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Aims\ud \ud There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research.\ud \ud \ud \ud Methods\ud \ud Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air–liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins.\ud \ud \ud \ud Results\ud \ud Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17β [E2] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E2 concentrations (100–400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression.\ud \ud \ud \ud Conclusion\ud \ud In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT.

INTRODUCTION AND OBJECTIVES Soft tissue interposition (STI) using local and/or regional flaps is often necessary in urogenital reconstruction to stimulate wound healing and prevent recurrence. Harvesting STI flaps can cause donor site morbidity and may not be available in some patients. In this study, we designed estradiol (E2) releasing hydrogel that could be used as an alternative to a STI flap and to investigate its ability to stimulate tissue production and angiogenesis. MATERIALS AND METHODS A hydrogel was constructed by crosslinking a solution of estradiol, methacrylated gelatin (15%, w/v), and methacrylated hyaluronic acid (1%, w/v). The release of estradiol was measured using a UV-spectrophotometer (λmax = 220 nm). Angiogenesis was evaluated by an ex ovo chicken embryo chorioallantoic membrane (CAM) assay. RESULTS Estradiol was gradually released from the hydrogel over 21 days. The hydrogels could be easily manipulated with surgical forceps without any deformation. The hydrogels significantly increased collagen production of human dermal fibroblasts (HDFs). Scanning electron microscopic examination demonstrated that HDFs produced significantly more extracellular matrix (ECM) on estradiol releasing hydrogels compared with the controls. Estradiol releasing hydrogels doubled the number of blood vessels growing toward the hydrogel compared with the controls (vasculogenic index, 59.6 [±6.4] and 25.6 [±4.0], respectively; [P

The absorption capacity of cotton dressings is a critical factor in their widespread use where they help absorb wound exudate. Cotton wax dressings, in contrast, are used for wounds where care is taken to avoid adhesion of dressings to sensitive wounds such as burn injuries. Accordingly, we explored the loading of 2‐deoxy‐D‐ribose (2dDR), a small sugar, which stimulates angiogenesis and wound healing in normal and diabetic rats, into both types of dressings and measured the release of it over several days. The results showed that approximately 90% of 2dDR was released between 3 and 5 days when loaded into cotton dressings. For wax‐coated cotton dressings, several methods of loading of 2dDR were explored. A strategy similar to the commercial wax coating methodology was found the best protocol which provided a sustained release over 5 days.\ud \ud Cytotoxicity analysis of 2dDR loaded cotton dressing showed that the dressing stimulated metabolic activity of fibroblasts over 7 days confirming the non‐toxic nature of this sugar‐loaded dressings. The results of the chick chorioallantoic membrane (CAM) assay demonstrated a strong angiogenic response to both 2dDR loaded cotton dressing and to 2dDR loaded cotton wax dressings. Both dressings were found to increase the number of newly formed blood vessels significantly when observed macroscopically and histologically.\ud \ud We conclude this study offers a simple approach to developing affordable wound dressings as both have the potential to be evaluated as pro‐active dressings to stimulate wound healing in wounds where management of exudate or prevention of adherence to the wounds are clinical requirements.

Degradable implant membranes designed to separate hard and soft tissues and to trigger the growth of underlying bone and with antimicrobial properties are needed for the management of bone defects, ridge augmentation and to facilitate dental implants. In this study, the composite scaffolds of poly(lactic acid) (PLA), poly(caprolactone) (PCL) blended with nano hydroxyapatite and cefixime‐β cyclodextrin (Cfx‐βCD) inclusion complexes were synthesized by electrospinning. The prepared electrospun fibrous membranes were characterized by scanning electron microscopy and Fourier transform infrared (FTIR) spectroscopy. Membranes were microporous with random fibers in the range of 0.2–0.37 µm. The data from FTIR spectral analysis helped to characterize the presence of PCL, PLA, Cfx, and βCD in the electrospun membranes. In addition, the mechanical properties (i.e., elastic modulus and tensile strength) of the scaffolds were investigated. The mechanical strength and suture retention ability of the membranes was comparable to that of skin grafts. Drug release assays confirmed the slow release of Cfx from the membranes in the presence of βCD and antimicrobial studies showed that the membranes possessed antibacterial properties. The interaction of cells with membranes was evaluated by culturing them with the mouse pre‐osteoblast cell line MC3T3 and assessment of bone formation was done using Alizarin Red Assay. Culturing MC3T3 cells on the scaffolds showed that cells attached and entered the membranes and increased in number over time. In summary, these membranes are flexible, strong, bactericidal and osteogenic, which are the ideal implant properties for dental and maxillofacial surgery.

Aims Pelvic organ prolapse and stress urinary incontinence affect 40-50% of postmenopausal women worldwide. Polypropylene meshes have been extensively used for the surgical intervention of these disorders; however, these meshes can lead to severe complications in some patients. The need for synthetic materials more suited for use in pelvic floor repair is widely accepted. This study aims to develop an electrospun 17-β-estradiol releasing polyurethane (PU) scaffold that not only provides the appropriate mechanical support but can also stimulate new extracellular matrix (ECM) production and angiogenesis. Methods PU scaffolds with and without 17-β-estradiol (25 and 50 mg/g) were prepared by blend electrospinning. Mechanical properties of scaffolds were assessed by uniaxial cyclic and non-cyclic testing. The viability and ECM production of human adipose derived mesenchymal stem cells (hADMSCs) cultured on 17-β-estradiol releasing PU scaffolds was evaluated. Angiogenic potential of estradiol releasing scaffolds was demonstrated by using an ex ovo chick chorioallantoic membrane (CAM) assay. Results The inclusion of estradiol in PU scaffolds did not change the ultrastructure but it significantly increased the ultimate tensile strength of scaffolds. hADMSCs on estradiol-releasing PU scaffolds showed more ECM production. The CAM assay revealed a significantly higher angiogenic potential of estradiol-releasing PU scaffolds with an additive effect seen when hADMSCs cultured on estradiol scaffolds. Histological examination of CAM tissue sections showed extensive cellular infiltration and a good tissue integration for all constructed scaffolds. Conclusions This study shows the angiogenic potential of estradiol-releasing PU scaffolds with appropriate strength and elasticity desirable to support the pelvic floor.

Synthetic or biological materials can be used for the surgical repair of pelvic organ prolapse (POP) or stress urinary incontinence (SUI). While non-degradable synthetic mesh has a low failure rate, it is prone to complications such as infection and erosion, particularly in the urological/gynecological setting when subject to chronic influences of gravity and intermittent, repetitive strain. Biological materials have lower complication rates, although allografts and xenografts have a high risk of failure and the theoretical risk of infection. Autografts are used successfully for the treatment of SUI and are not associated with erosion; however, can lead to morbidity at the donor site. Tissue engineering has thus become the focus of interest in recent years as researchers seek an ideal tissue remodeling material for urogynecological repair. Herein, we review the directions of current and future research in this exciting field. Electrospun poly-L-lactic acid (PLA) and porcine small intestine submucosa (SIS) are two promising scaffold material candidates. Adipose-derived stem cells (ADSCs) appear to be a suitable cell type for scaffold seeding, and cells grown on scaffolds when subjected to repetitive biaxial strain show more appropriate biomechanical properties for clinical implantation. After implantation, an appropriate level of acute inflammation is important to precipitate moderate fibrosis and encourage tissue strength. New research directions include the use of bioactive materials containing compounds that may help facilitate integration of the new tissue. More research with longer follow-up is needed to ascertain the most successful and safe methods and materials for pelvic organ repair and SUI treatment.

Oesophageal exposure to duodenogastroesophageal refluxate is implicated in the development of Barrett's metaplasia (BM), with increased risk of progression to oesophageal adenocarcinoma. The literature proposes that reflux exposure activates NF-κB, driving the aberrant expression of intestine-specific caudal-related homeobox (CDX) genes. However, early events in the pathogenesis of BM from normal epithelium are poorly understood. To investigate this, our study subjected a 3D model of the normal human oesophageal mucosa to repeated, pulsatile exposure to specific bile components and examined changes in gene expression. Initial 2D experiments with a range of bile salts observed that taurochenodeoxycholate (TCDC) impacted upon NF-κB activation without causing cell death. Informed by this, the 3D oesophageal model was repeatedly exposed to TCDC in the presence and absence of acid, and the epithelial cells underwent gene expression profiling. We identified ~300 differentially expressed genes following each treatment, with a large and significant overlap between treatments. Enrichment analysis (Broad GSEA, DAVID and Metacore™; GeneGo Inc) identified multiple gene sets related to cell signalling, inflammation, proliferation, differentiation and cell adhesion. Specifically NF-κB activation, Wnt signalling, cell adhesion and targets for the transcription factors PTF1A and HNF4α were highlighted. Our data suggest that HNF4α isoform switching may be an early event in Barrett's pathogenesis. CDX1/2 targets were, however, not enriched, suggesting that although CDX1/2 activation reportedly plays a role in BM development, it may not be an initial event. Our findings highlight new areas for investigation in the earliest stages of BM pathogenesis of oesophageal diseases and new potential therapeutic targets.

Objectives/Hypothesis Effective treatments for hollow organ stenosis, scarring, or agenesis are suboptimal or lacking. Tissue-engineered implants may provide a solution, but those performed to date are limited by poor mucosalization after transplantation. We aimed to perform a systematic review of the literature on tissue-engineered airway mucosa. Our objectives were to assess the success of this technology and its potential application to airway regenerative medicine and to determine the direction of future research to maximize its therapeutic and commercial potential. Data Sources and Review Methods A systematic review of the literature was performed searching Medline (January 1996) and Embase (January 1980) using search terms “tissue engineering” or “tissue” and “engineering” or “tissue engineered” and “mucous membrane” or “mucous” and “membrane” or “mucosa.” Original studies utilizing tissue engineering to regenerate airway mucosa within the trachea or the main bronchi in animal models or human studies were included. Results A total of 719 papers matched the search criteria, with 17 fulfilling the entry criteria. Of these 17, four investigated mucosal engineering in humans, with the remaining 13 studies investigating mucosal engineering in animal models. The review demonstrated how an intact mucosal layer protects against infection and suggests a role for fibroblasts in facilitating epithelial regeneration in vitro. A range of scaffold materials were used, but no single material was clearly superior to the others. Conclusion The review highlights gaps in the literature and recommends key directions for future research such as epithelial tracking and the role of the extracellular environment. Laryngoscope, 124:961–968, 2014

Aims: Synthetic non-absorbable meshes are widely used to augment surgical repair of stress urinary incontinence (SUI) and pelvic organ prolapse (POP); however, there is growing concern such meshes are associated with serious complications. This study compares the potential of two autologous cell sources for attachment and extra-cellular matrix (ECM) production on a biodegradable scaffold to develop tissue engineered repair material (TERM). Methods: Human oral fibroblasts (OF) and human adipose-derived stem cells (ADSC) were isolated and cultured on thermo-annealed polyL-lactic acid (PLA) scaffolds for two weeks under either unrestrained conditions or restrained (either with or without intermittent stress) conditions. Samples were tested for cell metabolic activity (AlamarBlue 1 assay), contraction (serial photographs analyzed with image J software), total collagen production (Sirius red assay), and production of ECM components (immunostaining for collagen I, III, and elastin; and scanning electron microscopy) and biomechanical properties (BOSE tensiometer). Differences were statistically tested using two sample t-test. Results: Both cells showed good attachment and proliferation on scaffolds. Unrestrained scaffolds with ADSC produced more total collagen and a denser homogenous ECM than OF under same conditions. Restrained conditions (both with and without intermittent stress) gave similar total collagen production, but improved elastin production for both cells, particularly OF. The addition of any cell onto scaffolds led to an increase in biomechanical properties of scaffolds compared to unseeded scaffolds. Conclusions: OF and ADSC both appear to be suitable cell types to combine with biodegradablescaffolds,in thedevelopmentofa TERMfor thetreatmentofSUI andPOP.Neurourol.Urodynam. 33:531– 537, 2014. # 2013 Wiley Periodicals, Inc.

Objectives To identify candidate materials which have sufficient potential to be taken forward for an in vivo tissue-engineering approach to restoring the tissue structure of the pelvic floor in women with stress urinary incontinence (SUI) or pelvic organ prolapse (POP). Materials and Methods Oral mucosal fibroblasts were seeded onto seven different scaffold materials, AlloDerm ( LifeCell Corp., Branchburg, NJ, USA), cadaveric dermis, porcine dermis, polypropylene, sheep forestomach, porcine small intestinal submucosa (SIS) and thermoannealed poly(L) lactic acid (PLA) under both free and restrained conditions. The scaffolds were assessed for: cell attachment using AlamarBlue and 4,6-diamidino-2phenylindole (DAPI); contraction using serial photographs; and extracellular matrix production using Sirius red staining, immunostaining and scanning electron microscopy. Finally the biomechanical properties of all the scaffolds were assessed. Results Of the seven, there were two biodegradable scaffolds, synthetic PLA and natural SIS, which supported good cell attachment and proliferation. Immunostaining confirmed the presence of collagen I, III and elastin which was highest in SIS and PLA. The mechanical properties of PLA were closest to native tissue with an ultimate tensile strength of 0.72 ± 0.18 MPa, ultimate tensile strain 0.53 ± 0.16 and Young's modulus 4.5 ± 2.9 MPa. Scaffold restraint did not have a significant impact on the above properties in the best scaffolds. Conclusion These data support both PLA and SIS as good candidate materials for use in making a tissue-engineered repair material for SUI or POP.

Aim: The purpose of this study was to assess the effectiveness of combining freshly excised oral mucosa tissue with different biological and synthetic scaffolds for production of graft material for one-stage soft-tissue reconstruction. Material and Methods: Small oral mucosa biopsies were finely minced and transplanted onto a larger circular subcutaneous wound bed in rats. Test groups included explants seeded onto (1) an electrospun scaffold, (2) acellular dermis, and (3) directly on the muscle fascia. The soft-tissue implants were examined using histology and immunohistochemical analysis for cytokeratins (CKs) 5, 10, 13, and 19. Results: Mucosal explants grew into fully developed epithelium with keratinisation. Surprisingly, instead of spreading over the surface of the membranes, the epithelial explants curled up and formed cyst-like structures in all test groups. The epithelial layer of the cysts originated from mucosal explants grown on the electrospun scaffold showed weak positive staining for CK5, CK10, and CK19 similar to native rat oral mucosa. Other groups showed negative staining for the CKs. Conclusions: Subcutaneous transplantation of freshly excised oral mucosa explants resulted in the growth of the explants and formation of inclusion cyst-like structures. The phenotype of the cyst epithelium was affected by the underlying connective tissue substrate.

Aims 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. Methods We performed a systematic review of the literature to identify the biomechanical properties of paravaginal tissue and of prostheses used in the treatment of SUI and POP. We examined to what extent these findings correlated to their reported success and erosion rates. Results Biomechanical properties have been determined for prostheses used in the treatment of POP and SUI both pre- and post-implantation in animal models. Implantation generally led to a decrease in ultimate tensile strength and Young's modulus. We were unable to find any simple correlation between the uniaxial mechanical properties of materials and their success or erosion rates. Conclusions Mechanical properties of prostheses, as measured by uniaxial testing, only appears to form part of the picture. We suggest implant integration and host responses to materials, as well as the biomechanical properties of the materials need to be considered to design materials to achieve lasting clinical benefit. Neurourol. Urodynam. 31:13–21, 2012. © 2011 Wiley Periodicals, Inc.

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.

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.

What’s known on the subject? and What does the study add? The use of tissue engineered buccal mucosa in substitution urethroplasty removes some of the potential drawbacks of harvesting buccal mucosa however it introduces the risk of using donor tissue (allodermis) in its creation. Biocompatible biodegradable non-woven fabrics created by electrospinning can be used as entirely synthetic matrices for seeding with autologous cells, creating tissues for implantation. This would both remove the donor tissue disease transmission risk and reduce the potential risks of harvesting buccal mucosa. While removing the risks of donor tissue, we showed that we can indeed make a replacement tissue which has similar biomechanical properties to buccal mucosa. We also found that each processing step in the creation of such a tissue is critical, for example the initial sterilisation can have a profound effect on the tissue created. OBJECTIVE 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. MATERIAL AND METHODS Three methods of sterilization, peracetic acid (PAA), γ-irradiation and ethanol, were evaluated for their effects on a biodegradable electrospun scaffold of polylactide-co-glycolide (PLGA, 85 : 15), to identify a sterilization method with minimal adverse effects on the scaffolds. Two protocols for seeding oral cells on the scaffold were compared, co-culture of fibroblasts and keratinocytes on the scaffolds for 14 days, and seeding fibroblasts for 5 days then adding keratinocytes for a further 10 days. Cell viability and proliferation on the scaffolds, scaffold contraction and mechanical properties of the scaffolds with and without cells were examined. RESULTS γ-irradiation and PAA sterilized scaffolds remained sterile for >3 months when incubated in antibiotic-free culture medium, while ethanol sterilized and unsterilized samples became infected within 2–14 days. All scaffolds showed extensive contraction (up to 50% over 14 days) irrespective of the method of sterilization or the presence of cells. All methods of sterilization, particularly ethanol, reduced the tensile strength of the scaffolds. The addition of cells tended to further reduce mechanical properties but increased elasticity. The cell-seeding protocol of adding fibroblasts for 5 days followed by keratinocytes for 10 days was the most promising, achieving a mean (sem) ultimate tensile stress of 1.20 (0.24) × 105 N/m2 compared to 3.77 (1.05) × 105 N/m2 for native buccal mucosa, and a Young’s modulus of 2.40 (0.25) MPa, compared to 0.73 (0.09) MPa for the native buccal mucosa. CONCLUSION This study adds to our understanding of how sterilization and cell seeding affect the physical properties of scaffolds. Both PAA and γ-irradiation appear to be suitable methods for sterilizing PLGA scaffolds, although both reduce the tensile properties of the scaffolds. Cells grow well on the sterilized scaffolds, and with our current protocol produce constructs which have ≈30% of the mechanical strength and elasticity of the native buccal mucosa. We conclude that sterilized PLGA 85 : 15 is a promising material for producing tissue-engineered buccal mucosa.

Polymethacrylate hydrogels are useful scaffolds for tissue engineering and cell-based therapies provided they can be modified to give good cell adhesion characteristics. They have advantages over non-swollen materials, such as good diffusion properties, tunable moduli and porosities. However, conventional non-charged hydrogels are essentially non-adhesive for cells and they are poor substrates for cell culture. In a previous volume in this series of symposia from the Polymer Networks Group, we described how both amphiphilic conetworks and alkyl-aminated hydrogels were excellent substrates for cell culture.([1]) In this article we report our most recent work on understanding how to modify hydrogels for cell attachment. We report how the choice of photoinitiator affects the viability of human dermal fibroblasts (HDFs). Next, we provide further results involving the use of an enzyme deprotection strategy that can be carried out after copolymerization of a GRGDS methacrylate monomer. We show that the peptide enhanced the culture of HDFs in serum when the peptide was attached to poly(1,2-propane diol methacrylate-co-ethane diol dimethacrylate). On the other hand, poly(n-butyl methacrylate-co-ethane diol dimethacrylate) networks adsorb larger amounts of protein non-specifically, and they are reasonable substrates for adhesion of HDFs without peptide modification. Attachment of GRGDS to an example of these networks did not increase the attachment and proliferation of HDFs in serum containing media. We also describe the attachment of fibrinogen/fibrin coatings by the transglutaminase mediated reaction of alkyl amines attached to a poly(1,2-propane diol methacrylate-co-ethane diol dimethacrylate-co-dodecyl methacrylate) network. This work illustrates the reactivity of alkyl-aminated hydrogels in transglutaminase catalyzed reactions, although the addition of the fibrin coating did not improve the performance of the material for cell culture which was already good on these hydrogels.

Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal microscopy techniques. The results showed that using fluorescence microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence microscopy with Volocity and ImageJ.

The development of a facile method with general applicability and mild reaction conditions for grafting PEG onto surfaces to reduce bio-adhesion is described. The approach taken was to use CAN to graft PEG to plasma polymers coatings selected to give a high concentration of alcohol groups. The study showed that grafting required functional groups on surfaces, a minimum concentration of CAN initiator (0.05 M) and was time and initiator concentration dependent. Adhesion of fibroblasts and endothelial cells was reduced to negligible levels compared to the adhesion to tissue culture polystyrene and untreated plasma polymers of allyl alcohol following CAN induced PEG grafting.

Recently, the ability of natural phospholipids to self-assemble into organized membrane-enclosed structures has been mimicked by amphiphilic copolymers. These synthetic macromolecular amphiphiles form assemblies that are remarkably similar to biological analogues, such as vesicles, tubular myelins, and tissuelike structures. Polymeric amphiphiles have much higher molecular weights than phospholipids and can self-assemble into more entangled membranes, imparting improved mechanical properties to the final structure. Polymer vesicles or polymersomes can combine several different polymeric compositions provided that they have the correct hydrophile/hydrophobe ratio. Particularly interesting for biomedical applications are those copolymers that combine hydrophobic blocks with the nonfouling and nonantigenic properties of either poly(ethylene glycol) (PEG) or biomimetic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC). Indeed, the macromolecular nature of such polymersomes offers several advantages compared to low-molecular-mass amphiphilic systems such as liposomes. PEG or PMPC polymersomes can be decorated by denser and higher molecular mass hydrophilic polymeric corona, with consequent longer circulation times than more traditional delivery systems such as stealth liposomes and other nanoparticles. In addition, amphiphilic copolymers have critical aggregation concentrations (i.e., the minimum copolymer concentration necessary to form colloidal aggregates) that are very low and, in some cases, essentially zero. Therefore, polymeric amphiphiles have very slow chain exchange dynamics, implying that they assemble into locally isolated, nonergodic structures. This means that such copolymer assemblies have very slow rates of dissociation, allowing the retention of the payload for very long time periods. Further, the absence of molecularly dissolved amphiphilic copolymers in solution avoids cytotoxic interactions with biological phospholipid membranes. These can range from complete biomembrane dissolution and therefore cell death, in the case of small-molecule surfactants, to more benign effects such as up-regulating the gene expression and altering cell genetic responses for low-molecular-weight copolymers. Responsive formulations have also been created to exploit the sensitivity of specific hydrophobic polymers to external stimuli such as pH, oxidative species, and enzyme degradation. These properties have recently encouraged the application of polymer vesicles as delivery vectors for bioactive molecules. One of the most technically challenging hurdles is the intracellular delivery of genetic materials (DNA, RNA, and other nucleic acids). These species are used to treat diseases by modifying gene expression within specific cells. Gene therapy requires vectors that can deliver genetic materials within the cell without compromising its integrity. To date, the most efficient gene vectors have exploited the natural ability of viruses to transfect cells. However, viral vectors can produce adverse systemic immune responses, including patient fatalities. Moreover, without further genetic engineering, the targeting efficiency is limited to the specificity of the virus. In vitro alternatives to viruses are based on controlled cellular membrane permeabilization via electroporation, ultrasound poration, or particle bombing. In contrast, in vivo synthetic vectors such as cationic lipids, cationic polymers, inorganic particles, and polymer-inorganic particle hybrids have been designed to condense DNA via electrostatic interactions. Unfortunately, not only do these condensation methods have very low transfection efficiencies compared to viruses but, since the nucleic acid encapsulation is based on electrostatic complexation, highly charged plasma proteins can compete with the cationic vector for nucleic acid binding, potentially displacing it. This translates into very short plasma circulation times with rapid hepatic uptake. Recently, it has been observed that both lipids and PEGylated lipids are not able to protect nucleic acids from enzymatic degradation, either outside or inside the cell. Herein we present the use of biomimetic polymersomes for gene delivery. We have used pH-sensitive poly(2-(methacryloyloxy)ethyl-phosphorylcholine)-co-poly(2-(diisopropylamiC O M M U N IC A TI O N

methacrylate networks have a long history of applications in medical technology and much is known of their non-fouling properties. However, in recent times it has become clear that the swollen nature of these materials may provide some advantages if they are used as scaffolds in tissue engineering. In general however these hydrogels are resistant to protein adsorption and human cells do not easily adhere. in this work we provide an overview of several strategies that are designed to improve the cell-adhesive properties of hydrogels while maintaining their useful properties, mainly ease of diffusion of nutrients and growth factors. We describe our early attempts at modifying hydrogels based on 2,3-propandiol -1-methacrylate, with either hydrophobic units or acid groups. Modification with lauryl methacrylate produced an improvement but acid modification failed to provide surfaces that were conducive to cell culture. Much better scaffolds were prepared by amination of epoxy functional 2,3-propandiol-1-methacrylate networks. Optimized materials in this class were shown to be good substrates for the co-culture of bovine keratocytes with human corneal epithelial cells. We also describe the synthesis and biological properties of methacrylate conetworks, which phase separate during synthesis to give porous amphiphilic materials. Optimization of these materials produces materials that perform as well as tissue culture plastic so that confluent sheets of human dermal fibroblasts can be produced using standard culture techniques.

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. Biotechnol. Bioeng. 2007; 97: 1617–1625. © 2007 Wiley Periodicals, Inc.

This contribution chronicles the research history over the past ten years behind a cell therapy product for the treatment of chronic wounds. The product is a plasma coated bandage, which delivers autologous cells (keratinoctyes) to non-healing wound beds. The account given is chronological, starting with the initial investigations of cell (keratinocyte) attachment to a range of plasma polymer surfaces. It details the subsequent selection of acrylic acid plasma polymer surfaces, on which the growth, proliferation and transfer of keratinoctyes to an in vitro wound model are studied. It concludes with the translation of this technology from the laboratory to the clinic, describing the first "proof of concept" study undertaken with non-healing diabetic foot ulcers.

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.

Aims To evaluate the use of a new cell-tailored carrier surface (TranCell) for delivery of autologous keratinocytes to promote wound healing in patients with chronic neuropathic foot ulcers. Methods TranCell is a sterile medical grade polymer coated with a plasma-polymerized functional surface containing 20% carboxylic acid which enables keratinocytes to attach and proliferate. Six diabetic patients with neuropathic ulcers resistant to conventional therapy were treated with weekly applications of autologous keratinocytes delivered on TranCell. A split-thickness skin biopsy was taken from each patient followed by isolation, expansion and freezing down of keratinocytes. Keratinocytes were thawed and seeded on TranCell 48 h prior to application. This procedure was repeated weekly in addition to conventional therapy until wound healing was achieved. Results Complete healing was achieved in six out of nine ulcers in six patients, a reduction in ulcer size was achieved in one ulcer and no response was seen in one ulcer. Treatment was discontinued in one patient due to development of Methicillin-Resistant Staphylococcus aureus (MRSA) after only three applications of TranCell. Wound healing took 6–17 applications over 6–20 weeks. There were no recurrences in the healed ulcers after a follow-up of 6 months. Conclusions TranCell delivery of autologous cells is a promising treatment for chronic diabetic foot ulcers with no side-effects and no recurrence in the healed ulcers.

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.

The purpose of this study was to investigate the efficacy of organic ligands in cleaning human skin contaminated with nickel. 4 ligands were investigated, 5-chloro-7-iodoquinolin-8-ol (either as HL(1) or NaL(1)), ethylenediaminetetraacetic acid (either as H4L(2) or Na2H2L(2)), sodium diethyldithiocarbamate (NaL(3)) and L-histidine (HL(4)). The cytotoxicity of these ligands was assessed using HaCaT cells (a transformed human keratinocyte cell line). The cytotoxicity order of the ligands was NaL(1) >Na2H2L(2)>NaL(3)>HL(4). An in vitro methodology for examining nickel removal from viable human skin was developed. This methodology was then used to compare the efficiency of the ligands in removing nickel from skin, both alone and in combination with soap solutions. HL(1) and NaL(3) were no more effective than control solutions in removing nickel over the pH range 2-11. In contrast, both H4L(2) and HL(4) removed between 74 and 87% (mean=82+/-3%) of nickel from human skin over the same pH range. Nickel removal from skin by sodium lauryl ethoxy sulfate (SLES, the active ingredient in most liquid skin cleansers) was independent of concentration and no more effective than phosphate-buffered saline (PBS). The amount of nickel removed by PBS solutions of Na2H2L(2) and HL(4) was significantly greater than the amount removed by SLES and was concentration dependent. An evaluation of nickel removal from skin by commercial solid soap, liquid soap and PBS, both alone and with added Na2H2L(2) or HL(4), was conducted. Commercial liquid soap with added HL(4) was more effective than the untreated soap. PBS with either added Na2H2L(2) or HL(4) was more effective than PBS alone.

Alpha-MSH, a proopiomelanocortin (POMC)-derived peptide, is known to be produced in the pituitary, the skin, and melanoma tumors and to possess many biological effects, mainly on melanocyte pigmentation and growth. Moreover, the melanocyte expresses adhesion molecules, including ICAM-1. The latter has been reported to play a role in melanoma spread and associated metastatic process. We conducted a study in order to evaluate the possible effect of MSH on ICAM-1 expression in human cultured malignant and normal melanocytes. Our data show that alpha-MSH inhibits ICAM-1 expression stimulated by TNF in a concentration-dependent manner, both at the protein and gene expression level. Ninety percent inhibition was obtained with 10 nM MSH, while 50% inhibition was achieved with 1 nM. Endogenous cAMP elevation with forskolin as well as an exogenous cAMP stable analogue (Sp-cAMPS) produced the same inhibitory effect. A screening of malignant melanocytes showed that inhibition of ICAM-1 expression could be achieved only in those cells expressing detectable MSH receptors and seemed to correlate with the number of binding sites. In conclusion, our data strongly suggest alpha-MSH as a potent inhibitor of ICAM-1 expression in malignant melanocytes acting through MSH receptor stimulation and subsequent cAMP increase.

Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with alpha-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by alpha-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; alpha-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or alpha-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinase-related genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.

Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physiological regulation of melanocyte proliferation and differentiation is not yet fully defined and this study summarises several separate lines of evidence which suggest that, in vivo, some of the signals required for melanocyte proliferation and differentiation may derive from extracellular matrix (ECM) proteins adjacent to these cells. Culture of cutaneous and uveal melanocytes on cell-derived and individual ECM proteins was found to influence cell morphology with such effects being most noticeable in mitogen-deficient media. Similarly, cell-derived and individual ECM proteins increased tyrosinase activity in normal cutaneous melanocytes and effects of these ECM proteins were seen most consistently in mitogen-deficient media. Uveal melanocytes (as has been reported for cutaneous melanocytes) showed preferential attachment to fibronectin over other ECM substrates. This attachment was particularly sensitive to drugs which affected intracellular calcium or calmodulin activity. Acute addition of fibronectin to coverslips of uveal melanocytes loaded with Fura-2 produced an acute and transient increase in intracellular calcium which was more prevalent in low density than higher density cells. We conclude that ECM proteins in vitro are capable of influencing melanocyte morphology, tyrosinase activity, and proliferation and that an ECM-induced elevation in intracellular calcium may be part of the signalling system that transmits ECM information into the cell.

Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti-estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen-receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.

Bleeding complications in uraemia are not uncommon. The pathogenesis of haemorrhage in uraemia is still a matter of controversy and the pattern of bleeding suggests a defect of primary haemostasis. Platelet aggregation and biochemistry, including calcium levels, have been studied; however, the results are controversial. We have examined platelet aggregation, platelet-free calcium and calmodulin in platelet-rich plasma because of the significant role of calcium and calmodulin in regulating platelet and other cells' functions. Platelet aggregation in uraemic subjects was similar to that of controls. Platelet basal free cytosolic calcium and platelet calcium in response to 10 microM Ca++ ionophore A23187 in eight subjects with uraemia were 117 +/- 33 nM and 2025 +/- 398 nM (mean +/- SEM) respectively. By contrast in seven matched healthy controls basal calcium and ionophore-stimulated calcium values were 47 +/- 14 nM and 1354 +/- 414 nM, significantly less than in the patients with uraemia (P less than 0.05). The sensitivity of uraemic platelets to A23187 was similar to that of controls. Calmodulin activity in platelet-rich plasma of 12 subjects with uraemia showed no significant difference from that of controls [1.86 +/- 0.29 micrograms/ml (mean +/- SEM) and 2.0 +/- 0.37 micrograms/ml (mean +/- SEM) respectively]. We conclude that despite elevation of platelet calcium in uraemia, which may be due to a plasma factor such as parathyroid hormone, platelet aggregation is normal and bleeding in uraemia is more likely to be due to other factors, including the effect of reduced haematocrit on platelet endothelial interaction. Disturbances in platelet calcium cannot explain the bleeding manifestations in uraemia but warrant further investigation in order to identify the pathogenic mechanisms responsible.

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) release and vascular tone was measured in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution. The effects of 3 vasodilators, acetylcholine (ACh), atrial natriuretic factor (ANF) and sodium nitroprusside (SNP) on the renal release of cyclic GMP and vascular tone were examined. The ability of the endothelial-derived relaxing factor (EDRF) inhibitors, haemoglobin and gossypol, to modify vasodilatation and vasodilator-induced changes in cyclic GMP releases from the kidney was also investigated. 2. Renal cyclic GMP release was elevated 8 fold by ANF (0.01 microM), 5 fold by SNP (1 microM) and 3 fold by ACh (0.3 microM). 3. For ACh, both the increase in renal cyclic GMP release and the vasodilatation were reduced by the EDRF inhibitors, haemoglobin (1 microM) and gossypol (15 microM). For SNP, neither the increase in renal cyclic GMP release nor vasodilatation were inhibited by gossypol (15 microM). 4. For ANF, neither the increase in cyclic GMP release from the kidney nor its vasodilator activity were affected by haemoglobin (1 microM). 5. EDRF inhibitors reduced the basal release of cyclic GMP from 0.32 +/- 0.06 pmol min-1 to 0.18 +/- 0.03 pmol min-1, gossypol being more effective than haemoglobin. 6. The results are consistent with the ability of ACh to induce EDRF-mediated vasodilatation in the isolated perfused kidney of the rat. Basal EDRF release appears to contribute approximately 50% to the basal release of cyclic GMP from this preparation. The renal vasodilator action of ANF however, is independent of EDRF, although the renal vascular endothelium cannot be discounted as a site at which ANF stimulates cyclic GMP production.

α-Melanocyte-stimulating hormone (α-MSH) has previously been identified as a potent anti-inflammatory agent in various tissues including the skin. It operates by binding to the melanocortin-1 receptor (MC-1R) which results in the elevation of cyclic AMP. α-MSH opposes the action of several proinflammatory cytokines including tumour necrosis factor-α (TNF-α). We have shown that α-MSH can inhibit TNF-α-stimulated activation of nuclear factor-κB (NF-κB) in human cultured melanocytes, melanoma cells, keratinocytes, fibroblasts, Schwann cells and olfactory ensheathing cells. It also inhibits TNF-α-stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in many of these cells and can inhibit peroxide-stimulated activation of glutathione peroxidase, suggesting an antioxidant role. α-MSH is also able to stimulate intracellular calcium release in keratinocytes and fibroblasts (which do not readily show detectible cyclic AMP elevation) but only in the presence of PIA (an adenosine agonist). The carboxyl terminal tripeptides KPV/KP-D-V are reported to be the minimal sequences necessary to convey anti-inflammatory potential, but evidence on how they act is not fully known. Stable transfection of Chinese hamster ovary cells with MC-1R suggests that the KPV peptides operate by this receptor, at least by elevating intracellular calcium. Elevation of cyclic AMP by these tripeptides has not been detected in any cell type studied; however, calcium elevation can inhibit TNF-α-stimulated NF-κB activity (as for cyclic AMP). In conclusion, the MSH peptides convey anti-inflammatory and antioxidant activity in many cell types in skin and nerve, by counteracting proinflammatory cytokine signalling. The KPV peptides appear to act functionally via the MC-1R and can also elevate intracellular calcium.