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AimsTo report the global uptake of simple limbal epithelial transplantation (SLET) and compare the economic, clinical and social outcomes of SLET with those of cultured limbal epithelial transplantation (CLET).MethodsA comprehensive literature review and an online survey of eye surgeons were conducted to understand the efficacy and current uptake of SLET surgery. A de novo economic model was developed to estimate the cost savings with SLET compared with CLET. Our economic analysis is conducted from an Indian perspective, as this is where the technique originated. A scenario analysis using the UK cost data and a user-friendly Excel model is included to allow users to input the costs from their setting to estimate the cost savings with using SLET compared with using CLETResultsThe anatomical success with SLET in adults (72.6% (range 62%–80%)) was the same as CLET (70.4% (range 68%–80.9%)). For children, the outcome for SLET (77.8% (range 73%–83%)) was better than with CLET (44.5% (range 43%–45%)). In response to our informal questionnaire, 99 surgeons reported to have performed SLET on 1174 patients in total. They appreciated that SLET negates the requirement for costly tissue engineering facilities. Results of economic analysis suggested that SLET provided an estimated cost-savings of US$6470.88 for adults and US$6673.10 for children. In broad terms, the cost of SLET is approximately 10% of the cost of CLET for adults and 8% for children.ConclusionSLET offers a more accessible and financially attractive alternative to CLET to treat limbal stem cell deficiency.

PURPOSE OF REVIEW To review the current clinical management of stress urinary incontinence and pelvic organ prolapse following the adverse complications seen in the use of polypropylene mesh to treat both. RECENT FINDINGS Materials developed for use in abdominal hernia repair have not proven risk-free when used to support pelvic organs particularly when inserted via the vagina. Following unacceptably high levels of severe complications when high-density polypropylene mesh is inserted via the vagina to treat pelvic organ prolapse, reported over the last decade, there is now an agreed consensus between surgeons about surgical approaches and materials, which should be recommended for use in stress urinary incontinence and pelvic organ prolapse. SUMMARY There is a need for new biomaterials and tissue engineered/regenerative medicine approaches to treat stress urinary incontinence and pelvic organ prolapse. New materials need to be evaluated critically in both preclinical and clinical studies before being adopted into routine clinical use.

Biomaterials need to be vigorously tested at every stage of preclinical development. As demand for in vivo culture environments continues to increase, traditional animal models are often technically complex, ethically undesirable, time-consuming, and resource intensive and thus present a barrier to high throughput screening. The chick chorioallantoic membrane (CAM) assay has long been used to study the effects of drugs on angiogenesis in vivo, providing researchers with a readily available, accessible, self-sustaining, and high throughput screen without requiring animal facilities. It has also been recognized as an in vivo assay to test initial tissue response to biomaterials; however it has not yet gained widespread acceptance. This could be due to lack of specific protocols on how to optimize this assay to specifically test biomaterials. Here we describe how the ex ovo (shell-less) CAM assay can be effectively used to study the angiogenic potential and initial tissue response to biomaterials. In comparison to alternative in vivo approaches, this technique provides additional advantages to the researcher as it allows better visualization of implanted biomaterials and the ability to implant several samples simultaneously enabling combinatorial biomaterial assays to be conducted.

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

Background Polypropylene meshes cause severe complications in some patients. Materials that are biomechanically compatible and can better integrate into host tissues are urgently needed. Objective To design an oestradiol-releasing electrospun poly- l -lactic acid (PLA) mesh and evaluate its ability to stimulate new extracellular matrix and new blood vessel formation. Design, setting, and participants Human adipose derived mesenchymal cells (ADMSC) were isolated from fat. PLA meshes with micro- to nano-sized fibres containing 1%, 5%, and 10% oestradiol were constructed and used for in vitro and in vivo experiments. Intervention The angiogenic potential of the fibrous meshes was evaluated using an in vivo chorioallantoic membrane and an in vitro chick aortic arch assays. Outcome measurements and statistical analysis Oestradiol release was measured fluorometrically. The effect of fibrous meshes on proliferation and extracellular matrix (ECM) production of ADMSC was assessed using immunohistology. Mechanical properties were tested using a tensiometer. Results and limitations The ultrastructure of the mesh was not affected by the inclusion of oestradiol and mechanical properties were only slightly modified. Oestradiol was released from PLA meshes over a 5-mo period. ADMSCs cultured on oestradiol-releasing PLA meshes produced more ECM involving collagen I, collagen III, and elastin. Oestradiol-releasing meshes doubled new blood vessel formation in the chorioallantoic membrane assay ( p = 0.001) and outgrowth of pro-angiogenic cells in the aortic arch assay ( p = 0.001). Further studies in longer-term animal models are required to confirm these results. Conclusions Oestradiol-releasing PLA meshes increase ECM production and stimulate angiogenesis. As such, they are promising candidate materials to be used in pelvic floor repair and to improve the initial healing phase of a repair material following implantation. Patient summary In this study, we designed a tissue engineered material to be used to support weakened pelvic floor tissues in women to avoid the complications associated with current surgical mesh. Our results showed that this material can stimulate new blood vessel formation in simple chick assays and tissue production in vitro. Both properties should help with the integration of this material into patients’ tissues and merit further study in physiologically relevant animal models.

The surgical mesh material used in the surgical treatment of stress urinary incontinence (SUI) and pelvic organ prolapse (POP) in women is associated with significant complications in some women. This has recently become a public health issue with involvement of national parliaments and regulatory bodies. The occurrence of mesh complications is thought to be a result of multifactorial processes involving problems related to the material design, the surgical techniques used and disease, and patient-related factors. However, the infectious complications and mesh-tissue interactions are least studied. The aim of this article is to review any previous clinical and basic scientific evidence about the contribution of infectious and inflammatory processes to the occurrence of mesh-related complications in SUI and POP. A literature search for the relevant publications without any time limits was performed on the Medline database. There is evidence to show that vaginal meshes are associated with an unfavourable host response at the site of implantation. The underlying mechanisms leading to this type of host response is not completely clear. Mesh contamination with vaginal flora during surgical implantation can be a factor modifying the host response if there is a subclinical infection that can trigger a sustained inflammation. More basic science research is required to identify the biological mechanisms causing a sustained inflammation at the mesh-tissue interface that can then lead to contraction, mesh erosion, and pain.

ObjectiveThe aim of this study was to assess the safety of poly-lactic co-glycolic acid (PLGA) electrospun membranes as carriers for limbal tissue explants for treatment of limbal stem cell deficiency (LSCD).Methods and analysisApproval was obtained for a first in-man study from the Drug Controller General of India. PLGA membranes were applied to the affected eye of five patients after removal of the vascular pannus. Simple limbal epithelial transplantation was performed and limbal explants were secured on the membrane using fibrin glue followed by a bandage contact lens. Patients were followed up for 1 year with ocular exams including slit lamp exam, corneal thickness measurements, intraocular pressure measurements and recording of corneal vascularisation and visual acuity. Systemic examinations included pain grading, clinical laboratory assessment, blood chemistry and urine analysis at baseline, 3 and 6 months after surgery.ResultsPLGA membranes completely degraded by 8 weeks post-transplantation without any infection or inflammation. In all five patients, the epithelium regenerated by 3 months. In two in five patients, there was a sustained two-line improvement in vision. In one in five patients, the vision improvement was limited due to an underlying stromal scarring. There was recurrence of pannus and LSCD in two in five patients 6 months after surgery which was not attributable to the membrane. The ocular surface remained clear with no epithelial defects in three in five subjects at 12 months.ConclusionPLGA electrospun membranes show promise as carrier for limbal epithelial cells in the treatment of LSCD.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

Background There is an urgent need to develop better materials to provide anatomical support to the pelvic floor without compromising its function. Objective Our aim was to assess outcomes after simulated vaginal prolapse repair in a sheep model using three different materials: (1) ultra-lightweight polypropylene (PP) non-degradable textile (Restorelle) mesh, (2) electrospun biodegradable ureidopyrimidinone-polycarbonate (UPy-PC), and (3) electrospun non-degradable polyurethane (PU) mesh in comparison with simulated native tissue repair (NTR). These implants may reduce implant-related complications and avoid vaginal function loss. Design, setting, and participants A controlled trial was performed involving 48 ewes that underwent NTR or mesh repair with PP, UPy-PC, or PU meshes (n = 12/group). Explants were examined 60 and 180 d (six per group) post-implantation. Intervention Posterior rectovaginal dissection, NTR, or mesh repair. Outcome measurements and statistical analysis Implant-related complications, vaginal contractility, compliance, and host response were assessed. Power calculation and analysis of variance testing were used to enable comparison between the four groups. Results There were no visible implant-related complications. None of the implants compromised vaginal wall contractility, and passive biomechanical properties were similar to those after NTR. Shrinkage over the surgery area was around 35% for NTR and all mesh-augmented repairs. All materials were integrated well with similar connective tissue composition, vascularization, and innervation. The inflammatory response was mild with electrospun implants, inducing both more macrophages yet with relatively more type 2 macrophages present at an early stage than the PP mesh. Conclusions Three very different materials were all well tolerated in the sheep vagina. Biomechanical findings were similar for all mesh-augmented repair and NTR. Constructs induced slightly different mid-term inflammatory profiles. Patient summary Product innovation is needed to reduce implant-related complications. We tested two novel implants, electrospun and an ultra-lightweight polypropylene textile mesh, in a physiologically relevant model for vaginal surgery. All gave encouraging outcomes., European Urology Focus, 6 (1), ISSN:2405-4569

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.

Vaginal meshes used in the treatment of stress urinary incontinence (SUI) and pelvic organ prolapse (POP) have produced highly variable outcomes, causing life-changing complications in some patients while providing others with effective, minimally invasive treatments. The risk:benefit ratio when using vaginal meshes is a complex issue in which a combination of several factors, including the inherent incompatibility of the mesh material with some applications in pelvic reconstructive surgeries and the lack of appropriate regulatory approval processes at the time of the premarket clearance of these products, have contributed to the occurrence of complications caused by vaginal mesh. Surgical mesh used in hernia repair has evolved over many years, from metal implants to knitted polymer meshes that were adopted for use in the pelvic floor for treatment of POP and SUI. The evolution of the material and textile properties of the surgical mesh was guided by clinical feedback from hernia repair procedures, which were also being modified to obtain the best outcomes with use of the mesh. Current evidence shows how surgical mesh fails biomechanically when used in the pelvic floor and materials with improved performance can be developed using modern material processing and tissue engineering techniques.

Simple limbal epithelial transplantation (SLET) is an innovative limbal stem cell transplantation technique that has gained increasing popularity over the last few years. Different groups from across the world have published the clinical results of SLET in large case series with varying types and severities of limbal stem cell deficiency (LSCD). This review attempts to place all the available knowledge on SLET together in one place for the benefit of not only cornea specialists and trainees but also for residents and general ophthalmologists. It follows a balanced approach of blending evidence with experience by providing an objective analysis of published results along with helpful insights from subject experts, starting from preoperative considerations including the role of newer imaging modalities to the technical aspects of the surgery itself and the management of possible complications. Original data and novel insights on allogeneic SLET for bilateral LSCD are included in the review to address the few remaining lacunae in the existing literature on this topic. This review intends to inform, educate, and empower all aspiring and practicing SLET surgeons to optimize their clinical outcomes and to have maximal positive impact on the lives of the individuals affected by unilateral or bilateral chronic LSCD.

Following recent work from our group on a small ribose-derived sugar 2-deoxy-d-ribose which stimulates angiogenesis in a chick chorionic allantoic membrane bioassay and in skin wounds in healthy rats, we tested the effect of this sugar when added to a common alginate wound dressing in a well-accepted diabetic skin wound model in rats. Dressings were simply loaded with filter-sterilized sugar solutions that were proved stable at room temperature for at least two weeks. Prior to undertaking the animal study, we assessed in vitro release of sugar loaded from alginate dressings loaded with either 5 or 10% 2-deoxy-d-ribose. Both showed more than 90% release of sugar over three days followed by a lesser and sustained release for up to eight days. Diabetes was induced in rats by streptozotocin injection followed by confirmation through measuring elevated blood glucose levels all over the period of the study. Full thickness skin wounds of 20 mm diameter showed slow healing and were still unhealed at 20th day in the control animals. The addition of alginate hydrogel did significantly stimulate wound healing compared to the untreated animals, but the addition of 2-deoxy-d-ribose at either 5 or 10% further and significantly improved the rate of wound healing. The most striking result was seen in the effect of sugar on angiogenesis in the wound beds where alginate alone did not improve the extent of CD34 identified blood vessels in the wound beds but the addition of sugar significantly increased these - this could be seen as early as seven days post addition of the sugar-loaded alginate dressing and was still evident at day 20. Thus, these results suggest that the controlled use of 2-deoxy-d-ribose could be a novel and cost-effective therapy for the management of impaired wound healing in diabetic patients.

Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) are conditions which result in significant physical, mental and social consequences for women worldwide. The high rates of recurrence reported with primary repair for POP led to the use of synthetic mesh to augment repairs in both primary and secondary cases following failed previous POP repair. The widely reported, unacceptably high rates of complications associated with the use of synthetic, transvaginal mesh in pelvic floor repair have severely limited the treatment options that surgeons can offer. This article summarises the recent advances in pelvic floor repair, such as improved quantification and modelling of the biomechanics of the pelvic floor and the developing technology within the field of tissue engineering for treatment of SUI/POP, including biomaterials and cell-based therapies. Finally, we will discuss the issues surrounding the commercial introduction of synthetic mesh for use within the pelvic floor and what lessons can be learned for the future as well as the current guidance surrounding treatment for SUI/POP.

PURPOSE OF REVIEW Interactions between biomaterials and biomaterial-delivering cells and the host tissues are complexly affected by the material itself, the ultrastructure of the overall construct and cells and other bioactive factors involved. The aim of this review is to review the current understanding on the definitions of biocompatibility and current advances in improving biocompatability of tissue-engineered constructs. RECENT FINDINGS Some synthetic materials are associated with more foreign body reactions compared with natural materials; however, they allow fabrication of materials with a great diversity of physical and mechanical properties. Material design strategies can be tailored to mimic the natural extracellular matrix topography. There are also advancements in the pharmacological functionalization of materials with improved angiogenic potential that can lead to better tissue response. Stem cells are also used to improve the tissue response of tissue-engineered materials; however, the recent regulations on regenerative medicine products necessitate significant regulatory approval processes for these. SUMMARY The biggest challenge faced in translation of tissue-engineered constructs into clinical practice relates to their engraftment and poor tissue integration into the challenging wound bed of the pelvic floor. Biocompatibility of tissue engineered constructs can theoretically be improved by the incorporation of bioactive agents, such as vitamins C or oestradiol.

This review identifies the clinical complications associated with the design of the current polyproplylene mesh materials used for the treatment of stress urinary incontinence and pelvic organ prolapse. Following on from this, new alternative materials under development for pelvic floor repair are reviewed.It is well accepted that the textile properties of the current polypropylene surgical meshes are not suitable for the pelvic floor environment. This together with the chemical nature of the current mesh leads to complications whenever implanted in the pelvic floor of women. New alternative materials for the repair of the pelvic floor have been developed with properties designed to be more appropriate for the biomechanical requirements and implantation requirements for the pelvic floor to reduce these clinical complications. To support this, these newer materials are being rigorously tested using more appropriate in-vitro regimes and animal models.This chapter summarizes developments in the design of new materials for pelvic floor repair. These are being subjected to preclinical testing to exclude materials, which might fail to work in this dynamic environment by either showing a poor mechanical match to the requirements of the tissue or by provoking sustained inflammation. The hope is that new materials will prove effective without causing the high incidence of unacceptable side-effects currently seen with polypropylene mesh implants.

Advancements in biomedical engineering and advanced therapies including tissue engineering products necessitate revisions to the regulation and governance of their production and use to ensure patient safety. In this review, the current regulations and recent improvements on the governance of biomedical devices are reviewed.Current regulations on approval of biomedical devices failed to address some important aspects related to the definition of biocompatibility of medical implants. The main issue was that the failure to establish design requirements for a specific application - in this case, the pelvic floor. Another issue was the lack of knowledge on disease mechanisms leading to an inability to define clear targets for surgical treatment. A clear example of this is the recent vaginal mesh scandal.Surgical innovations are inherently challenging. It is no surprise that the regulatory landscape lags behind advancements in biomedical technologies. Very recent modifications to the available regulations particularly in Europe aim to establish a robust, transparent, predictable and sustainable regulatory framework for medical devices, which ensures patient safety while supporting innovation.

Purpose of review To set in context the challenge of developing tissue-engineered constructs for use in the female pelvic floor compared with at least 30 years of research progress in tissue engineering for other tissues. Recent findings The relative lack of information on the mechanical requirements of the pelvic floor in women who have suffered damage to these tissues is a major challenge to designing tissue-engineered materials for use in this area. A few groups are now using autologous cells and biomaterials to develop constructs for repair and regeneration of the pelvic floor. Progress with these has reached early stage evaluation in small animal models. Meanwhile the regulatory challenge of introducing laboratory-expanded cell therapy into the clinic is prompting groups to look at alternatives, such as using lipoaspirate retrieved in theatre as a source of adult stem cells for a number of tissues. In our group, we have begun to look at lipoaspirate for repair of the pelvic floor. Summary There is a need for research to harvest the advances made over the last 30 years in developing tissue-engineered constructs for several tissues to now tackle the problems of the weakened pelvic floor. At present, there are relatively few groups engaged in this challenge despite the growing clinical need.

Serious complications can develop with the mesh implants used for stress urinary incontinence and pelvic organ prolapse surgery. We evaluated 2 materials currently in clinical use and 2 alternative materials using a rabbit abdominal model to assess host response and biomechanical properties of the materials before and after implantation.Poly-L-lactic acid and polyurethane meshes were electrospun to be compared to commercially available polypropylene and polyvinylidene fluoride meshes. A total of 40 immunocompetent full-thickness abdominal wall defect rabbit models were used, including 8 in each of the poly-L-lactic acid, polyurethane, polyvinylidene fluoride and polypropylene experimental groups, and sham controls. Two 20 mm defects were created per animal and primarily repaired. The experimental groups then underwent onlay of each repair material while sham controls did not. Four rabbits per group were sacrificed at days 30 and 90. Abdominal wall specimens containing the defect with or without repair material were explanted to be assessed by histology (hematoxylin and eosin staining, and immunohistochemistry) and biomechanical testing at 30 and 90 days.At 90 days of implantation tissues repaired with all 4 materials showed biomechanical properties without significant differences. However, polypropylene and polyvinylidene fluoride meshes demonstrated a sustained chronic inflammatory response profile by 90 days. In contrast, poly-L-lactic acid and polyurethane meshes integrated well into host tissues with a decreased inflammatory response, indicative of constructive remodeling.Poly-L-lactic acid and polyurethane alternative materials achieved better host integration in rabbit models than current synthetic repair materials.

Numerous bone and soft tissue grafting techniques are followed to repair cleft of lip and palate (CLP) defects. In addition to the gold standard surgical interventions involving the use of autogenous grafts, various allogenic and xenogenic graft materials are available for bone regeneration. In an attempt to discover minimally invasive and cost effective treatments for cleft repair, an exceptional growth in synthetic biomedical graft materials have occurred. This study gives an overview of the use of dental materials to repair cleft of lip and palate (CLP). The eligibility criteria for this review were case studies, clinical trials and retrospective studies on the use of various types of dental materials in surgical repair of cleft palate defects. Any data available on the surgical interventions to repair alveolar or palatal cleft, with natural or synthetic graft materials was included in this review. Those datasets with long term clinical follow-up results were referred to as particularly relevant. The results provide encouraging evidence in favor of dental and other related biomedical materials to fill the gaps in clefts of lip and palate. The review presents the various bones and soft tissue replacement strategies currently used, tested or explored for the repair of cleft defects. There was little available data on the use of synthetic materials in cleft repair which was a limitation of this study. In conclusion although clinical trials on the use of synthetic materials are currently underway the uses of autologous implants are the preferred treatment methods to date.

Cardiovascular disease is the leading cause of death worldwide, with this trend predicted to continue for the foreseeable future. Common disorders are associated with the stenosis or occlusion of blood vessels. The preferred treatment for the long-term revascularization of occluded vessels is surgery utilizing vascular grafts, such as coronary artery bypass grafting and peripheral artery bypass grafting. Currently, autologous vessels such as the saphenous vein and internal thoracic artery represent the gold standard grafts for small-diameter vessels (

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease. Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence. Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air–liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation. Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models., Collagen IV- and laminin-rich decellularised dermis scaffolds support a mucociliary airway epithelial graft but in vivo transplantation in pre-clinical models is challenging http://bit.ly/2IdQp5d

Preterm premature rupture of fetal membranes is a leading cause of preterm delivery. Preterm labour can compromise fetal survival, and even if a pregnancy affected by preterm premature rupture of fetal membrane continues, major complications associated with leakage of amniotic fluid and risk of infection can affect the normal development and survival of the baby. There are limited management options for preterm premature rupture of fetal membrane other than delivery of the baby if ascending infection (chorioamnionitis) is suspected. We have previously reported the development and characterisation of an implantable membrane with the aim of using it to occlude the internal os of the cervix, in order to prevent amniotic fluid loss, allow fluid reaccumulation and reduce the risk of chorioamnionitis. For this, an electrospun biocompatible and distensible bilayer membrane was designed with mechanical properties similar to the human amniotic membrane. In this study, we consider the effects of sterilization on the membrane, how to insert the membrane and visualise it using routine clinical methods. To do this, we used e-beam sterilisation and examined the ability of the membrane to adhere to ex vivo human cervical tissues. We also studied its insertion into a custom-synthesised model of a 20-week pregnant uterus and imaged the membrane using ultrasound. Sterilisation produced minor effects on physical and mechanical properties, but these did not affect the capacity of the membrane to be sutured or to provide a fluid barrier. We demonstrated that fibrin glue can successfully adhere the bilayer membrane to cervical tissues. Finally, we demonstrated that the membrane can be inserted through the cervix as well as visualized in place using ultrasound imaging and an endoscope. In summary, we suggest this membrane is a candidate for further development in an appropriate animal model, supported by appropriate imaging, to precede possible future human studies if judged to demonstrate satisfactory safety and efficacy profiles.

AIMS: Stress urinary incontinence and pelvic organ prolapse are very common conditions with a proportion of patients requiring implantation of synthetic materials for a durable repair. However increasing numbers of post-surgical complications have been reported related to the use of polypropylene meshes. One hypothesis for the adverse response is poor mechanical matching of the relatively stiff polypropylene mesh particularly as materials in the pelvic floor will need to cope with decades of distension as occurs with increase of intraabdominal pressure on coughing, laughing, or sneezing. METHODS: In this study we have undertaken a very simple fatigue testing regime to compare the mechanical abilities of six materials. Four commercial meshes in clinical use and two novel electrospun materials not yet evaluated in the clinic were assessed using a uniaxial tensile test. This was performed on six samples of each dry material and on another six samples of each material after just 3 days of fatigue conditions using a dynamic bioreactor. RESULTS: The four commercial materials showed permanent mechanical deformation after just 3 days of stretching these materials by 25% elongation on a regular dynamic cycle, whereas the two new materials presented more elastic properties without deformation. CONCLUSIONS: We suggest that a test as simple as this 3-day fatigue testing is sufficient to distinguish between materials which have already been found to cause complications clinically and newer materials yet to be tested clinically which will hopefully prove more mechanically appropriate for implantation in the pelvic floor. ispartof: NEUROUROLOGY AND URODYNAMICS vol:38 issue:1 pages:107-115 ispartof: location:United States status: published

Urethral stricture disease is increasingly common occurring in about 1% of males over the age of 55. The stricture tissue is rich in myofibroblasts and multi-nucleated giant cells which are thought to be related to stricture formation and collagen synthesis. An increase in collagen is associated with the loss of the normal vasculature of the normal urethra. The actual incidence differs based on worldwide populations, geography, and income. The stricture aetiology, location, length and patient's age and comorbidity are important in deciding the course of treatment. In this review we aim to summarise the existing knowledge of the aetiology of urethral strictures, review current treatment regimens, and present the challenges of using tissue-engineered buccal mucosa (TEBM) to repair scarring of the urethra. In asking this question we are also mindful that recurrent fibrosis occurs in other tissues—how can we learn from these other pathologies? Keywords: Urethral strictures, Fibrosis, Tissue-engineered buccal mucosa, Augmentation urethroplasty

AimThe aim of this study was to assess the local and systemic response to poly-lactic co-glycolic acid (PLGA) 50:50 membranes, developed as synthetic biodegradable alternatives to the use of human donor amniotic membrane in the treatment of limbal stem cell deficiency.MethodsPLGA membranes of 2 cm diameter and 50 µm thickness were placed on one eye of rabbits and secured in place using fibrin glue and a bandage contact lens, suturing the eye close with a single stitch. Control animals were treated identically, with the absence of the membranes. Plain and microfabricated electrospun membranes (containing micropockets which roughly emulate the native limbal niche) were examined over 29 days. All animals were subjected to a detailed gross and histopathological observation as well as a detailed examination of the eye.ResultsApplication of the membranes both with and without microfabricated pockets did not adversely affect animal welfare. There was complete degradation of the membranes by day 29. The membranes did not induce any significant local or systemic toxicity. Conjunctival congestion and corneal vascularisation were noted in a few control and PLGA-treated animals. Intraocular pressure was normal and the retinal status was unaltered. The ocular surface was clear and intact in all animals by the end of 29 days.ConclusionMembranes of 50:50 PLGA can be safely applied to rabbit corneas without inducing any local or systemic toxicity and these break down completely within 29 days.

Women commonly experience pelvic floor damage during pregnancy and vaginal childbirth and later develop stress urinary incontinence and prolapse. Polypropylene (PPL) mesh can repair abdominal hernias, but complications result when used in the female pelvic floor. In October, the Royal Society hosted a workshop to address issues surrounding pelvic floor disorders.

Introduction: We aimed to compare alternative synthetic scaffolds suitable for future implantation and to examine the use of an inhibitor of lysyl oxidase (beta-amino-propionitrile [β-APN]) to reduce contraction in these implants.Methods: Three synthetic scaffolds were compared to natural dermis as substrates for the production of tissue-engineered skin. For natural dermis, Euroskin was used to provide a cell-free cadaveric dermis. Synthetic scaffolds consisted of microfibrous poly-L-lactic acid (PLA), nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and a micro-/nanofibrous trilayer of PLA-PHBV-PLA. The latter were all electrospun and then all four scaffolds (three synthetic, one natural) were placed in six well plates. A culture well was formed on the scaffold using a 1 cm diameter stainless steel ring and 1.5x105 oral fibroblasts were seeded one side; after two days of culture, the ring was placed on the other side of the scaffolds and 3x105 oral keratinocytes were seeded on to the scaffolds and cultured with keratinocytes uppermost. After a further two days of culture, scaffolds were cut to 1 cm2 and raised to an air-liquid interface on stainless steel grids; some were treated with 200 μg/ mL β-APN throughout the culture period (28 days). Contraction in vitro was assessed by serial digital photography of cell-seeded scaffolds and cell-free scaffolds three times a week for 28 days. All cell-seeded scaffolds were assessed for cell metabolic activity, mechanical properties, histology, and morphology by scanning electron microscopy (SEM).Results: The mean fibre diameters and pore sizes of PLA and PHBV scaffolds were 2.4±0.77, 0.85±0.21 μm (p0.05).Conclusions: This study shows that a trilayer micro-nano-3D porous synthetic scaffold is suitable for oral keratinocyte and fibroblast growth with good cell viability and minimal contraction. This material also has good mechanical properties and histological analyses showed its ability to mimic normal human oral mucosal morphology. Furthermore, synthetic trilayer scaffolds have advantages over biological scaffolds — there is no risk of disease transmission or immunological rejection and they appear resistant to contraction. We suggest they present a good alternative to allodermis for future use in urethral reconstruction.

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.

Purpose Autologous buccal mucosa is commonly utilized in the surgical treatment of urethral strictures. Extensive strictures require a larger quantity of tissue, which may lead to donor site morbidity. This review assesses progress in producing tissue engineered buccal mucosa as an alternative graft material. Results Few clinical studies have introduced cells onto biological or synthetic scaffolds and implanted resulting constructs in patients. The available studies show that buccal mucosa cells on acellular human dermis or on collagen matrix lead to good acute stage tissue integration. Urothelial cells on a synthetic substrate also perform well. However while some patients do well many years post-grafting, others develop stricture recurrence. Acellular biomaterials used to treat long urethral defects in animals commonly lead to fibrosis. Conclusions Tissue engineered buccal mucosa shows promise as a substitute for native tissue. The fibrosis which occurs months post-implantation may reflect the underlying disease process recurring in these patients.

Advances in tissue engineering have permitted the three-dimensional (3D) reconstruction of human oral mucosa for various in vivo and in vitro applications. Tissue-engineered oral mucosa have been further optimized in recent years for clinical applications as a suitable graft material for intra-oral and extra-oral repair and treatment of soft-tissue defects. Novel 3D in vitro models of oral diseases such as cancer, Candida, and bacterial invasion have been developed as alternatives to animal models for investigation of disease phenomena, their progression, and treatment, including evaluation of drug delivery systems. The introduction of 3D oral mucosal reconstructs has had a significant impact on the approaches to biocompatibility evaluation of dental materials and oral healthcare products as well as the study of implant-soft tissue interfaces. This review article discusses the recent advances in tissue engineering and applications of tissue-engineered human oral mucosa. This record was migrated from the OpenDepot repository service in June, 2017 before shutting down.

Purpose The reconstruction of congenital diaphragmatic hernia or other congenital soft tissue defects often requires implants. These can be either degradable or permanent, each having their advantages. Whatever type is being used, the host response induced by implants plays a crucial role to determine the outcome. Macrophages are pivotal during implant remodeling; they are plastic and acquire in response to environmental stimuli either an inflammatory status and mediate subsequent fibrosis or a regulatory status and facilitate functional remodeling. Matrices engineered with mesenchymal stem cells (MSCs) have the capacity to modulate the host immune reaction. MSCs are believed to promote constructive remodeling of the implant through a regulatory macrophage response among others. Herein, we evaluate this potential of MSC derived from the amniotic fluid (AF-MSC), an interesting MSC type for neonatal reconstruction, on electrospun polylactic acid (PLA) scaffolds. Methods We seeded AF-MSC at a density of 1.105/cm2 on electrospun PLA matrices and determined cell viability. In vivo, we used cell-seeded or cell-free PLA matrices for subcutaneous implantation in immune competent rats. The host immune response was evaluated by histomorphometry at 14 days postoperatively. Results The PLA matrix supported adherence and proliferation of AF-MSC. Fourteen days after implantation, PLA matrices were well penetrated by inflammatory cells, new blood vessels, and collagen fibers. AF-MSC–seeded scaffolds were associated with a similar response yet with a decreased number of eosinophils, increased matrix degradation and collagen fiber deposition compared with controls. The amount of total macrophages and of M2-subtype was similar for all animals. Conclusion Electrospun PLA matrices are a suitable substrate for short-term culture of AF-MSC. In rats, addition of AF-MSC to PLA matrices modulates the host response after subcutaneous implantation, yet without a difference in macrophage profile compared with control.

Patients with laryngeal disorders may have severe morbidity relating to swallowing, vocalization, and respiratory function, for which conventional therapies are suboptimal. A tissue-engineered approach would aim to restore the vocal folds and maintain respiratory function while limiting the extent of scarring in the regenerated tissue. Under Good Laboratory Practice conditions, we decellularized porcine larynges, using detergents and enzymes under negative pressure to produce an acellular scaffold comprising cartilage, muscle, and mucosa. To assess safety and functionality before clinical trials, a decellularized hemilarynx seeded with human bone marrow-derived mesenchymal stem cells and a tissue-engineered oral mucosal sheet was implanted orthotopically into six pigs. The seeded grafts were left in situ for 6 months and assessed using computed tomography imaging, bronchoscopy, and mucosal brushings, together with vocal recording and histological analysis on explantation. The graft caused no adverse respiratory function, nor did it impact swallowing or vocalization. Rudimentary vocal folds covered by contiguous epithelium were easily identifiable. In conclusion, the proposed tissue-engineered approach represents a viable alternative treatment for laryngeal defects.

Context: Surgical nonautologous meshes have been used for several decades to repair abdominal wall herniae. Implantable materials have been adopted for the treatment of female and male stress urinary incontinence (SUI) and female pelvic organ prolapse (POP). Objective: A consensus review of existing data based on published meta-analyses and reviews. Evidence acquisition: This document summarises the deliberations of a consensus group meeting convened by the European Association of Urology (EAU) and the European Urogynecological Association, to explore the current evidence relating to the use of polypropylene (PP) materials used for the treatment of SUI and POP, with reference to the 2016 EAU guidelines (European Association of Urology 2016), the European Commission's SCENIHR report on the use of surgical meshes (SCENIHR 2015), other available high-quality evidence, guidelines, and national recommendations. Evidence synthesis: Current data suggest that the use of nonautologous durable materials in surgery has well-established benefits but significant risks, which are specific to the condition and location they are used for. Various graft-related complications have been described-such as infection, chronic pain including dyspareunia, exposure in the vagina, shrinkage, erosion into other organs of xenografts, synthetic PP tapes (used in SUI), and meshes (used in POP)-which differ from the complications seen with abdominal herniae. Conclusions: When considering surgery for SUI, it is essential to evaluate the available options, which may include synthetic midurethral slings (MUSs) using PP tapes, bulking agents, colposuspension, and autologous sling surgery. The use of synthetic MUSs for surgical treatment of SUI in both male and female patients has good efficacy and acceptable morbidity. Synthetic mesh for POP should be used only in complex cases with recurrent prolapse in the same compartment and restricted to those surgeons with appropriate training who are working in multidisciplinary referral centres. Patient summary: Synthetic slings can be safely used in the surgical treatment of stress incontinence in both male and female patients. Patients need to be aware of the alternative therapy and potential risks and complications of this therapy. Synthetic mesh for treating prolapse should be used only in complex cases with recurrent prolapse in specialist referral centres. (C) 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved

It has only been a quarter of a century since the discovery of adult stem cells at the human corneo-scleral limbus. These limbal stem cells are responsible for generating a constant and unending supply of corneal epithelial cells throughout life, thus maintaining a stable and uniformly refractive corneal surface. Establishing this hitherto unknown association between ocular surface disease and limbal dysfunction helped usher in therapeutic approaches that successfully addressed blinding conditions such as ocular burns, which were previously considered incurable. Subsequent advances in ocular surface biology through basic science research have translated into innovations that have made the surgical technique of limbal stem cell transplantation simpler and more predictable. This review recapitulates the basic biology of the limbus and the rationale and principles of limbal stem cell transplantation in ocular surface disease. An evidence-based algorithm is presented, which is tailored to clinical considerations such as laterality of affliction, severity of limbal damage and concurrent need for other procedures. Additionally, novel findings in the form of factors influencing the survival and function of limbal stem cells after transplantation and the possibility of substituting limbal cells with epithelial stem cells of other lineages is also discussed. Finally this review focuses on the future directions in which both basic science and clinical research in this field is headed.

Urethral stricture disease is increasingly common occurring in about 1% of males over the age of 55. The stricture tissue is rich in myofibroblasts and multi-nucleated giant cells which are thought to be related to stricture formation and collagen synthesis. An increase in collagen is associated with the loss of the normal vasculature of the normal urethra. The actual incidence differs based on worldwide populations, geography, and income. The stricture aetiology, location, length and patient's age and comorbidity are important in deciding the course of treatment. In this review we aim to summarise the existing knowledge of the aetiology of urethral strictures, review current treatment regimens, and present the challenges of using tissue-engineered buccal mucosa (TEBM) to repair scarring of the urethra. In asking this question we are also mindful that recurrent fibrosis occurs in other tissues—how can we learn from these other pathologies?

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.

Background During labour, the cervix undergoes a series of changes to allow the passage of the fetoplacental unit. While this visible transformation is well-described, the underlying and causative microscopic changes, in which collagen plays a major role, are poorly understood and difficult to visualise. Recent studies in mice and humans have shown that Second Harmonic Generation (SHG) microscopy, a non-destructive imaging technique, can detect changes in the cervical collagen. However, the question of whether SHG can identify changes in the arrangement of cervical collagen at different physiological stages still needs addressing. Therefore, this study aimed to compare the cervical collagen alignment between pre- and postmenopausal women using SHG and to generate proof-of-concept data prior to assessing this technique in pregnancy. Methods Cervical biopsies from premenopausal (n = 4) and postmenopausal (n = 4) multiparous women undergoing hysterectomy for benign conditions were cross-sectionally scanned using an upright confocal microscope. SHG images were collected in Z-stacks and qualitatively evaluated using semi-quantitative scoring (0–3 in ascending degree of alignment) by assessors who were unaware of the classification of the SHG images, and quantitatively, using 2D Fourier transformation analysis. The dominant orientation and difference in dispersion of collagen fibres in each z-stack (X ± SD) was calculated and compared between groups. Results Qualitatively, collagen fibres appeared more organised in postmenopausal women, [premenopausal: median 0, range (0–1), postmenopausal: median 1.25, range (1–3); X 2 (df = 5) = 19.35, p = 0.002]. Quantitatively, there was a statistically significant difference in collagen fibre dispersion between premenopausal (5.39° ± 12.68°) and postmenopausal women (−1.58° ± 8.24°), [Welch’s t-test (245.54) = 5.54, p

The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells are continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. In the event that the cornea is damaged and the limbal stem cell population is severely reduced, this condition known as Limbal Stem Cell Deficiency and can lead to blindness. There are numerous treatments but most have high long term failure rates. Most treatment methods include the transplantation of limbal stem cells into damaged limbus with hope of repopulating the region and regenerating at healthy corneal epithelium. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images. A bespoke OCT has been built to investigate the trajectories of these limbal stem cells after transplantation to see whether if they do repopulate the damaged limbus or not. In the experimentation magneto-labelling was used to track the limbal stem cells. For the magneto-labelling a mixture of limbal stem cells and cornea epithelium are cultured with super paramagnetic iron (Fe3O4) nanoparticles (20-30nm in size) for 24hours, to allow for uptake. The cells are then transplanted onto the denuded cornea. The transplanted cell mixture with the encapsulated magnetic nanoparticles is actuated with an external magnetic field 0.08T leading to a phase modulation on the signal. A Phase sensitive Magneto-motive OCT is used to locate the transplanted cells. The location of the cells with embed SPIOs were located both in 2D and 3D.

We describe a method to prepare patterned environments for eukaryotic cells by inkjet printing paraffin wax onto glass. This technique bypasses the requirement to create a master mould, typically required with the use of polydimethylsiloxane techniques and the printed structure could be immediately used to guide cell proliferation. In a space of 2-3 hours, the desired pattern could be created with computer assisted design, printed and have cells seeded onto the scaffold, which could reduce the cycle time of prototyping micropattern designs. Human dermal fibroblasts and RN22 Schwann cells were seen to proliferate within the fabricated patterns and survive for more than 7 days. Additionally, the wax constructs could be readily removed from the substrate at any stage after cell seeding with the cells continuing to proliferate. Thus, we report on a simple but novel approach for the controlled physical positioning of live cells by wax inkjet printing.

Background Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation. Aims and Objectives Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration. Methods Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds. Results Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC. Conclusions Therefore, prolonged mechanical distension in vitro causes polypropylene to fail. Materials with more appropriate mechanical properties for use as sling materials can be produced using PU. Combining PLA with PU greatly improves interaction of cells with this material.

An increasing number of studies suggest inflammation stimulates tumour invasion. In melanoma, despite recent advances in targeted therapy and immunomodulatory therapies, this cancer remains difficult to treat. Our previous studies show melanoma cells interact with skin cells in their invasion into tissue engineered skin and suggest inflammation stimulates invasion. The aim of this study was to investigate the use of an anti-inflammatory on melanoma invasion. To do this we developed a wounded and inflamed in vitro 3D melanoma model in which to investigate the use of an anti-inflammatory on melanoma invasion. The tissue engineered skin model was based on human de-epidermised acellular dermis to which keratinocytes, fibroblasts and three different melanoma cell lines were added in various combinations. A simple incisional wound was made in the model and TNF-α and fibrin were added to simulate conditions of inflammation. Topical ibuprofen in a hydrogel was added and the extent of melanoma invasion into the dermis was assessed under the various conditions. The results showed that penetration of two of the cell lines (HBL and A375SM) into the tissue engineered skin was exacerbated by wounding and ibuprofen significantly decreased invasion of A375SM cells and slightly reduced invasion of HBL cells. A third cell line, C8161, was aggressively invasive under all conditions to an extent that was not influenced by wounding, TNF-α or the addition of ibuprofen. In summary, the results for one these cell lines (and a trend for a second cell line) support the hypothesis that a wound environment is conducive to melanoma invasion but the local addition of an anti-inflammatory drug such as ibuprofen may attenuate invasion.

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.