Fibroblasts Play a Regulatory Role in the Control of Pigmentation in Reconstructed Human Skin from Skin Types I and II

Pigment Cell Research / Sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society. Feb, 2002  |  Pubmed ID: 11837456

Human melanocytes in monolayer culture are extremely dependent on a wide range of soluble signals for their proliferation and melanogenesis. The advent of three-dimensional models of reconstructed skin allows one to ask questions of how these cells are regulated within a setting which more closely approximates normal skin. The purpose of this study was to investigate to what extent melanocytes within a reconstructed skin model are sensitive to regulation by dermal fibroblasts, basement membrane (BM) proteins and the addition of alpha-melanocyte-stimulating hormone (alpha-MSH). Sterilized acellular de-epidermized dermis (prepared to retain BM proteins or deliberately denuded of BM by enzymatic treatment) from skin type I or II was reconstituted with fibroblasts, melanocytes and keratinocytes. In all but one case (9/10), cell donors were skin type I or II. The presence of BM antigens was found to be necessary for positional orientation of the melanocytes; in the absence of BM, melanocytes moved into the upper keratinocyte layer pigmenting spontaneously. Addition of fibroblasts suppressed the extent of spontaneous pigmentation of melanocytes within this model. Neither alpha-MSH nor cholera toxin induced pigmentation in this model despite the fact that melanocytes clearly had the ability to synthesize pigment.

Melanoma Cell Attachment, Invasion, and Integrin Expression is Upregulated by Tumor Necrosis Factor Alpha and Suppressed by Alpha Melanocyte Stimulating Hormone

The Journal of Investigative Dermatology. Nov, 2002  |  Pubmed ID: 12445207

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.

Inhibition of Tumor Necrosis Factor-alpha Stimulated NFkappaB/p65 in Human Keratinocytes by Alpha-melanocyte Stimulating Hormone and Adrenocorticotropic Hormone Peptides

The Journal of Investigative Dermatology. Dec, 2002  |  Pubmed ID: 12485424

Alpha-melanocyte stimulating hormone (alpha-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-alpha in a number of tissues, including skin. We have previously shown that alpha-MSH can inhibit tumor necrosis factor-alpha stimulated intercellular adhesion molecule 1 upregulation and nuclear factor kappaB (NFkappaB) transcription factor activation in melanocyte and melanoma cells. It is thought, however, that this MSH biology may also extend to other cells of the skin and in this study we extend our work to keratinocytes. We have investigated in detail the ability of three alpha-MSH peptides to inhibit tumor necrosis factor alpha stimulated NFkappaB activation in nonpigmentary HaCaT keratinocytes (alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val) and two adrenocorticotropic hormone (ACTH) peptides (1-17 and 1-39), reported to be present in skin tissue. NFkappaB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor alpha stimulated NFkappaB activation, whereas ACTH 1-17 and 1-39 did not, in the HaCaT keratinocytes. MSH peptides and ACTH 1-39 were effective, however, at inhibiting NFkappaB activation in normal human keratinocytes. Immunolabeling of inhibitor kappaBalpha of NFkappaB (IkappaBalpha) revealed an abnormal localization to the nucleus of HaCaT cells, which was unaffected by MSH/ACTH peptides. In contrast, normal human keratinocytes showed a normal IkappaBalpha distribution that responded to MSH/ACTH with nuclear translocation. Our data support previous work on the role of MSH/ACTH peptides as immunomodulatory/anti-inflammatory regulators, and extend this work to keratinocytes identifying a novel IkappaBalpha mechanism and extends findings to ACTH peptides, identifying an abnormal IkappaBalpha mechanism in the immortal HaCaT versus normal keratinocyte.

Tumor Necrosis Factor Alpha Increases and Alpha-melanocyte-stimulating Hormone Reduces Uveal Melanoma Invasion Through Fibronectin

The Journal of Investigative Dermatology. Sep, 2003  |  Pubmed ID: 12925215

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.

Development of a Plasma-polymerized Surface Suitable for the Transplantation of Keratinocyte-melanocyte Cocultures for Patients with Vitiligo

Tissue Engineering. Dec, 2003  |  Pubmed ID: 14670100

The purpose of this study was to develop a convenient methodology for the coculture of autologous melanocytes and keratinocytes for grafting of patients with vitiligo. While grafting of pure melanocytes may achieve repigmentation, the inclusion of keratinocytes ensures rapid reepithelialization. Previously we have used confluent sheets of keratinocytes (with melanocytes present) to transfer cells. However, we found that as the keratinocyte density increased, melanocyte number and function were downregulated. Accordingly in this study we explored combinations of three culture surfaces and three media, seeking to achieve subconfluent culture of primary keratinocytes with a reasonable density of melanocytes, using cells immediately after isolation from skin. For this in vitro study, the surfaces studied were uncoated glass coverslips, and glass coverslips coated with collagen I or a nitrogen-containing plasma polymer. The results show that both the substrate surface and the medium composition influence the proliferation and survival of melanocytes. Keratinocytes and melanocytes could be successfully cocultured on a chemically defined plasma polymer substrate using a serum-free medium.

Alpha-Melanocyte-stimulating Hormone, MSH 11-13 KPV and Adrenocorticotropic Hormone Signalling in Human Keratinocyte Cells

The Journal of Investigative Dermatology. Apr, 2004  |  Pubmed ID: 15102092

alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

Use of Peracetic Acid to Sterilize Human Donor Skin for Production of Acellular Dermal Matrices for Clinical Use

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. May-Jun, 2004  |  Pubmed ID: 15225206

We previously reported methods for sterilizing human skin for clinical use. In a comparison of gamma-irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity. The aim of this study was to evaluate peracetic acid as an alternative sterilizing agent. Skin sterilized with peracetic acid was compared with skin sterilized using glycerol alone or glycerol with ethylene oxide. The effect of subsequently storing peracetic acid sterilized skin in glycerol or propylene glycol was also examined. Acellular dermal matrices were produced after removal of the epidermis and cells in the dermis, processed for histological and ultrastructural analysis, and the biological function was evaluated by reconstitution with keratinocytes and fibroblasts. Results showed that sterilized acellular matrices retained the integrity of dermal structure and major components of the basement membrane. There were no overall significant differences in the ability of these matrices to form reconstructed skin, but peracetic acid alone gave a lower histologic score than when combined with glycerol or propylene glycol. We conclude that peracetic acid sterilization followed by preservation in glycerol or propylene glycol offers a convenient alternative protocol for processing of human skin. It is suggested that this sterile acellular dermis may be suitable for clinical use.

Developments in Xenobiotic-free Culture of Human Keratinocytes for Clinical Use

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2004  |  Pubmed ID: 15555054

We have recently reported that irradiated human fibroblasts can be used as a feeder layer, to expand keratinocytes under serum-free conditions, on a chemically defined plasma polymer surface developed for the culture and transfer of keratinocytes for clinical use. While this is a significant advance in developing a serum-free keratinocyte culture approach, the need to irradiate fibroblasts to growth-arrest them and prevent them from overgrowing the keratinocytes introduces another small, but potential, risk for the patient. The aim of the present study was to develop conditions for the coculture of normal human keratinocytes with nonirradiated normal human fibroblasts under serum-free conditions. We examined the fibroblast/keratinocyte relationship on three separate surfaces: tissue culture plastic, non-tissue culture plastic, and a plasma polymer surface designed for clinical use. We report that it is possible to achieve rapid and successful expansion of human keratinocytes under serum-free conditions on all three surfaces providing one uses a keratinocyte-friendly media, a minimum seeding density of keratinocytes, and a ratio of fibroblasts to keratinocytes that does not exceed 1:1. These results provide us with a rapid laboratory expansion of proliferative human keratinocytes, under completely defined culture conditions, without any xenobiotic cells (mouse fibroblasts) or material (bovine serum), for the treatment of patients with extensive skin loss.

Approaches to Improve Angiogenesis in Tissue-engineered Skin

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2004  |  Pubmed ID: 15555055

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

Dopa Oxidase Activity in the Hair, Skin and Ocular Melanocytes is Increased in the Presence of Stressed Fibroblasts

Experimental Dermatology. May, 2005  |  Pubmed ID: 15854130

We previously reported that mesenchymal cells (dermal fibroblasts and dermal papilla cells) can stimulate dopa oxidase activity in the skin melanocytes. This study extends the investigation of the influence of the fibroblast in a comparative study of melanogenesis in melanocytes from the hair, the skin and the eye. Culture of melanocytes with normal proliferative dermal fibroblasts slightly increased dopa oxidase activity of the hair, skin and ocular melanocytes (by 17, 11 and 28%, respectively), but co-culture with fibroblasts recovering from storage in liquid nitrogen or growth-arrested by means of gamma radiation showed much greater effects. Most dramatic results were obtained with fibroblasts, which had been both gamma-irradiated and then frozen in liquid nitrogen, where increases in dopa oxidase activity of 125, 227 and 185% for melanocytes of the hair, the skin and the eye, respectively, were seen. Experiments by using transwell cultures of melanocytes and fibroblasts and by using fibroblast-conditioned medium showed that a large proportion of this fibroblast influence could be mediated by diffusible factors, of which a good proportion was attributable to basic Fibroblast Growth Factor (bFGF). The addition of bFGF significantly increased dopa oxidase activity of the skin melanocytes, when fibroblasts were present, but not in their absence. These data show that fibroblasts in vitro, particularly when deliberately stressed, have the ability to increase dopa oxidase activity in melanocytes of the hair, the skin and the eye and further suggest that this effect is mediated by bFGF acting in combination with some other fibroblast-derived factors.

Alpha-melanocyte Stimulating Hormone Cytoprotective Biology in Human Dermal Fibroblast Cells

Peptides. Jul, 2005  |  Pubmed ID: 15949633

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory peptide effective in various tissues including skin. It acts by inhibiting the production and action of several pro-inflammatory stimuli including TNF-alpha, IL-1beta and LPS in a number of cell types. The role of such stimuli in inducing cellular apoptosis is also well described; however the precise role of alpha-MSH in apoptosis is presently unclear, with studies reporting both anti- and pro-apoptotic activity. The present study demonstrates that cultured human dermal fibroblasts respond to serum depletion and TNF-alpha, IL-1beta and LPS with an increase in membrane permeability, a decrease in viability and an increase in phosphatidylserine externalization (indicative of apoptosis) over 48-96 h. alpha-MSH (at 10(-6) M, but not 10(-9) M) was found to inhibit the serum free and pro-inflammatory mediated reduction in membrane permeability and cellular viability and also inhibited increases in apoptosis. In conclusion, data support a cytoprotective and anti-apoptotic role of the alpha-MSH peptide in human dermal fibroblast cells.

Self-organization of Skin Cells in Three-dimensional Electrospun Polystyrene Scaffolds

Tissue Engineering. Jul-Aug, 2005  |  Pubmed ID: 16144438

Much research in tissue engineering focuses on the synthesis of complex three-dimensional polymer scaffolds containing functional biomolecules to which cells are introduced. Typical scaffolds for skin tissue engineering are macroscopically porous with struts or fibers approximately 10 microm thick at a packing fraction of approximately 0.1. We made a polystyrene scaffold without cell signaling or spatial information by electrospinning and studied the growth of skin fibroblasts, keratinocytes, and endothelial cells, as single and cocultured populations in the presence and absence of fetal calf serum. In the absence of serum, keratinocytes, fibroblasts, and endothelial cells did not grow when cultured alone. However, when fibroblasts were cocultured with keratinocytes and endothelial cells, expansion of keratinocytes and endothelial cells occurred even in the absence of serum. Furthermore, cells displayed native spatial three-dimensional organization when cultured at an air-liquid interface, even when all three cell types were introduced at random to the scaffold. This study shows that coculture with fibroblasts enables keratinocytes and endothelial cells to proliferate without serum, but also to self-organize according to the native epidermal-dermal structure given the symmetry-breaking field of an air-liquid interface.

Development of a Closed Bioreactor System for Culture of Tissue-engineered Skin at an Air-liquid Interface

Tissue Engineering. Nov-Dec, 2005  |  Pubmed ID: 16411828

A bioreactor has been developed for the production of tissue-engineered skin at an air-liquid interface for clinical and experimental use. In this closed system, scaffold and bioreactor sterilization, cell seeding, and medium perfusion were all performed with a peristaltic pump. Natural and synthetic dermal substitutes were seeded directly with skin cells without opening the bioreactor and fed either by continuous medium perfusion or by batch-feed. The system was validated by monoculture of human dermal fibroblasts and keratinocytes and the coculture of both cell types in acellular human dermis, Azowipes, electrospun polystyrene, and an electrospun composite of polystyrene and poly-DL-lactide fibers. A comparison was made of culture at an air-liquid interface versus submerged culture and of medium change by continuous perfusion versus batch-feed. Fibroblast and endothelial cells showed greater viability under submerged rather than air-liquid conditions whereas keratinocytes favored culture at an air-liquid interface as did cocultured keratinocytes and fibroblasts. Total cellular viability for reconstructed skin with keratinocytes and fibroblasts was greatest with continuous perfusion rather than batch-feed and with electrospun scaffolds compared with acellular human dermis. The bioreactor could also be easily configured to give replicate small areas for experimental use or one continuous area of construct for clinical use.

Examination of the Effects of Poly(N-vinylpyrrolidinone) Hydrogels in Direct and Indirect Contact with Cells

Biomaterials. May, 2006  |  Pubmed ID: 16426677

Poly(N-vinylpyrrolidinone) (PNVP) has been used in various biomedical applications for many years. This study explores two PNVP hydrogels for their biocompatibility with skin cells and their ability to support the growth of skin cells in direct and indirect contact with the cells. Two crosslinked PNVP's were investigated, one crosslinked with ethylene glycol dimethacrylate (EGDMA) and the other crosslinked with diethylene glycol bisallylcarbonate (DEGBAC). The different crosslinkers lead to hydrogels with different mechanical and slightly different biological properties. While neither hydrogel proved to be a suitable substrate for culturing cells (based on fibroblasts and a range of other cells), indirect contact with both showed them to be biocompatible and even stimulatory to fibroblasts. The P(NVP-co-DEGBAC) hydrogel stimulated fibroblast viability more reliably than the P(NVP-co-EGDMA) hydrogel when in indirect contact with cells. This effect was shown to be independent of the presence of foetal calf serum in the culture media, and could not be explained by any hydrogel breakdown products during the course of these experiments. Rather the phenomenon was observed to be the result of a dynamic interaction between the hydrogels and the cells.

Sodium Salicylate Inhibits TNF-alpha-induced NF-kappaB Activation, Cell Migration, Invasion and ICAM-1 Expression in Human Melanoma Cells

Melanoma Research. Feb, 2006  |  Pubmed ID: 16432451

We have previously shown that tumour necrosis factor-alpha (TNF-alpha) upregulates human melanoma cell integrin expression, migration and invasion in vitro. The aim of this study was to investigate the effect of the non-steroidal anti-inflammatory agent sodium salicylate on TNF-alpha-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and upregulation of intercellular adhesion molecule-1 (ICAM-1), and TNF-alpha-stimulated cell migration and invasion through fibronectin. HBL human melanoma cells were pre-incubated with sodium salicylate prior to stimulation with TNF-alpha for 24 h. NF-kappaB activation was measured using an assay that detects changes in the expression of a luciferase reporter gene under the direct control of NF-kappaB transcriptional activity. The effect of sodium salicylate and TNF-alpha on HBL cell invasion over 20 h and migration over 24 h was studied using fibronectin invasion and 'scratch wound' migration models in vitro, as described previously. Sodium salicylate inhibited TNF-alpha-stimulated NF-kappaB activation in melanoma cells in a concentration-dependent manner, and this was achieved with pre-incubation times as short as 15 min. TNF-alpha-stimulated ICAM-1 expression in HBL cells was also downregulated by sodium salicylate, although in a manner inversely related to the concentration of this agent. In functional assays, TNF-alpha stimulated migration and invasion, and sodium salicylate significantly reduced the extent of melanoma invasion and migration in both the presence and absence of TNF-alpha. In conclusion, sodium salicylate effectively inhibited TNF-alpha-induced upregulation of NF-kappaB, ICAM-1 expression, in-vitro migration and invasion in human melanoma cells, indicating that non-steroidal anti-inflammatory drugs may be a useful therapeutic approach to oppose inflammation-induced melanoma invasion and metastasis in vivo.

Culture of Skin Cells in 3D Rather Than 2D Improves Their Ability to Survive Exposure to Cytotoxic Agents

Journal of Biotechnology. Apr, 2006  |  Pubmed ID: 16446003

In this study, we asked the question of whether cells in 3D culture cope more effectively with cytotoxic agents than cells in 2D. The sensitivities of human skin cells (keratinocytes, dermal fibroblasts and endothelial cells) to oxidative stress (hydrogen peroxide) and to a potentially toxic heavy metal (silver) when cultured under 2D and 3D conditions were investigated. The results show a marked resistance of cells to a given dose of hydrogen peroxide or silver nitrate causing a 50% loss of viability in 3D cultures, when compared to the same cells grown in 2D. There was also an improvement in the ability of cells to withstand both stresses when cells were in co-culture rather than in mono-culture. Foetal calf serum was found to have a mild protective effect in 2D culture but this was not extended to findings in 3D culture. This study suggests that dermatotoxicity testing using 3D co-cultures might be more likely to reflect true physiological responses to xenobiotic materials than existing models that rely on 2D mono-cultures.

In Situ Image Analysis of Interactions Between Normal Human Keratinocytes and Fibroblasts Cultured in Three-dimensional Fibrin Gels

Biomaterials. Jun, 2006  |  Pubmed ID: 16510181

The non-invasive investigation of different cells to interact and become spatially organised in a three-dimensional (3D) environment or scaffold is an important challenge in tissue engineering and tissue physiology. The aim of the present study was to develop 3D cell culture systems using fibrin gels, which would allow for the single and co-culture of different cell types with in situ image analysis. Two chambers were constructed for mono-culture and co-culture of human dermal fibroblasts and keratinocytes. During cell culture, in situ imaging and morphological characterisation of cells was assessed using brightfield light and/or fluorescence microscopy, and later confirmed by staining of fixed cells using immunofluorescence microscopy. The results showed that it was possible to investigate fibroblast and keratinocyte interactions in a fibrin scaffold for at least 12 days. Using this model system it was found that when a co-culture of fibroblasts and keratinocytes were plated on top of the fibrin gels, fibroblasts were seen to migrate into the gels within 2-3 days in contrast to keratinocytes, which did not enter. However, keratinocytes were found to retard fibroblast migration into gels when compared to fibroblasts cultured on their own, illustrating the dependency of intracellular communication on cell position for reconstructive approaches.

Use of Human Fibroblasts in the Development of a Xenobiotic-free Culture and Delivery System for Human Keratinocytes

Tissue Engineering. Feb, 2006  |  Pubmed ID: 16548683

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.

Development of a Three-dimensional Organ Culture Model for Corneal Wound Healing and Corneal Transplantation

Investigative Ophthalmology & Visual Science. Jul, 2006  |  Pubmed ID: 16799023

To develop and evaluate a three-dimensional organ culture system of the cornea anterior chamber that could replicate the in vivo processes occurring during corneal wound healing and corneal transplantation.

Modeling the Effect of Exogenous Calcium on Keratinocyte and HaCat Cell Proliferation and Differentiation Using an Agent-based Computational Paradigm

Tissue Engineering. Aug, 2006  |  Pubmed ID: 16968170

In this study we sought to develop a computational modeling paradigm in order to describe the influence of calcium on normal and transformed keratinocyte proliferation and differentiation. Keratinocytes and HaCat cells were grown in monolayer cultures with low and physiologic calcium concentrations, and levels of proliferation and involucrin expression were assessed. Both types of cells grew as monolayers under a low-calcium environment, and stratified in media with physiologic levels of calcium. However, keratinocytes were more proliferative in low rather than physiologic levels of calcium, whereas the opposite was true for HaCat cells. Normal keratinocytes differentiated as calcium levels increased. HaCat cells showed little differentiation at any calcium concentration. However, while the computer simulation could be modified to describe the effect of calcium on the growth of normal keratinocytes, our findings did not support the hypothesis that simply "turning off" the ability of HaCat cells to differentiate would account for the growth characteristics of these transformed cells. This demonstrates the application of computational modeling to hypothesis testing in biological systems.

Further Validation of the Interpersonal Exchange Model of Sexual Satisfaction

Journal of Sex & Marital Therapy. Jan-Feb, 2006  |  Pubmed ID: 16234226

We conducted two studies to further test the validity of the Interpersonal Exchange Model of Sexual Satisfaction for long-term opposite-sex relationships (IEMSS; Lawrance & Byers, 1995). Study 1 examined, in a sample of 79 individuals, the extent to which the history of sexual exchanges is associated with sexual satisfaction as well as whether changes in sexual rewards and costs are associated with change in sexual satisfaction. Using a sample of 104 couples, Study 2 examined whether partner rewards and costs add to individuals' own sexual satisfaction over and above own sexual rewards and costs for men or women. The results provided further evidence for the validity of the IEMSS, including support for the propositions that in long-term relationships: (a) sexual satisfaction is influenced by the history of sexual rewards and costs rather than by rewards and costs at a particular point in time; (b) decreases in sexual satisfaction are associated with sexual exchanges becoming less favorable; and (c) satisfaction is influenced by dyadic factors for both men and women.

Alpha-Melanocyte Stimulating Hormone, Inflammation and Human Melanoma

Peptides. Feb, 2006  |  Pubmed ID: 16274844

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.

Clinical Experience Using Cultured Epithelial Autografts Leads to an Alternative Methodology for Transferring Skin Cells from the Laboratory to the Patient

Regenerative Medicine. Nov, 2006  |  Pubmed ID: 17465762

We report a 10-year audit using cultured epithelial autografts (CEAs) for patients with extensive burns. Clinical take using CEAs averaged only 45% (as has been reported by others) but over half of all cells cultured for these patients had to be discarded owing to difficulties of timing the production of CEA sheets to the needs of the patients. CEAs could not be used until they had reached confluence and formed an integrated sheet, which took, on average, 12 days. However, once formed, they needed to be used within 2-3 days or they lost the ability to attach to wound beds. In response to this we developed a simpler carrier dressing methodology for transferring cultured subconfluent keratinocytes from the laboratory to the wound bed. This methodology offers an increase in speed of delivery but its major contribution is the greater flexibility in timing the transfer of cells from the laboratory to the changing needs of the patients.

Use of an in Vitro Model of Tissue-engineered Skin to Investigate the Mechanism of Skin Graft Contraction

Tissue Engineering. Nov, 2006  |  Pubmed ID: 17518627

Skin graft contraction leading to loss of joint mobility and cosmetic deformity remains a major clinical problem. In this study we used a tissue-engineered model of human skin, based on sterilized human adult dermis seeded with keratinocytes and fibroblasts, which contracts by up to 60% over 28 days in vitro, as a model to investigate the mechanism of skin contraction. Pharmacologic agents modifying collagen synthesis, degradation, and cross-linking were examined for their effect on contraction. Collagen synthesis and degradation were determined using immunoassay techniques. The results show that skin contraction was not dependent on inhibition of collagen synthesis or stimulation of collagen degradation, but was related to collagen remodelling. Thus, reducing dermal pliability with glutaraldehyde inhibited the ability of cells to contract the dermis. So did inhibition of matrix metalloproteinases and inhibition of lysyl oxidase-mediated collagen cross-linking, but not transglutaminase-mediated cross-linking. In summary, this in vitro model of human skin has allowed us to identify specific cross-linking pathways as possible pharmacologic targets for prevention of graft contracture in vivo.

Epithelialization of Hydrogels Achieved by Amine Functionalization and Co-culture with Stromal Cells

Biomaterials. Dec, 2007  |  Pubmed ID: 17888510

The aim of this study was to develop a hydrogel which would be suitable for corneal cell re-epithelialization when used as a corneal implant. To achieve this, a series of hydrogels were functionalized with primary amines by post-polymerization reactions between amine compounds and glycidyl ether groups attached to the hydrogels. We report a strong correlation between the structure of the amine and the viability of stromal cells and epithelial cells cultured on these hydrogels. Subsequent co-culture of epithelial and stromal cells on the amine modified hydrogels allowed successful expansion of epithelial cells on surfaces functionalized with alkyl alpha-omega diamines with carbon chain lengths of between 3 and 6. Analysis of variance showed that corneal epithelial cells had a strong preference for surfaces functionalized by the reaction of excess 1,3 diaminopropane with units of glycidyl methacrylate compared to the reaction products of other amines (ammonia; 1,2-diaminoethane; 1,4-diaminobutane or 1,6-diaminohexane). We suggest this approach of amine functionalization combined with stromal/epithelial co-culture offers a promising new approach to achieving a secure corneal epithelium.

Enhancement of Keratinocyte Performance in the Production of Tissue-engineered Skin Using a Low-calcium Medium

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Sep-Oct, 2007  |  Pubmed ID: 17971018

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.

Randomized, Controlled, Single-blind Study on Use of Autologous Keratinocytes on a Transfer Dressing to Treat Nonhealing Diabetic Ulcers

Regenerative Medicine. Nov, 2007  |  Pubmed ID: 18034628

To compare the rate of healing of diabetic neuropathic ulcers using cultured autologous keratinocytes delivered on chemically defined transfer discs (Myskin) (active treatment) versus healing obtained with cell-free discs (placebo).

A Xenobiotic-free Culture System for Human Limbal Epithelial Stem Cells

Regenerative Medicine. Nov, 2007  |  Pubmed ID: 18034630

Murine 3T3 feeder cells are commonly used for stem cell expansion. Although 'feeder-free' systems are being developed for a variety of stem cells including embryonic, the use of feeder cells currently remains optimal for the expansion of epithelial stem cells. In this study, MRC-5, a human embryonic fibroblast cell line, has been investigated for its potential use as a feeder layer in human limbal epithelial (HLE) cell expansion under serum-free conditions, with the aim of developing a xenobiotic-free culture system for therapeutic corneal regeneration applications.

The Effect of Induced Biphasic Pulsed Currents on Re-epithelialization of a Novel Wound Healing Model

Bioelectromagnetics. Jan, 2007  |  Pubmed ID: 16988995

The coordinated migration of keratinocytes is crucial to cutaneous wound healing; failure of keratinocytes to migrate into a wound can lead to chronic non-healing wounds. Keratinocyte migration can be influenced by applied electrical fields. Our aim was to investigate whether keratinocyte migration could be accelerated by applying an induced biphasic pulsed electrical field. We developed two in vitro biological systems models for this purpose: a keratinocyte colony-forming model and a reconstituted skin wound healing model with biphasic pulsed currents. Our in vitro skin models were capable of generating trans-epithelial potentials (TEP) similar to in vivo mammalian skin. Histological examination of the wound healing model also indicated that re-epithelialization occurred in a similar manner to that seen in vivo, although no evidence of a reconstitution of a basement membrane was seen during the 14 days in vitro experimental period. We found that growth of keratinocyte colonies and keratinocyte migration in an in vitro wound bed were not significantly affected by induced short duration biphasic pulsed currents at a frequency of 0.5 Hz of 100 and 200 mV/mm.

Development of a 3D Cell Culture System for Investigating Cell Interactions with Electrospun Fibers

Biotechnology and Bioengineering. Aug, 2007  |  Pubmed ID: 17171721

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.

Accelerated Formation of Multicellular 3-D Structures by Cell-to-cell Cross-linking

Biotechnology and Bioengineering. Aug, 2007  |  Pubmed ID: 17304565

The three-dimensional (3-D) arrangement of cells within tissues is integral to their development and function. Advances in stem cell science and regenerative medicine have stimulated interest in the replication of this architecture in vitro. We have developed a versatile method for controlling short-term cell-cell and cell-matrix interactions via a facile cell surface engineering process that enables the rapid formation of specific 3-D interactions for a range of cell types. We demonstrate that chemical modification of cell surfaces and matrix proteins can artificially accelerate the cell adhesion process and confirm the ability to control the formation of multicellular aggregates with defined architectures and heterotypic cell types. Direct comparison with a natural aggregation process seen during differentiation of embryonic stem (ES) cells revealed increased expression of developmental regulatory proteins and a concomitant enhancement of ES cell differentiation. Furthermore, this new methodology has numerous applications in generating layered structures. For example, we demonstrate improved transfer of therapeutic human keratinocytes onto a dermal layer in a skin repair model.

Progress and Opportunities for Tissue-engineered Skin

Nature. Feb, 2007  |  Pubmed ID: 17314974

Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.

Plasma Polymer Coated Surfaces for Serum-free Culture of Limbal Epithelium for Ocular Surface Disease

Journal of Materials Science. Materials in Medicine. Feb, 2007  |  Pubmed ID: 17323166

The potential use of plasma polymer coatings as substrates for serum-free expansion of limbal epithelial cells was investigated. Preliminary studies using a human corneal epithelial cell line showed that acrylic acid-coated surfaces performed better than allyl amine and allyl alcohol coated surfaces in terms of cell metabolic activity and confluence as assessed using the MTT assay. Subsequently, the proliferation and maturity of primary human limbal epithelial cells in co-culture with growth arrested 3T3 fibroblasts on a range of acrylic acid plasma coated surfaces, octadiene plasma coated surfaces and tissue culture plastic was investigated using MTT and cytokeratin 3 immunostaining. The cells performed better in the presence of serum on all surfaces. However, the acrylic acid coated surfaces successfully sustained a serum-free fibroblast/epithelial cell co-culture. The metabolic activity of the epithelial cells was superior on the acrylic acid coated surfaces than on tissue culture plastic in serum-free conditions and their levels of differentiation were not significantly higher than in the presence of serum. These results suggest that these surfaces can be used successfully for the serum-free expansion of human limbal epithelial cells.

The Effects of Nickel and Chromium on Human Keratinocytes: Differences in Viability, Cell Associated Metal and IL-1alpha Release

Toxicology in Vitro : an International Journal Published in Association with BIBRA. Aug, 2007  |  Pubmed ID: 17368827

This study was carried out to assess the effects of chromium and nickel upon isolated keratinocytes as an in vitro model of human skin. Keratinocytes were isolated from healthy volunteer skin samples of unknown metal sensitivity (n=10) and were compared with cells from patient biopsies of known metal sensitivity (n=7). Cells were dosed with a concentration range of nickel and chromium (0-10,000 microM) and cellular mitochondrial activity, viability, metal uptake and cytokine release were measured. Responses of primary versus passaged keratinocytes were also compared. Toxicity data from primary and passaged keratinocytes was statistically analysed by the non-linear Hill Plot model. Results showed that hexavalent chromium was significantly more cytotoxic, associated more with keratinocytes and induced a dose dependant release of IL-1alpha compared to nickel. Significant differences were observed between primary and passaged keratinocytes with regard to the toxicity of chromium and nickel and variation of response. No differences were observed in the cytotoxicity or cytokine release induced by chromium or nickel for the known sensitised biopsy patient samples (n=4) compared to patch test negative controls (n=3). The results from this study suggest human keratinocytes in vitro respond very differently to chromium and nickel.

An Integrated Systems Biology Approach to Understanding the Rules of Keratinocyte Colony Formation

Journal of the Royal Society, Interface / the Royal Society. Dec, 2007  |  Pubmed ID: 17374590

Closely coupled in vitro and in virtuo models have been used to explore the self-organization of normal human keratinocytes (NHK). Although it can be observed experimentally, we lack the tools to explore many biological rules that govern NHK self-organization. An agent-based computational model was developed, based on rules derived from literature, which predicts the dynamic multicellular morphogenesis of NHK and of a keratinocyte cell line (HaCat cells) under varying extracellular Ca++ concentrations. The model enables in virtuo exploration of the relative importance of biological rules and was used to test hypotheses in virtuo which were subsequently examined in vitro. Results indicated that cell-cell and cell-substrate adhesions were critically important to NHK self-organization. In contrast, cell cycle length and the number of divisions that transit-amplifying cells could undergo proved non-critical to the final organization. Two further hypotheses, to explain the growth behaviour of HaCat cells, were explored in virtuo-an inability to differentiate and a differing sensitivity to extracellular calcium. In vitro experimentation provided some support for both hypotheses. For NHKs, the prediction was made that the position of stem cells would influence the pattern of cell migration post-wounding. This was then confirmed experimentally using a scratch wound model.

Real-time Detection of Stress in 3D Tissue-engineered Constructs Using NF-kappaB Activation in Transiently Transfected Human Dermal Fibroblast Cells

Tissue Engineering. May, 2007  |  Pubmed ID: 17430089

The main objective of this study was to develop a nondestructive reporter system for assessing the response of human cells contained within a three-dimensional (3D) tissue-engineered construct to exogenous stress. Dermal fibroblasts were transiently transfected with a reporter construct linked to nuclear factor kappaB (NF-kappaB) activation which led to expression of a nonstable form of enhanced green fluorescent protein (d2EGFP) after stimulation. This led to a temporary production of fluorescence, which could be readily detected but was not intrinsically toxic, as cells were able to metabolize the initial cycle of d2EGFP produced. This permitted the model to be used for restimulation post recovery. To investigate the performance and predictive ability of this method for assessing cellular response to stress in 3D, we used a range of compounds known to have pro-inflammatory or oxidative properties. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) were selected for having a direct cytokine action; lipopolysaccharide (LPS) was selected for modeling bacterial-mediated inflammation; and hydrogen peroxide was selected as a crude method for delivering an oxidative stress. Transfected cells were stimulated with the above compounds in 3D and the synthesis of d2EGFP was detected as a measure of NF-kappaB activation. The resultant fluorescence was scored using a series of photomicrographs taken by epifluorescence microscopy. All agents activated NF-kappaB when cells were grown in 3D scaffolds but did not cause any significant reduction in cell viability as measured by a standard MTT-ESTA viability test. Parallel NF-kappaB activation and MTT measurements was also conducted in two-dimension (2D) and confirmed findings in 3D. The 3D model described using a fluorescent reporter gene is a highly sensitive and reliable method for detecting cellular stress and represents a key step in developing tissue engineering models with the potential for screening pharmaceutical and cosmetic compounds, as an alternative to existing in vitro and in vivo methods.

The Mechanism of Skin Graft Contraction: an Update on Current Research and Potential Future Therapies

Burns : Journal of the International Society for Burn Injuries. Mar, 2008  |  Pubmed ID: 18226455

Skin graft contraction is a common and intractable problem. The current treatments focus on mechanical opposition of contractile forces using splints and on compression of the grafted skin with pressure garments. For the patients, this causes significant morbidity with restriction of joint mobility and often requires multiple episodes of corrective surgery. Despite 50 years of research in this area, treatment and prevention of graft contraction have progressed very little and understanding of the underlying mechanism remains poor. This article reviews the clinical problem and the approaches used to prevent or treat graft contracture. It also considers to what extent we currently understand the cellular basis of graft contracture, based on in vitro models of skin contraction and in vivo observation of patients.

Tissue-engineered Buccal Mucosa Urethroplasty-clinical Outcomes

European Urology. Jun, 2008  |  Pubmed ID: 18262717

Whilst buccal mucosa is the most versatile tissue for urethral replacement, the quest continues for an ideal tissue replacement for the urethra when substantial tissue transfer is needed. Previously we described the development of autologous tissue-engineered buccal mucosa (TEBM). Here we report clinical outcomes of the first human series of its use in substitution urethroplasty.

Decellularization and Sterilization of Porcine Urinary Bladder Matrix for Tissue Engineering in the Lower Urinary Tract

Regenerative Medicine. Mar, 2008  |  Pubmed ID: 18307398

Several synthetic and natural matrices have been described for tissue engineering of bladder but there is little information on the effects of processing on their subsequent mechanical performance or interaction with human cells.

Inhibition of Keratinocyte-driven Contraction of Tissue-engineered Skin in Vitro by Calcium Chelation and Early Restraint but Not Submerged Culture

Journal of Burn Care & Research : Official Publication of the American Burn Association. Mar-Apr, 2008  |  Pubmed ID: 18354296

Skin graft contracture remains a significant cause of patient morbidity with reduction in joint mobility and cosmetic deformity. Despite recent advances, its mechanism is largely unknown. The authors have previously demonstrated the importance of the keratinocyte in the contraction of tissue-engineered skin in vitro. In this study, they investigate the effect of reducing keratinocyte differentiation on contraction by adding 0.8 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA) to the culture medium and by culturing tissue-engineered skin submerged in medium rather than at air-liquid interface. They also simulate the effect of early mechanical splinting in vitro to study its effect on contraction. Here the study shows that removal of the epidermis after 16 days culture at air-liquid interface results in immediate dermal relaxation with a return to the original dermal surface area. Lowering extracellular calcium concentration with EGTA reduces keratinocyte differentiation and reduces the rate of contraction. Submerged culture does not significantly reduce differentiation of tissue-engineered skin and does not reduce contraction. However, following an initial short period of mechanical constraint, the rate of contraction of tissue-engineered skin is reduced. Reducing keratinocyte differentiation by lowering extracellular calcium with EGTA, reduces contraction. However, submerged culture of tissue-engineered skin is ineffective. A short period of splinting of meshed skin grafts during the initial phase of epithelialization and keratinocyte differentiation may be most effective in the prevention of subsequent contractures in vivo but additional studies are needed to establish this.

Development of Biodegradable Electrospun Scaffolds for Dermal Replacement

Biomaterials. Jul, 2008  |  Pubmed ID: 18448164

Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA+10% oligolactide; (3) PLLA+rhodamine and (4-6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.

Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

PloS One. 2008  |  Pubmed ID: 18461132

Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this.

Biocompatible Wound Dressings Based on Chemically Degradable Triblock Copolymer Hydrogels

Biomacromolecules. Aug, 2008  |  Pubmed ID: 18598077

The synthesis of a series of thermo-responsive ABA triblock copolymers in which the outer A blocks comprise poly(2-hydroxypropyl methacrylate) and the central B block is poly(2-(methacryloyloxy)ethyl phosphorylcholine) is achieved using atom transfer radical polymerization. These novel triblock copolymers form thermo-reversible physical gels with critical gelation temperatures and mechanical properties that are highly dependent on the copolymer composition and concentration. TEM studies on dried dilute copolymer solutions indicate the presence of colloidal aggregates, which is consistent with micellar gel structures. This hypothesis is consistent with the observation that incorporating a central disulfide bond within the B block leads to thermo-responsive gels that can be efficiently degraded using mild reductants such as dithiothreitol (DTT) over time scales of minutes at 37 degrees C. Moreover, the rate of gel dissolution increases at higher DTT/disulfide molar ratios. Finally, these copolymer gels are shown to be highly biocompatible. Only a modest reduction in proliferation was observed for monolayers of primary human dermal fibroblasts, with no evidence for cytotoxicity. Moreover, when placed directly on 3D tissue-engineered skin, these gels had no significant effect on cell viability. Thus, we suggest that these thermo-responsive biodegradable copolymer gels may have potential applications as wound dressings.

Cell Adhesive Hydrogels Synthesised by Copolymerisation of Arg-protected Gly-Arg-Gly-Asp-Ser Methacrylate Monomers and Enzymatic Deprotection

Chemical Communications (Cambridge, England). Dec, 2008  |  Pubmed ID: 19030549

This work reports the synthesis of protected Gly-Arg-Gly-Asp-Ser functionalised hydrogels, which are deprotected (and activated for cell adhesion) by reaction with glutathione-S-transferase.

Non-cytotoxic Polymer Vesicles for Rapid and Efficient Intracellular Delivery

Faraday Discussions. 2008  |  Pubmed ID: 19048994

We have recently achieved efficient cytosolic delivery by using pH-sensitive poly(2-(methacryloyloxy)ethylphosphorylcholine)-co-poly(2-(diisopropylamino)ethylmethacrylate) (PMPC-PDPA) diblock copolymers that self-assemble to form vesicles, known as polymersomes, in aqueous solution. It is particularly noteworthy that these diblock copolymers form stable polymersomes at physiological pH but rapidly dissociate below pH 6 to give molecularly-dissolved copolymer chains (unimers). These PMPC-PDPA polymersomes are used to encapsulate nucleic acids for efficient intracellular delivery. Confocal laser scanning microscopy and fluorescence flow cytometry are used to quantify cellular uptake and to study the kinetics of this process. Finally, we examine how PMPC-PDPA polymersomes affect the viability of primary human cells (human dermal fibroblasts (HDF)), paying particular regard to whether inflammatory responses are triggered.

Development of a Mini 3D Cell Culture System Using Well Defined Nickel Grids for the Investigation of Cell Scaffold Interactions

Journal of Materials Science. Materials in Medicine. Jul, 2009  |  Pubmed ID: 19225869

A bioreactor system was developed using a series of fine mesh nickel grids as free standing scaffolds to investigate the behaviours of fibroblasts and keratinocytes in tissue culture. It was found that the mesh size of the suspended grids, but not of the grids that attached to tissue culture surface, had significant influences on cell behaviour and there was a maximum size for fibroblast to span within the defined culture period. Time lapse video microscopy demonstrated fibroblasts cultured on these grids initially migrated onto the struts but then worked together to fill in the voids between struts with a membranous sheet of tissue. In contrast keratinocytes barely migrated from the initial site of cell deposition and when they moved (to a modest extent) it was as an integrated sheet of cells. Similar results were observed when both types of cells were co-cultured in the system.

Development of a Surface-modified Contact Lens for the Transfer of Cultured Limbal Epithelial Cells to the Cornea for Ocular Surface Diseases

Tissue Engineering. Part A. Oct, 2009  |  Pubmed ID: 19265461

Our aim was to develop an improved cell transfer system for delivering laboratory-cultured human limbal epithelial cells to the cornea, which would be low risk for the patient and convenient to use for the surgeon. We took a standard contact lens and developed a plasma polymer layer for coating this for attachment of cells to the lens and subsequent transfer of cells to the cornea. A range of plasma polymer surfaces were examined for initial cell attachment using three different combinations of human and rabbit epithelial and stromal cells, initially expanding cells both with and without bovine serum. The most promising surfaces, based on acrylic acid, were then coated onto contact lenses. Cell transfer from the lenses to the denuded surface of a 3D rabbit organ culture model was then used to make a second selection of substrates, which permitted reliable cell transfer. Primary rabbit and human corneal cells attached and proliferated well on acrylic acid-coated surfaces. Reliable transfer of primary epithelial cells from the coated contact lenses to a rabbit cornea was achieved by coating lenses with acrylic acid at 5 W/10 cm(3)/min and using cell densities of 1 x 10(5)/lens and above.

Development of Three-dimensional Tissue-engineered Models of Bacterial Infected Human Skin Wounds

Tissue Engineering. Part C, Methods. Sep, 2009  |  Pubmed ID: 19292658

While infected skin wounds are on the increase because of ageing populations, rising incidence of diabetes, and antibiotic resistance, we lack relevant in vivo or in vitro models to study many aspects of bacterial interaction with skin. The aim of this study was to develop three-dimensional models of normal human skin to study bacterial infection. The common dermatological pathogens Staphylococcus aureus and Pseudomonas aeruginosa were used to infect tissue-engineered skin, and the course of infection in the skin was examined over several days. Two forms of model were developed-one in which bacteria were introduced directly to 10 mm wounds in the epidermis, and another in which wounds were created by burning a 4 mm hole in the center of the tissue before inoculation. The bacteria flourished within the engineered skin, and colonized the upper epidermal layers before invasion into the dermis. Infection with P. aeruginosa caused a loss of epidermis and de-keratinization of the skin constructs, as well as partial loss of basement membrane. These novel complex human skin infection models could be used to investigate microbial invasion of normal skin epithelium, basement membrane, and connective tissue, and as a model to study approaches to reduce bacterial burden in skin wounds.

Diffusion Studies of Nanometer Polymersomes Across Tissue Engineered Human Oral Mucosa

Pharmaceutical Research. Jul, 2009  |  Pubmed ID: 19387800

To measure the diffusion of nanometer polymersomes through tissue engineered human oral mucosa.

Preparation and Aqueous Solution Properties of Thermoresponsive Biocompatible AB Diblock Copolymers

Biomacromolecules. Jul, 2009  |  Pubmed ID: 19499888

The synthesis of a series of amphiphilic AB diblock copolymers in which the A block comprises poly(2-(methacryloyloxy)ethyl phosphorylcholine) [PMPC] and the B block comprises poly(2-hydroxypropyl methacrylate) [PHPMA] by atom transfer radical polymerization (ATRP) is reported. The aqueous solution properties of these new diblock copolymers were examined using dynamic light scattering and variable temperature (1)H NMR spectroscopy. Copolymers with shorter thermoresponsive PHPMA blocks formed relatively large aggregates, while copolymers with longer PHPMA blocks formed smaller aggregates. This apparently "anomalous" self-assembly behavior occurs because the PHPMA block becomes more hydrophobic as its degree of polymerization is increased. Therefore, shorter PHPMA blocks lead to the formation of loose highly hydrated aggregates, whereas longer blocks formed more compact dehydrated aggregates. In addition, these new PMPC-PHPMA diblock copolymers are highly biocompatible and can mediate the relatively rapid efficient uptake of a fluorescent dye by human dermal fibroblast cells. Interestingly, dye uptake kinetics appear to depend on the hydrophobic/hydrophilic balance of the copolymer. This not only bodes well for in vitro imaging of live cells for biomedical applications but also highlights the importance of copolymer design to ensure efficient drug delivery.

Phosphate Functional Core-shell Polymer Nanoparticles for the Release of Vascular Endothelial Growth Factor

Chembiochem : a European Journal of Chemical Biology. Sep, 2009  |  Pubmed ID: 19644996

Exploring Hypotheses of the Actions of TGF-beta1 in Epidermal Wound Healing Using a 3D Computational Multiscale Model of the Human Epidermis

PloS One. 2009  |  Pubmed ID: 20046881

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units (keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged (by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing.

Role of Sexual Self-disclosure in the Sexual Satisfaction of Long-term Heterosexual Couples

Journal of Sex Research. Jan-Feb, 2009  |  Pubmed ID: 19012061

This study examined two proposed pathways between sexual self-disclosure (SSD) and sexual satisfaction in a sample of 104 heterosexual couples in long-term relationships. According to the proposed instrumental pathway, disclosure of sexual preferences increases a partner's understanding of those preferences resulting in a sexual script that is more rewarding and less costly. A more favorable balance of sexual rewards to sexual costs, in turn, results in greater sexual satisfaction for the disclosing individual. According to the proposed expressive pathway, mutual self-disclosure contributes to relationship satisfaction, which in turn leads to greater sexual satisfaction. Support was found for the instrumental pathway for both men and women. Support also was found for an expressive pathway between own SSD and partner nonsexual self-disclosure (NSD) and men's sexual satisfaction, and between own NSD and women's sexual satisfaction. These results are interpreted in terms of mechanisms for establishing and maintaining sexual satisfaction in long-term relationships in men and women.

Development of a Three Dimensional Multiscale Computational Model of the Human Epidermis

PloS One. 2010  |  Pubmed ID: 20076760

Transforming Growth Factor (TGF-beta1) is a member of the TGF-beta superfamily ligand-receptor network. and plays a crucial role in tissue regeneration. The extensive in vitro and in vivo experimental literature describing its actions nevertheless describe an apparent paradox in that during re-epithelialisation it acts as proliferation inhibitor for keratinocytes. The majority of biological models focus on certain aspects of TGF-beta1 behaviour and no one model provides a comprehensive story of this regulatory factor's action. Accordingly our aim was to develop a computational model to act as a complementary approach to improve our understanding of TGF-beta1. In our previous study, an agent-based model of keratinocyte colony formation in 2D culture was developed. In this study this model was extensively developed into a three dimensional multiscale model of the human epidermis which is comprised of three interacting and integrated layers: (1) an agent-based model which captures the biological rules governing the cells in the human epidermis at the cellular level and includes the rules for injury induced emergent behaviours, (2) a COmplex PAthway SImulator (COPASI) model which simulates the expression and signalling of TGF-beta1 at the sub-cellular level and (3) a mechanical layer embodied by a numerical physical solver responsible for resolving the forces exerted between cells at the multi-cellular level. The integrated model was initially validated by using it to grow a piece of virtual epidermis in 3D and comparing the in virtuo simulations of keratinocyte behaviour and of TGF-beta1 signalling with the extensive research literature describing this key regulatory protein. This research reinforces the idea that computational modelling can be an effective additional tool to aid our understanding of complex systems. In the accompanying paper the model is used to explore hypotheses of the functions of TGF-beta1 at the cellular and subcellular level on different keratinocyte populations during epidermal wound healing.

Binding Bacteria to Highly Branched Poly(N-isopropyl Acrylamide) Modified with Vancomycin Induces the Coil-to-globule Transition

Journal of the American Chemical Society. Feb, 2010  |  Pubmed ID: 20099898

Binding of highly branched poly(N-isopropylacrylamide) with vancomycin end groups to Staphylococcus aureus induced a coil-to-globule phase transition. The polymers aggregated this gram-positive bacteria (but not gram-negative bacteria) over a wide range of temperatures, but cooling to 24-26 degrees C progressed the polymer-bound bacteria through a globule-to-coil phase transition, after which the bacteria were released.

A Micro-incubator for Cell and Tissue Imaging

BioTechniques. Feb, 2010  |  Pubmed ID: 20359297

A low-cost micro-incubator for imaging dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment that can be altered to study responses of cell monolayers for several days as well as relatively thick tissue samples and tissue-engineered epithelial tissues in experiments lasting several hours. Samples are contained in a sterile cavity closed by a gas-permeable membrane. The incubator can be positioned in any direction and used on an inverted or upright microscope. Temperature is regulated using a Peltier module controlled by a sensor positioned close to the sample, enabling compensation for any changes in temperature. Rapid changes in a sample's surrounding environment can be achieved due to the fast response of the Peltier module. These features permit monitoring of sample adaptation to induced environmental changes.

Enhanced Fluorescence Imaging of Live Cells by Effective Cytosolic Delivery of Probes

PloS One. 2010  |  Pubmed ID: 20454666

Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery.

Using Poly(lactide-co-glycolide) Electrospun Scaffolds to Deliver Cultured Epithelial Cells to the Cornea

Regenerative Medicine. May, 2010  |  Pubmed ID: 20455650

To assess the potential of electrospun poly(lactide-co-glycolide) membranes to provide a biodegradable cell carrier system for limbal epithelial cells.

Using Swept-source Optical Coherence Tomography to Monitor the Formation of Neo-epidermis in Tissue-engineered Skin

Journal of Tissue Engineering and Regenerative Medicine. Dec, 2010  |  Pubmed ID: 20603865

There is an increasing need for a robust, simple to use, non-invasive imaging technology to follow tissue-engineered constructs as they develop. Our aim was to evaluate the use of swept-source optical coherence tomography (SS-OCT) to image tissue-engineered skin as it developed over several weeks. Tissue-engineered skin was produced using both de-epithelialized acellular dermis (DED) and amorphous collagen gels. In both cases the epidermis could be readily distinguished from the neodermis, based on a comparison with standard destructive histology of samples. Constructs produced with DED showed more epidermal/dermal maturation than those produced using collagen. The development of tissue-engineered skin based on DED was accurately monitored with SS-OCT over 3 weeks and confirmed with conventional histology.

Internalization and Biodistribution of Polymersomes into Oral Squamous Cell Carcinoma Cells in Vitro and in Vivo

Nanomedicine (London, England). Sep, 2010  |  Pubmed ID: 20874018

The prognosis for oral squamous cell carcinoma (OSCC) is not improving despite advances in surgical treatment. As with many cancers, there is a need to deliver therapeutic agents with greater efficiency into OSCC to improve treatment and patient outcome. The development of polymersomes offers a novel way to deliver therapy directly into tumor cells. Here we examined the internalization and biodistribution of two different fluorescently labeled polymersome formulations; polyethylene oxide (PEO)-poly 2-(diisopropylamino)ethyl methacrylate (PDPA) and poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)-PDPA, into SCC4 OSCC cells in vitro and in vivo. In vitro SCC4 monolayers internalized PMPC-PDPA and PEO-PDPA at similar rates. However, in vivo PMPC-PDPA polymersomes penetrated deeper and were more widely dispersed in SCC4 tumors than PEO-PDPA polymersomes. In the liver and spleen PMPC-PDPA mainly accumulated in tissue macrophages. However, in tumors PMPC-PDPA was found extensively in the nucleus and cytoplasm of tumor cells as well as in tumor-associated macrophages. Use of PMPC-PDPA polymersomes may enhance polymersome-mediated antitumor therapy.

Development of an Ibuprofen-releasing Biodegradable PLA/PGA Electrospun Scaffold for Tissue Regeneration

Biotechnology and Bioengineering. Feb, 2010  |  Pubmed ID: 19731254

Our aim was to develop a biodegradable fibrous dressing to act as a tissue guide for in situ wound repair while releasing Ibuprofen to reduce inflammation in wounds and reduce pain for patients on dressing changes. Dissolving the acid form of Ibuprofen (from 1% to 10% by weight) in the same solvent as 75% polylactide, 25% polyglycolide (PLGA) polymers gave uniformly loaded electrospun fibers which gave rapid release of drug within the first 8 h and then slower release over several days. Scaffolds with 10% Ibuprofen degraded within 6 days. The Ibuprofen released from these scaffolds significantly reduced the response of fibroblasts to major pro-inflammatory stimulators. Fibroblast attachment and proliferation on scaffolds was unaffected by the addition of 1-5% Ibuprofen. Scaffolds loaded with 10% Ibuprofen initially showed reduced cell attachment but this was restored by soaking scaffolds in media for 24 h. In summary, addition of Ibuprofen to electrospun biodegradable scaffolds can give acute protection of adjacent cells to inflammation while the scaffolds provide an open 3D fibrous network to which cells can attach and migrate. By 6 days, such scaffolds will have completely dissolved into the wound bed obviating any need for dressing removal.

The Development and Characterization of an Organotypic Tissue-engineered Human Esophageal Mucosal Model

Tissue Engineering. Part A. Mar, 2010  |  Pubmed ID: 19845463

There is a demand for a reliable three-dimensional tissue-engineered model of the esophageal mucosa for use as an experimental platform for investigating esophageal epithelial biology and the pathogenesis of esophageal neoplasia and precursor lesions such as Barrett's metaplasia. A number of models have been described, but there has been little systematic assessment of the different approaches, making selection of a preferred platform difficult. This study assesses the properties of organotypic cultures using four different scaffolds (human esophageal matrix, porcine esophageal matrix, human dermal matrix, and collagen) and two different epithelial cell types (primary human esophageal squamous cells and the Het-1A esophageal squamous cell line). Human esophageal matrix and dermis did not give consistent results, but porcine esophageal matrix and collagen proved more reliable and were studied in greater detail. Both matrices supported the formation of a mature stratified epithelium that was similar to that of the normal human esophagus, demonstrated by Ki67, CK4, CK14, and involucrin staining. However, collagen showed reduced epithelial adherence, while fibroblast penetration into the porcine matrix was poor. Composite cultures using Het-1A cells formed a hyperproliferative epithelium with no evidence of differentiation. We propose human esophageal squamous cells seeded onto porcine esophageal matrix as the preferred model of the normal human esophagus.

A Novel Route for the Production of Chitosan/poly(lactide-co-glycolide) Graft Copolymers for Electrospinning

Biomedical Materials (Bristol, England). Dec, 2010  |  Pubmed ID: 21079284

Both chitosan and polylactide/polyglycolide have good biocompatibility and can be used to produce tissue engineering scaffolds for cultured cells. However the synthetic scaffolds lack groups that would facilitate their modification, whereas chitosan has extensive active amide and hydroxyl groups which would allow it to be subsequently modified for the attachment of peptides, proteins and drugs. Also chitosan is very hydrophilic, whereas PLGA is relatively hydrophobic. Accordingly there are many situations where it would be ideal to have a copolymer of both, especially one that could be electrospun to provide a versatile range of scaffolds for tissue engineering. Our aim was to develop a novel route of chitosan-g-PLGA preparation and evaluate the copolymers in terms of their chemical characterization, their performance on electrospinning and their ability to support the culture of fibroblasts as an initial biological evaluation of these scaffolds. Chitosan was first modified with trimethylsilyl chloride, and catalyzed by dimethylamino pyridine. PLGA-grafted chitosan copolymers were prepared by reaction with end-carboxyl PLGA (PLGA-COOH). FT-IR and(1)H-NMR characterized the copolymer molecular structure as being substantially different to that of the chitosan or PLGA on their own. Elemental analysis showed an average 18 pyranose unit intervals when PLGA-COOH was grafted into the chitosan molecular chain. Differential scanning calorimetry results showed that the copolymers had different thermal properties from PLGA and chitosan respectively. Contact angle measurements demonstrated that copolymers became more hydrophilic than PLGA. The chitosan-g-PLGA copolymers were electrospun to produce either nano- or microfibers as desired. A 3D fibrous scaffold of the copolymers gave good fibroblast adhesion and proliferation which did not differ significantly from the performance of the cells on the chitosan or PLGA electrospun scaffolds. In summary this work presents a methodology for making a hybrid material of natural and synthetic polymers which can be electrospun and reacts well as a substrate for cell culture.

A Comparison of Imaging Methodologies for 3D Tissue Engineering

Microscopy Research and Technique. Dec, 2010  |  Pubmed ID: 20981758

Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current "gold standard" for imaging three-dimensional tissue engineered constructs, histology.

Tracking Nanoparticles in Three-dimensional Tissue-engineered Models Using Confocal Laser Scanning Microscopy

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21042964

Here we describe a method for imaging the position of nanoparticles within a 3D tissue-engineered model using confocal laser scanning microscopy (CLSM). The ability to track diffusion of nanoparticles in vitro is an important part of trans-dermal and trans-mucosal drug delivery development as well as for intra-epithelial drug delivery. Using 3D tissue-engineered models enables us to image diffusion in vitro in a physiologically relevant way; not possible in two-dimensional monolayer cultures (MacNeil, Nature 445:874-880, 2007; Hearnden et al., Pharmaceutical Res. 26(7):1718-1728, 2009). CLSM enables imaging of viable in vitro models in three dimensions with good spatial and axial resolution (Georgakoudi et al., Tissue Eng 14:1-20, 2008; Schenke-Layland et al., Adv. Drug Del. Rev. 58:878-896, 2006). Here we show that fluorescently labelled nanoparticles can be visualised, quantified, and their position within the cell can be determined using CLSM.

Production of Tissue-engineered Skin and Oral Mucosa for Clinical and Experimental Use

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21042970

Since the early 1990s, our understanding of how epithelial and stromal cells interact in 3D tissue-engineered constructs has led to tissue-engineered skin and oral mucosa models, which are beginning to deliver benefit in the clinic (usually in small-scale reconstructive surgery procedures) but have a great deal to offer for in vitro investigations. These 3D tissue-engineered models can be used for a wide variety of purposes such as dermato- and mucotoxicity, wound healing, examination of pigmentation and melanoma biology, and in particular, a recent development from this laboratory, as a model of bacterially infected skin. Models can also be used to investigate specific skin disease processes. In this chapter, we describe the basic methodology for producing 3D tissue-engineered skin and oral mucosa based on de-epidermised acellular human dermis, and we give examples of how these models can be used for a variety of applications.

Evaluating the Use of Optical Coherence Tomography for the Detection of Epithelial Cancers in Vitro

Journal of Biomedical Optics. Nov, 2011  |  Pubmed ID: 22112120

Optical coherence tomography (OCT) is a noninvasive imaging methodology that is able to image tissue to depths of over 1 mm. Many epithelial conditions, such as melanoma and oral cancers, require an invasive biopsy for diagnosis. A noninvasive, real-time, point of care method of imaging depth-resolved epithelial structure could greatly improve early diagnosis and long-term monitoring in patients. Here, we have used tissue-engineered (TE) models of normal skin and oral mucosa to generate models of melanoma and oral cancer. We have used these to determine the ability of OCT to image epithelial differences in vitro. We report that while in vivo OCT gives reasonable depth information for both skin and oral mucosa, in vitro the information provided is less detailed but still useful. OCT can provide reassurance on the development of TE models of skin and oral mucosa as they develop in vitro. OCT was able to detect the gross alteration in the epithelium of skin and mucosal models generated with malignant cell lines but was less able to detect alteration in the epithelium of TE models that mimicked oral dysplasia or, in models where tumor cells had penetrated into the dermis.

Highly Branched Polymers with Polymyxin End Groups Responsive to Pseudomonas Aeruginosa

Biomacromolecules. Jan, 2011  |  Pubmed ID: 21126084

Polymyxin peptide conjugated to the end groups of highly branched poly(N-isopropyl acrylamide) was shown to bind to a Gram negative bacterium, Pseudomonas aeruginosa . The nonbound polymer had a lower critical solution temperature (LCST) above 60 °C. However, binding caused aggregation, which was disrupted on cooling of the bacteria and polymer mixture. The data indicate that polymer binding of bacteria occurred by interaction of the end groups with lipopolysaccharide and that the binding decreased the LCST to below 37 °C. Cooling then progressed the polymer/bacteria aggregate through a bound LCST into an open polymer coil conformation that was not adhesive to P. aeruginosa .

Corneal Epithelialisation on Surface-modified Hydrogel Implants: Artificial Cornea

Journal of Materials Science. Materials in Medicine. Mar, 2011  |  Pubmed ID: 21287242

The objective was to investigate corneal re-epithelialisation of surface-modified polymethacrylate hydrogel implants in order to evaluate them as potential materials for an artificial cornea. Polymethacrylate hydrogels were modified with amines and then coated with different extracellular matrix proteins (collagen I, IV, laminin and fibronectin). The modified hydrogels were surgically implanted into bovine corneas maintained in a 3-D culture system for 5 days. The epithelial growth across the implant surface was evaluated using fluorescent, light and electron microscopy. Full epithelialisation was achieved on 1,4-diaminobutane-modified hydrogels after coating with collagen IV. Hydrogels modified with 1,4-diaminobutane but without further coating only showed partial re-epithelialisation. Hydrogels modified with other amines (1,2-diaminoethane or 1,3-diaminopropane) showed only partial re-epithelialisation; further coating with extracellular matrix proteins improved epithelialisation of these surfaces but did not result in complete re-epithelialisation. Evaluation of the corneas implanted with the 1,4-diaminobutane-modified hydrogels coated with collagen IV showed that the artificial corneas remain clear, integrate well and become covered by a healthy stratified epithelium. In conclusion the 1,4-diaminobutane surface-modified hydrogel coated with collagen IV supported the growth of a stable stratified epithelium. With further refinement this hydrogel has the potential to be used clinically for an artificial cornea.

State of the Art in Non-invasive Imaging of Cutaneous Melanoma

Skin Research and Technology : Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). Feb, 2011  |  Pubmed ID: 21342292

Background: This review focuses on looking at recent developments in the non-invasive imaging of skin, in particular at how such imaging may be used at present or in the future to detect cutaneous melanoma. Methods: A MEDLINE search was performed for papers using imaging techniques to evaluate cutaneous melanoma, including melanoma metastasis. Results: Nine different techniques were found: dermoscopy, confocal laser scanning microscopy (including multiphoton microscopy), optical coherence tomography, high frequency ultrasound, positron emission tomography, magnetic resonance imaging, and Fourier, Raman, and photoacoustic spectroscopies. This review contrasts the effectiveness of these techniques when seeking to image melanomas in skin. Conclusions: Despite the variety of techniques available for detecting melanoma, there remains a critical need for a high-resolution technique to answer the question of whether tumours have invaded through the basement membrane.

Biocompatible Hydrogels Based on Hyaluronic Acid Cross-linked with a Polyaspartamide Derivative As Delivery Systems for Epithelial Limbal Cells

International Journal of Pharmaceutics. Jul, 2011  |  Pubmed ID: 21596121

The aim of this work was to evaluate the potential use of hydrogels based on hyaluronic acid (HA) chemically cross-linked with α,β-poly(N-2-hydroxyethyl) (2-aminoethylcarbamate)-D,L-aspartamide (PHEA-EDA) as substitutes for the amniotic membrane able to release limbal cells for corneal regeneration. Hydrogels, shaped as films, with three different molar ratios (X) between PHEA-EDA and HA (X = 0.5, 1.0 and 1.5) have been investigated. First, it has been evaluated their swelling ability, hydrolytic resistance in simulated physiological fluid and cell compatibility by using human dermal fibroblasts chosen as a model cell line. Then adhesion studies in comparison with collagen gel, have been performed by using immortalized cells, such as human corneal epithelial cells (HCEC) or primary cells, such as rabbit limbal epithelial cells (RLEC) and/or rabbit limbal fibroblasts (RLF). HA/PHEA-EDA hydrogels allow a moderate/poor adhesion of all investigated cells thus suggesting their potential ability to act as cell delivery systems. Finally, commercial contact lenses have been coated, in their inner surface, with each HA/PHEA-EDA film and it has been found that in these conditions, a greater cell adhesion occurs, particularly when RLEC are in co-culture with RLF. However, this adhesion is only transitory, in fact after three days, viable cells are released in the culture medium thus suggesting a potential application of HA/PHEA-EDA hydrogels, for delivering limbal cells in the treatment of corneal damage.

NF-κB is Activated in Oesophageal Fibroblasts in Response to a Paracrine Signal Generated by Acid-exposed Primary Oesophageal Squamous Cells

International Journal of Experimental Pathology. Oct, 2011  |  Pubmed ID: 21668534

Oesophageal exposure to duodenogastro-oesophageal refluxate leads to reflux oesophagitis and is implicated in the development of Barrett's metaplasia (BM). NF-κB signalling in epithelial cells is associated with the activation of transcription factors believed to be central to BM development, whilst NF-κB activation in fibroblasts plays a critical role in matrix remodelling. Our aim was to study the effects of acid exposure on NF-κB activation in primary human oesophageal fibroblasts (HOFs) and primary and immortalized oesophageal squames and to investigate any epithelial/stromal interactions in the response of these cells to acid. Primary HOFs and primary and immortalized oesophageal epithelial cells were exposed to acid (pH 7 - pH 4 ≤ 120 min) in single or pulsed treatments. Conditioned medium from epithelial cells following acid exposure was also applied to fibroblasts. Cell viability was determined by MTT-ESTA. NF-κB activation was determined by cellular localization of NF-κB/p65 visualized by immunofluorescence. Conditioned medium from oesophageal epithelial cells, subjected to pH 5 pulsatile exposure, activated NF-κB in fibroblasts, with some inter-patient variability, but these conditions did not directly activate NF-κB in the epithelial cells themselves. Significant NF-κB activation was seen in the epithelial cells but only with greater acidity and exposure times (pH 4, 60-120 min). Our findings show that acid exposure can cause indirect activation of stromal cells by epithelial-stromal interactions. This may contribute to the pathogenesis of oesophageal diseases, and the inter-patient variability may go some way to explain why some patients with reflux oesophagitis develop BM and others do not.

Methods to Reduce the Contraction of Tissue-engineered Buccal Mucosa for Use in Substitution Urethroplasty

European Urology. Oct, 2011  |  Pubmed ID: 21803482

We previously described the production and clinical outcomes of tissue-engineered buccal mucosa (TEBM) used to treat recurrent urethral strictures. In this study, two patients developed a recurrent stricture and there was also evidence of graft contraction.

Gadolinium Contrast Agent Associated Stimulation of Human Fibroblast Collagen Production

Investigative Radiology. Nov, 2011  |  Pubmed ID: 21826007

Nephrogenic systemic fibrosis occurs in patients with poor renal function who receive gadolinium-based contrast agents (Gd-CAs). Several reports suggest that this is more likely to occur with the less stable forms of Gd chelates, suggesting a release of cytotoxic free Gd ions from these. There is evidence that Gd can stimulate human fibroblast proliferation but the evidence is less clear concerning the production of collagen by these cells. Our aim was to assess effects of Gd chelates on human skin cell activity and collagen production.

Hyperbranched Poly(NIPAM) Polymers Modified with Antibiotics for the Reduction of Bacterial Burden in Infected Human Tissue Engineered Skin

Biomaterials. Jan, 2011  |  Pubmed ID: 20933276

The escalating global incidence of bacterial infection, particularly in chronic wounds, is a problem that requires significant improvements to existing therapies. We have developed hyperbranched poly(NIPAM) polymers functionalized with the antibiotics Vancomycin and Polymyxin-B that are sensitive to the presence of bacteria in solution. Binding of bacteria to the polymers causes a conformational change, resulting in collapse of the polymers and the formation of insoluble polymer/bacteria complexes. We have applied these novel polymers to our tissue engineered human skin model of a burn wound infected with Pseudomonas aeruginosa and Staphylococcus aureus. When the polymers were removed from the infected skin, either in a polymer gel solution or in the form of hydrogel membranes, they removed bound bacteria, thus reducing the bacterial load in the infected skin model. These bacteria-binding polymers have many potential uses, including coatings for wound dressings.

New Insights into Induction of Early-stage Neovascularization in an Improved Tissue-engineered Model of Psoriasis

Journal of Tissue Engineering and Regenerative Medicine. May, 2011  |  Pubmed ID: 20718049

We have previously shown that putrescine induces a psoriatic phenotype in tissue-engineered skin. The initial aim of this study was to further develop this in vitro model by introducing endothelial cells to mimic the increased vascularization found in psoriasis. Human keratinocytes and fibroblasts, which did not express CD34 or CD31 in 2D culture, were added to de-epidermised acellular human dermis and cultured for 4 weeks. For induction of a psoriatic phenotype, putrescine was added during this period. We report that after 4 weeks of culture, and particularly when exposed to putrescine, this model showed expression of vertically organised clusters of CD31 positive cells in the dermis in the absence of any exogenous endothelial cells. Further investigation in 2D cell cultures showed an indirect effect of putrescine on normal keratinocytes causing them to produce soluble factors that increased expression of CD133, CD34 and CD31 in cultured human dermal fibroblasts, previously negative for these antigens. This study reports a new and improved model of psoriasis for in vitro studies and offers a new insight into early stage neovascularization, which is of relevance not only to psoriasis, but to tissue engineering and wound healing in general.

Developing Biodegradable Scaffolds for Tissue Engineering of the Urethra

BJU International. Jan, 2011  |  Pubmed ID: 20477828

To develop a synthetic biodegradable alternative to using human allodermis for the production of tissue-engineered buccal mucosa for substitution urethroplasty, looking specifically at issues of sterilization and cell-seeding protocols and, comparing the results to native buccal mucosa

A Chemically Defined Carrier for the Delivery of Human Mesenchymal Stem/stromal Cells to Skin Wounds

Tissue Engineering. Part C, Methods. Feb, 2012  |  Pubmed ID: 21943098

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.

Are Biomechanical Properties Predictive of the Success of Prostheses Used in Stress Urinary Incontinence and Pelvic Organ Prolapse? A Systematic Review

Neurourology and Urodynamics. Jan, 2012  |  Pubmed ID: 22038890

Progressive weakness in pelvic floor tissues is extremely common and leads to the distressing problems of stress urinary incontinence (SUI) and pelvic organ prolapse (POP). There has been extensive work on a vast array of materials spanning synthetics, autografts, allografts, and xenografts. Uniaxial testing of materials has been used to predict their success and rates of erosion. We aimed to compare the uniaxial properties of prostheses to native paravaginal tissue and correlate these to their success and erosion rates.

Modelling Complex Biological Systems Using an Agent-based Approach

Integrative Biology : Quantitative Biosciences from Nano to Macro. Jan, 2012  |  Pubmed ID: 22052476

Many of the complex systems found in biology are comprised of numerous components, where interactions between individual agents result in the emergence of structures and function, typically in a highly dynamic manner. Often these entities have limited lifetimes but their interactions both with each other and their environment can have profound biological consequences. We will demonstrate how modelling these entities, and their interactions, can lead to a new approach to experimental biology bringing new insights and a deeper understanding of biological systems.

Simple Limbal Epithelial Transplantation (SLET): a Novel Surgical Technique for the Treatment of Unilateral Limbal Stem Cell Deficiency

The British Journal of Ophthalmology. Jul, 2012  |  Pubmed ID: 22328817

This study describes a novel surgical technique of limbal transplantation, which combines the benefits of existing techniques while avoiding their difficulties. Six patients with unilateral and total limbal stem cell deficiency following ocular surface burns underwent a single-stage procedure. A 2×2 mm strip of donor limbal tissue was obtained from the healthy eye and divided into eight to ten small pieces. After surgical preparation of the recipient ocular surface, these tiny limbal transplants were distributed evenly over an amniotic membrane placed on the cornea. After surgery, a completely epithelialised, avascular and stable corneal surface was seen in all recipient eyes by 6 weeks, and this was maintained at a mean±SD follow-up of 9.2±1.9 months. Visual acuity improved from worse than 20/200 in all recipient eyes before surgery to 20/60 or better in four (66.6%) eyes, while none of the donor eyes developed any complications. This technique requires less donor tissue than previously used for conventional autografting and does not need a specialist laboratory for cell expansion. Although long-term results are awaited, this simple limbal epithelial transplantation promises to be an easy and effective technique for treating unilateral limbal stem cell deficiency following ocular burns.

Transdermal Drug Delivery: from Micro to Nano

Nanoscale. Mar, 2012  |  Pubmed ID: 22334401

Delivery across skin offers many advantages compared to oral or intravenous routes of drug administration. Skin however is highly impermeable to most molecules on the basis of size, hydrophilicity, lipophilicity and charge. For this reason it is often necessary to temporarily alter the barrier properties of skin for effective administration. This can be done by applying chemical enhancers, which alter the lipid structure of the top layer of skin (the stratum corneum, SC), by applying external forces such as electric currents and ultrasounds, by bypassing the stratum corneum via minimally invasive microneedles or by using nano-delivery vehicles that can cross and deliver their payload to the deeper layers of skin. Here we present a critical summary of the latest technologies used to increase transdermal delivery.

Mesh Social Networking: a Patient-driven Process

BJU International. Jun, 2012  |  Pubmed ID: 22612339

Arginine Functionalization of Hydrogels for Heparin Binding-a Supramolecular Approach to Developing a Pro-angiogenic Biomaterial

Biotechnology and Bioengineering. Jul, 2012  |  Pubmed ID: 22753043

Our aim was to synthesize a biomaterial that stimulates angiogenesis for tissue engineering applications by exploiting the ability of heparin to bind and release vascular endothelial growth factor (VEGF). The approach adopted involved modification of a hydrogel with positively charged peptides (oligolysine or oligoarginine) to achieve heparin binding. Precursor hydrogels were produced from copolymerization of N-vinyl pyrolidone, diethylene glycol bis allyl carbonate and acrylic acid (PNDA) and functionalized after activation of the carboxylic acid groups with trilysine or triarginine peptides (PNDKKK and PNDRRR). Both hydrogels were shown to bind and release bioactive VEGF165 with arginine-modified hydrogel outperforming the lysine-modified hydrogel. Cytocompatibility of the hydrogels was confirmed in vitro with primary human dermal fibroblasts and human dermal microvascular endothelial cells (HUDMECs). Proliferation of HUDMECs was stimulated by triarginine-functionalized hydrogels, and to a lesser extent by lysine functionalized hydrogels once loaded with heparin and VEGF. The data suggests that heparin-binding hydrogels provide a promising approach to a pro-angiogenic biomaterial. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.