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1.
Int J Radiat Biol ; 89(11): 907-14, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23718176

ABSTRACT

PURPOSE: Oral mucositis is a severe and often dose-limiting side-effect of cancer therapy that occurs in patients receiving radiotherapy for head and neck cancers. Although radiation-induced effects on keratinocytes have been studied, little is known about its effect on fibroblasts or endothelial cells or, more importantly, when all these cells are combined in an engineered oral mucosal model. MATERIALS AND METHODS: Monolayer cultures of normal oral keratinocytes, normal oral fibroblasts, human dermal microvascular endothelial cells or tissue-engineered oral mucosa (TEOM) were exposed to 20 Gy irradiation. Cell damage and cytokine release was measured for 72 h for monolayer cultures and for up to 21 d for TEOM. RESULTS: Compared to non-irradiated cells, the viability of all monolayer and co-cultures was significantly reduced 72 h post-irradiation while levels of secreted interleukin IL-6 and CXCL8 were increased. The viability of irradiated TEOM models was significantly reduced compared to controls at all time-points. Histologically, irradiated TEOM displayed thinner epithelium, increased apoptosis and more extensive damage than non-irradiated models. IL-6, CXCL8 and granulocyte macrophage colony-stimulating factor release was reduced whereas IL-1α levels were increased in irradiated TEOM models compared to controls. CONCLUSIONS: TEOM models comprising of mixed cell populations may prove useful in examining the pathobiology of radiation-induced mucositis.


Subject(s)
Mouth Mucosa/radiation effects , Radiation Injuries/pathology , Radiotherapy/adverse effects , Stomatitis/etiology , Stomatitis/pathology , Tissue Engineering , Animals , Cell Proliferation/radiation effects , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelial Cells/radiation effects , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , Interleukin-6/metabolism , Interleukin-8/metabolism , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/radiation effects , Mouth Mucosa/pathology , Radiation Injuries/etiology
2.
Tissue Eng Part C Methods ; 18(2): 143-55, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21943098

ABSTRACT

Skin has a remarkable capacity for regeneration, but age- and diabetes-related vascular problems lead to chronic non-healing wounds for many thousands of U.K. patients. There is a need for new therapeutic approaches to treat these resistant wounds. Donor mesenchymal stem/stromal cells (MSCs) have been shown to assist cutaneous wound healing by accelerating re-epithelialization. The aim of this work was to devise a low risk and convenient delivery method for transferring these cells to wound beds. Plasma polymerization was used to functionalize the surface of medical-grade silicone with acrylic acid. Cells attached well to these carriers, and culture for up to 3 days on the carriers did not significantly affect their phenotype or ability to support vascular tubule formation. These carriers were then used to transfer MSCs onto human dermis. Cell transfer was confirmed using an MTT assay to assess viable cell numbers and enhanced green fluorescent protein-labeled MSCs to demonstrate that the cells post-transfer attached to the dermis. We conclude that this synthetic carrier membrane is a promising approach for delivery of therapeutic MSCs and opens the way for future studies to evaluate its impact on repairing difficult skin wounds.


Subject(s)
Acrylates/pharmacology , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Silicones/pharmacology , Skin/pathology , Wound Healing/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dermis/drug effects , Epithelium/drug effects , Green Fluorescent Proteins/metabolism , Humans , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic/drug effects , Phenotype , Photoelectron Spectroscopy , Skin/drug effects
3.
Tissue Eng Part C Methods ; 17(4): 375-82, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20973752

ABSTRACT

We have previously developed a cell delivery and transfer technology for delivering autologous keratinocytes and melanocytes to patients with vitiligo. However, for this technology to benefit many patients geographically distant from the cell culture facility transportation issues need to be overcome. In this study we begin to investigate this by looking at what role surface chemistry and medium supplements, including fetal calf serum, CO2 gassing, and temperature, play in influencing cell viability. Cells were maintained on carriers for up to 48 h outside of a CO2 incubator at 37 °C and their subsequent ability to adhere and become organized into a new epithelium with appropriately located melanocytes was assessed. Consistently good viability and performance on an in vitro wound bed model was achieved by maintaining cells for 48 h adherent to a 20% acrylic acid coated carrier at lower (around 23 °C rather than 37 °C) temperatures in the medium preperfused with CO2 before transport. Under these circumstances fetal calf serum was not required. In summary, the surface chemistry of the transport substrate and an appropriately CO2 buffered medium at near room temperature can extend the effective performance life of these cultured cells to at least 48 h from when they leave standard incubator conditions.


Subject(s)
Cell Transplantation/methods , Keratinocytes/cytology , Melanocytes/cytology , Vitiligo/therapy , Biological Transport/drug effects , Carbon Dioxide/pharmacology , Cell Count , Cell Survival/drug effects , Cells, Cultured , Coculture Techniques , Culture Media/pharmacology , Dermis/cytology , Dermis/drug effects , Humans , Keratinocytes/drug effects , Melanocytes/drug effects , Temperature
4.
Adv Exp Med Biol ; 681: 19-28, 2010.
Article in English | MEDLINE | ID: mdl-21222257

ABSTRACT

The melanocortin family are a series of very potent neuropeptides that derive from a parent propopiomelanocortin molecule (POMC). They are expressed predominantly in the brain, specifically the pituitary gland and also in the central nervous system. Interestingly, recent research also suggests the existence of regulatory functions outside of the brain, in a wide range of peripheral tissues. Several important melanocortin peptides with differing functions are created by the tissue-specific proteolytic cleavage of POMC, generating peptides including ACTH and α-MSH. For many years the major recognised function of α-MSH was an ability to stimulate melanocyte cells of the skin to pigment. However, a number of parallel functions unrelated to melanogenesis have been described in the literature for several years. A more complete understanding of this work arose after the discovery and cloning of the melanocortin receptors in 1992, which lead to the recognition of many wider roles of the melanocortin peptides. The knowledge of the tissue in which a given receptor subtype was expressed could now be combined with functional downstream studies. From these studies, we know that α-MSH has a very significant role in controlling inflammation and immunomodulation, with other roles including control over energy homeostasis and exocrine secretion, an ability to trigger erectile functions and the control of sexual behaviour. This chapter will briefly review the melanocortin system and melanocortin receptors, with a focus on the key signalling mechanisms of α-MSH and how these link receptors through to function.


Subject(s)
Melanocortins/metabolism , Signal Transduction , Animals , Humans , Inflammation/metabolism , Inflammation/pathology , Intracellular Space/drug effects , Intracellular Space/metabolism , Melanocortins/immunology , Pigmentation/drug effects , Receptors, Melanocortin/agonists , Receptors, Melanocortin/antagonists & inhibitors , Signal Transduction/drug effects
5.
J Invest Dermatol ; 128(6): 1554-64, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18200063

ABSTRACT

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.


Subject(s)
Keratinocytes/cytology , Melanocytes/cytology , Skin, Artificial , Vitiligo/pathology , Vitiligo/therapy , Bandages , Cell Adhesion , Coculture Techniques , Culture Techniques , Epidermis/metabolism , Humans , Keratinocytes/metabolism , Melanocytes/metabolism , Models, Biological , Polyvinyl Chloride/chemistry , Silicones/chemistry , Skin Transplantation , Tissue Engineering/methods
6.
Peptides ; 27(2): 444-52, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16274844

ABSTRACT

Alpha-melanocyte stimulating hormone (alpha-MSH) arises from the proteolytic cleavage of proopiomelanocortin (POMC) and is the most potent naturally occurring melanotropic peptide. The biological effects of alpha-MSH are mediated via melanocortin receptors (MCRs), which are expressed in virtually every cutaneous cell type. alpha-MSH has pleiotrophic functions including the modulation of a wide range of inflammatory stimuli such as proinflammatory cytokines, adhesion molecules and inflammatory transcription factors. All of the former would be consistent with a cytoprotective role for this hormone in protecting skin cells from exogenous stress, as would occur following UV exposure or exposure to agents inducing inflammation or oxidative stress. In addition to actions on normal skin cells it also modulates both cutaneous and uveal melanoma cell behavior. With respect to melanoma, alpha-MSH is intriguing as studies have shown that while alpha-MSH has the potential to retard metastatic spread (by reducing cell migration and invasion) it is also capable of reducing the ability of the immune system to detect tumor cells (by down regulating adhesion molecules that would normally assist in immune cell interaction with melanoma cells). This review considers the evolving biology of alpha-MSH and discusses its role in man that extend far beyond pigmentation of skin melanocytes, suggesting that the detoxifying role of alpha-MSH in inducing melanogenesis is only one aspect of the stress-coping role of this hormone. Indeed melanoma cells may owe at least some of their success to the 'protective' role of alpha-MSH.


Subject(s)
Inflammation , Melanoma/immunology , Skin Neoplasms/immunology , Uveal Neoplasms/immunology , alpha-MSH/physiology , Cell Line, Tumor , Humans , Melanoma/etiology , Models, Biological , Receptors, Melanocortin/metabolism , Skin/immunology , Skin/pathology , Skin Neoplasms/etiology , Uveal Neoplasms/etiology
7.
J Invest Dermatol ; 121(3): 557-63, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12925215

ABSTRACT

Iris melanomas are less likely to metastasize than posterior compartment melanomas. The anterior chamber of the eye is an immunosuppressed microenvironment where a wide range of immunosuppressive factors in aqueous humor contribute to the immune privilege. One such factor is alpha-melanocyte-stimulating hormone, a potent anti-inflammatory neuropeptide that exhibits efficacy in many studies of acute and chronic inflammation. The aim of this study was to investigate whether the different metastatic behavior of iris melanomas versus posterior compartment melanomas might be explained by the differing immunosuppressive/anti-inflammatory environments of these tumors in vivo. To investigate this hypothesis, we studied the effect of human aqueous and vitreous fluids, of the proinflammatory cytokine tumor necrosis factor alpha, and of the anti-inflammatory peptides alpha-melanocyte-stimulating hormone and melanocyte-stimulating hormone 11-13 (KP-D-V) on the invasion of three human uveal melanoma cell lines through human fibronectin. Fresh aqueous humor samples significantly decreased the invasion in two out of three uveal melanoma cell lines. In contrast, vitreous humor did not reduce invasion. Tumor necrosis factor alpha significantly increased the invasiveness of uveal melanoma cell lines by approximately 50%-80% over 20 h. Full-length alpha-melanocyte-stimulating hormone, at concentrations present in the aqueous humor (10-9 M), as well as melanocyte-stimulating hormone 11-13 (KP-D-V) reduced the invasion of cells through human fibronectin by 45%-50% and also protected uveal melanoma cells from the pro-invasive actions of tumor necrosis factor alpha. These data are consistent with inflammation playing a major role in affecting the metastatic ability of uveal melanomas. Thus, ocular microenvironments that differ in their immunosuppressive/anti-inflammatory properties may influence the invasiveness of developing tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Fibronectins/metabolism , Melanoma , Tumor Necrosis Factor-alpha/pharmacology , Uveal Neoplasms , alpha-MSH/pharmacology , Aqueous Humor , Humans , Iris Neoplasms , Melanocyte-Stimulating Hormones/pharmacology , Neoplasm Invasiveness , Peptide Fragments/pharmacology , Receptors, Corticotropin/metabolism , Receptors, Melanocortin , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Vitreous Body
8.
J Invest Dermatol ; 119(5): 1165-71, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12445207

ABSTRACT

We have previously shown alpha-melanocyte stimulating hormone to protect melanocytes and melanoma cells from the proinflammatory actions of tumor necrosis factor-alpha. The aim of the study was to extend this work to look into the influence of tumor necrosis factor-alpha on melanoma cell attachment, invasion, and integrin expression and ask to what extent alpha-melanocyte stimulating hormone might protect cells from tumor necrosis factor-alpha stimulation of increased integrin expression. HBL human melanoma cells were studied under resting and stressed conditions using tumor necrosis factor-alpha as a proinflammatory cytokine. Functional information on the actions of tumor necrosis factor-alpha on melanoma cells was obtained by examining the strength of attachment of melanoma cells to substrates and the ability of melanoma cells to invade through fibronectin. alpha3, alpha4, and beta1 integrin expression was detected by Western immunoblotting and the ability of alpha-melanocyte stimulating hormone to oppose the actions of tumor necrosis factor-alpha was studied on HBL cell attachment, invasion, and integrin subunit expression. Our results show that tumor necrosis factor-alpha increases the number of melanoma cells attaching to collagen (types I and IV) and tissue culture polystyrene, increases ability to invade through fibronectin, and upregulates the expression of alpha3 (28%), alpha4 (90%), and beta1 (65%) integrin subunit expression. In contrast, alpha-melanocyte stimulating hormone reduced cell attachment, invasion, and integrin expression and opposed the stimulatory effects of tumor necrosis factor-alpha. In conclusion this study provides further evidence of alpha-melanocyte stimulating hormone acting to "protect" melanoma cells from proinflammatory cytokine action. Our data support a hypothesis that an inflammatory environment would promote melanoma invasion and that the anti-invasive actions of alpha-melanocyte stimulating hormone are consistent with its working in an anti-inflammatory capacity.


Subject(s)
Antineoplastic Agents/pharmacology , Integrins/biosynthesis , Melanoma , Skin Neoplasms , Tumor Necrosis Factor-alpha/pharmacology , alpha-MSH/pharmacology , Cell Adhesion/drug effects , Fibronectins/metabolism , Humans , Hydrogen Peroxide/pharmacology , Integrin alpha3/biosynthesis , Integrin alpha4/biosynthesis , Integrin beta1/biosynthesis , Neoplasm Invasiveness , Oxidants/pharmacology , Tumor Cells, Cultured , Up-Regulation/drug effects
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