilayer epidermis sustained through multiple epidermal turnover cycles; this epidermis demonstrated retention of a spatially appropriate pattern of basal and suprabasal epidermal marker gene expression. These results confirm previous findings suggesting that viral promoter-driven gene expression is not durable and demonstrate that keratinocytes passaged in vitro can regenerate and sustain normal epidermis for prolonged periods.

Mast Cells Regulate Wound Healing in Diabetes.

Science.gov (United States)

Tellechea, Ana; Leal, Ermelindo C; Kafanas, Antonios; Auster, Michael E; Kuchibhotla, Sarada; Ostrovsky, Yana; Tecilazich, Francesco; Baltzis, Dimitrios; Zheng, Yongjun; Carvalho, Eugénia; Zabolotny, Janice M; Weng, Zuyi; Petra, Anastasia; Patel, Arti; Panagiotidou, Smaro; Pradhan-Nabzdyk, Leena; Theoharides, Theoharis C; Veves, Aristidis

2016-07-01

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

[Aesthetic effect of wound repair with flaps].

Science.gov (United States)

Tan, Qian; Zhou, Hong-Reng; Wang, Shu-Qin; Zheng, Dong-Feng; Xu, Peng; Wu, Jie; Ge, Hua-Qiang; Lin, Yue; Yan, Xin

2012-08-01

To investigate the aesthetic effect of wound repair with flaps. One thousand nine hundred and ninety-six patients with 2082 wounds hospitalized from January 2004 to December 2011. These wounds included 503 deep burn wounds, 268 pressure sores, 392 soft tissue defects caused by trauma, 479 soft tissue defects due to resection of skin cancer and mole removal, 314 soft tissue defects caused by scar excision, and 126 other wounds. Wound area ranged from 1.5 cm x 1.0 cm to 30.0 cm x 22.0 cm. Sliding flaps, expanded flaps, pedicle flaps, and free flaps were used to repair the wounds in accordance with the principle and timing of wound repair with flaps. Five flaps showed venous congestion within 48 hours post-operation, 2 flaps of them improved after local massage. One flap survived after local heparin wet packing and venous bloodletting. One flap survived after emergency surgical embolectomy and bridging with saphenous vein graft. One flap showed partial necrosis and healed after skin grafting. The other flaps survived well. One thousand three hundred and twenty-one patients were followed up for 3 months to 2 years, and flaps of them were satisfactory in shape, color, and elasticity, similar to that of normal skin. Some patients underwent scar revision later with good results. Application of suitable flaps in wound repair will result in quick wound healing, good function recovery, and satisfactory aesthetic effect.

Three-dimensional wound measurements for monitoring wound healing

DEFF Research Database (Denmark)

Bisgaard Jørgensen, Line; Møller Jeppesen, Sune; Halekoh, Ulrich

Telemedicine is increasingly used for monitoring wound healing. Three-dimensional (3D) measurement methods enable clinicians to assess wound healing with respect to all dimensions. However, the currently available methods are inaccurate, costly or complicated to use. To address these issues, a 3D......-WAM camera was developed. This camera is able to measure wound size (2D area, 3D area, perimeter and volume) and to assess wound characteristics....

Randomized clinical trial of intestinal ostomy takedown comparing pursestring wound closure vs conventional closure to eliminate the risk of wound infection.

Science.gov (United States)

Camacho-Mauries, Daniel; Rodriguez-Díaz, José Luis; Salgado-Nesme, Noel; González, Quintín H; Vergara-Fernández, Omar

2013-02-01

The use of temporary stomas has been demonstrated to reduce septic complications, especially in high-risk anastomosis; therefore, it is necessary to reduce the number of complications secondary to ostomy takedowns, namely wound infection, anastomotic leaks, and intestinal obstruction. To compare the rates of superficial wound infection and patient satisfaction after pursestring closure of ostomy wound vs conventional linear closure. Patients undergoing colostomy or ileostomy closure between January 2010 and February 2011 were randomly assigned to linear closure (n = 30) or pursestring closure (n = 31) of their ostomy wound. Wound infection within 30 days of surgery was defined as the presence of purulent discharge, pain, erythema, warmth, or positive culture for bacteria. Patient satisfaction, healing time, difficulty managing the wound, and limitation of activities were analyzed with the Likert questionnaire. The infection rate for the control group was 36.6% (n = 11) vs 0% in the pursestring closure group (p ostomy wound closure (shorter healing time and improved patient satisfaction).

Mitochondria-targeted antioxidant SkQ1 improves impaired dermal wound healing in old mice.

Science.gov (United States)

Demyanenko, Ilya A; Popova, Ekaterina N; Zakharova, Vlada V; Ilyinskaya, Olga P; Vasilieva, Tamara V; Romashchenko, Valeria P; Fedorov, Artem V; Manskikh, Vasily N; Skulachev, Maxim V; Zinovkin, Roman A; Pletjushkina, Olga Yu; Skulachev, Vladimir P; Chernyak, Boris V

2015-07-01

The process of skin wound healing is delayed or impaired in aging animals. To investigate the possible role of mitochondrial reactive oxygen species (mtROS) in cutaneous wound healing of aged mice, we have applied the mitochondria-targeted antioxidant SkQ1. The SkQ1 treatment resulted in accelerated resolution of the inflammatory phase, formation of granulation tissue, vascularization and epithelization of the wounds. The wounds of SkQ1-treated mice contained increased amount of myofibroblasts which produce extracellular matrix proteins and growth factors mediating granulation tissue formation. This effect resembled SkQ1-induced differentiation of fibroblasts to myofibroblast, observed earlierin vitro. The Transforming Growth Factor beta (TGFb) produced by SkQ1-treated fibroblasts was found to stimulated motility of endothelial cells in vitro, an effect which may underlie pro-angiogenic action of SkQ1 in the wounds. In vitro experiments showed that SkQ1 prevented decomposition of VE-cadherin containing contacts and following increase in permeability of endothelial cells monolayer, induced by pro-inflammatory cytokine TNF. Prevention of excessive reaction of endothelium to the pro-inflammatory cytokine(s) might account for anti-inflammatory effect of SkQ1. Our findings point to an important role of mtROS in pathogenesis of age-related chronic wounds.

Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma