Spatiotemporal release of VEGF from biodegradable polylactic-co-glycolic acid microspheres induces angiogenesis in chick chorionic allantoic membrane assay

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.While vascular endothelial growth factor (VEGF) is an acknowledged potent pro-angiogenic agent there is a need to deliver it at an appropriate concentration for several days to achieve angiogenesis. The aim of this study was to produce microspheres of biodegradable polylactic-co-glycolic acid (PLGA) tailored to achieve sustained release of VEGF at an appropriate concentration over seven days, avoiding excessive unregulated release of VEGF that has been associated with the formation of leaky blood vessels. Several formulations were examined to produce microspheres loaded with both human serum albumin (HSA) and VEGF to achieve release of VEGF between 3 and 10 ng per ml for seven days to match the therapeutic window desired for angiogenesis. In vitro experiments showed an increase in endothelial cell proliferation in response to microspheres bearing VEGF. Similarly, when microspheres containing VEGF were added to the chorionic membrane of fertilised chicken eggs, there was an increase in the development of blood vessels over seven days in response, which was significant for microspheres bearing VEGF and HSA, but not VEGF alone. There was an increase in both blood vessel density and branching – both signs of proangiogenic activity. Further, there was clearly migration of cells to the VEGF loaded microspheres. In summary, we describe the development of an injectable delivery vehicle to achieve spatiotemporal release of physiologically relevant levels of VEGF for several days and demonstrate the angiogenic response to this. We propose that such a treatment vehicle would be suitable for the treatment of ischemic tissue or wounds

Synthetic Materials Used in the Surgical Treatment of Pelvic Organ Prolapse: Problems of Currently Used Material and Designing the Ideal Material

Synthetic materials have long been used to provide structural support when surgically repairing pelvic organ prolapse (POP). The most widely used synthetic material is a mesh made of polypropylene (PPL). The use of mesh is intended to improve cure rates and prevent recurrences after POP surgery – however as more mesh materials have been implanted, it has become apparent that serious complications can occur in up to 30% of women, particularly when the mesh is implanted transvaginally. Over the years many different mesh kits have been marketed and used in the treatment of POP however polypropylene mesh was never designed or tested for use in pelvic floor. Instead it was approved for clinical use based on its biocompatibility and success in abdominal hernia repairs. It is now known that PPL meshes are neither compliant with the mechanical forces in the pelvic floor nor do they integrate well into paravaginal tissues. Better materials developed specifically for use in pelvic floor are urgently needed. The aim of this chapter is to define the requirements of an ideal mesh in terms of its material properties and to summarize the ongoing research on developing the next generation pelvic floor repair materials

Antimicrobial graft copolymer gels

In view of the growing worldwide rise in microbial resistance, there is considerable interest in designing new antimicrobial copolymers. The aim of the current study was to investigate the relationship between antimicrobial activity and copolymer composition/ architecture to gain a better understanding of their mechanism of action. Specifically, the antibacterial activity of several copolymers based on 2- (methacryloyloxy)ethyl phosphorylcholine [MPC] and 2-hydroxypropyl methacrylate (HPMA) toward Staphylococcus aureus was examined. Both block and graft copolymers were synthesized using either atom transfer radical polymerization or reversible addition−fragmentation chain transfer polymerization and characterized via 1H NMR, gel permeation chromatography, rheology, and surface tensiometry. Antimicrobial activity was assessed using a range of well-known assays, including direct contact, live/dead staining, and the release of lactate dehydrogenase (LDH), while transmission electron microscopy was used to study the morphology of the bacteria before and after the addition of various copolymers. As expected, PMPC homopolymer was biocompatible but possessed no discernible antimicrobial activity. PMPC-based graft copolymers comprising PHPMA side chains (i.e. PMPC-g-PHPMA) significantly reduced both bacterial growth and viability. In contrast, a PMPC−PHPMA diblock copolymer comprising a PMPC stabilizer block and a hydrophobic core-forming PHPMA block did not exhibit any antimicrobial activity, although it did form a biocompatible worm gel. Surface tensiometry studies and LDH release assays suggest that the PMPC-g-PHPMA graft copolymer exhibits surfactantlike activity. Thus, the observed antimicrobial activity is likely to be the result of the weakly hydrophobic PHPMA chains penetrating (and hence rupturing) the bacterial membrane

Detection of Tyrosinase Autoantibodies in Patients With Vitiligo Using 35S-Labeled Recombinant Human Tyrosinase in a Radioimmunoassay

Tyrosinase antibodies recently have been reported to occur frequently in patients with vitligo. We describe the detection of tyrosinase antibodies in vitiligo patients using in vitro 35S-labeled human tyrosinase in a radioimmunoassay. Of 46 vitiligo sera examined in the assay, five (10.9%) were found to be positive for tyrosinase antibodies. In contrast, 20 control sera and sera from 10 patients with Hashimoto's thyroiditis were negative. Four of the sera positive in the radioimmunoassay were also positive in an ELISA using mushroom tyrosinase as antigen. Absorption studies indicated that pre-incubation with mushroom tyrosinase absorbed out the immunoreactivity of the positive sera in the radioimmunoassay, suggesting cross-reactivity, but this absorption was never complete, indicating that there are tyrosinase antibodies in human sera that do not react with the mushroom protein. There was no obvious association between the presence of tyrosinase antibodies and the age of the patients (range: 22–62 y), their duration of disease (range: 5–20 y), or the type of vitiligo (one segmental, one symmetricallperiorificial, three symmetrical), although the three patients with the highest antibody levels also had an associated autoimmune disorder (one with Graves' disease; two with autoimmune hypothyroidism). The results confirm that tyrosinase autoantibodies are present in the sera of vitiligo patients but at a low frequency. The technique described is sensitive and quantitative and allows the detection of confirmational epitopes. It will be useful in longitudinal studies to determine the relation between the clinical features of vitiligo and tyrosinase antibody levels

A micro-incubator for cell and tissue imaging

International audienceA low-cost micro-incubator for the imaging of dynamic processes in living cells and tissues has been developed. This micro-incubator provides a tunable environment which can be altered to study the response of cell monolayers for several days as well as relatively thick tissue samples and tissue engineered epithelial tissues in experiments lasting several hours. Samples within the incubator are contained in a sterile cavity closed by a gas permeable membrane. The incubator can be positioned in any direction and used on an inverted as well as on an upright microscope. The temperature is regulated with a Peltier system controlled with a sensor positioned close to the sample to be able to compensate for any changes in temperature. Rapid changes in the environment can be applied to the sample because of the fast response of the Peltier system and the sample's adaptations to induced changes in the environment can be monitored. To evaluate the performance of the micro-incubator we report on studies using cultured cells in monolayers, on monolayers of cells stretched to breaking point on a distensible membrane, on cells in open 3D fibrous scaffolds and on fluorescently labelled polymersome penetration into 3D tissue engineered oral mucosa

Production and Characterization of a Novel, Electrospun, Tri-Layer Polycaprolactone Membrane for the Segregated Co-Culture of Bone and Soft Tissue

Composite tissue-engineered constructs combining bone and soft tissue have applications in regenerative medicine, particularly dentistry. This study generated a tri-layer, electrospun, poly-ε-caprolactone membrane, with two microfiber layers separated by a layer of nanofibers, for the spatially segregated culture of mesenchymal progenitor cells (MPCs) and fibroblasts. The two cell types were seeded on either side, and cell proliferation and spatial organization were investigated over several weeks. Calcium deposition by MPCs was detected using xylenol orange (XO) and the separation between fibroblasts and the calcified matrix was visualized by confocal laser scanning microscopy. SEM confirmed that the scaffold consisted of two layers of micron-diameter fibers with a thin layer of nano-diameter fibers in-between. Complete separation of cell types was maintained and calcified matrix was observed on only one side of the membrane. This novel tri-layer membrane is capable of supporting the formation of a bilayer of calcified and non-calcified connective tissue

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

Background Microscopic techniques enable real-space imaging of complex biological events and processes. They have become an essential tool to confirm and complement hypotheses made by biomedical scientists and also allow the re-examination of existing models, hence influencing future investigations. Particularly imaging live cells is crucial for an improved understanding of dynamic biological processes, however hitherto live cell imaging has been limited by the necessity to introduce probes within a cell without altering its physiological and structural integrity. We demonstrate herein that this hurdle can be overcome by effective cytosolic delivery. Principal Findings We show the delivery within several types of mammalian cells using nanometre-sized biomimetic polymer vesicles (a.k.a. polymersomes) that offer both highly efficient cellular uptake and endolysomal escape capability without any effect on the cellular metabolic activity. Such biocompatible polymersomes can encapsulate various types of probes including cell membrane probes and nucleic acid probes as well as labelled nucleic acids, antibodies and quantum dots. Significance We show the delivery of sufficient quantities of probes to the cytosol, allowing sustained functional imaging of live cells over time periods of days to weeks. Finally the combination of such effective staining with three-dimensional imaging by confocal laser scanning microscopy allows cell imaging in complex three-dimensional environments under both mono-culture and co-culture conditions. Thus cell migration and proliferation can be studied in models that are much closer to the in vivo situation

Developing A Synthetic Composite Membrane For Cleft Palate Repair

An oronasal fistula is a passage between the oral and nasal cavity. Currently, surgical procedures use mucosal flaps or collagen grafts to make a barrier between oral and nasal cavities. Our aim was to develop a cell-free synthetic repair material for closure of nasal fistulas. We surface functionalized electrospun polyurethane (PU) and poly-L-lactic acid (PLLA) and composite polymer (PU-PLLA) membranes with acrylic acid through plasma polymerization. Membranes were treated in a layer-by-layer approach to develop highly charged electrostatic layer that could bind heparin as a pro-angiogenic glycosaminoglycan. The properties were evaluated through physical, chemical, and mechanical characterization techniques. Cytotoxicity was tested with MC3T3 pre-osteoblast cell lines for 3, 7, and 14 days, and vasculogenesis was assessed by implantation into the chorio-allantoic membrane in chick embryos for 7 days. In vivo biocompatibility was assessed by subcutaneous implantation in rats for 1, 3, and 6 weeks. The membranes consisted of random fibers of PLLA-PU with fiber diameters of 0.47 and 0.12 μm, respectively. Significantly higher cell proliferation and migration of MC3T3 cells at 3, 7, and 14 days were shown on plasma-coated membranes compared with uncoated membranes. Further, it was found that plasma-coated membranes were more angiogenic than controls. In vivo implantation of membranes in rats did not reveal any gross toxicity to the materials, and wound healing was comparable with the native tissue repair (sham group). We therefore present a plasma-functionalized electrospun composite polymer membrane for use in the treatment of fistulas. These membranes are flexible, non-cytotoxic, and angiogenic, and we hope it should lead to permanent closure of oronasal fistula

A dinuclear ruthenium(II) phototherapeutic that targets duplex and quadruplex DNA

With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent dinuclear complex that functions as a DNA probe in live cells was modified to produce a new isostructural derivative containing RuII(TAP)2 fragments (TAP = 1,4,5,8- tetraazaphenanthrene). The structure of the new complex has been confirmed by a variety of techniques including single crystal X-ray analysis. Unlike its parent, the new complex displays RuL-based 3MLCT emission in both MeCN and water. Results from electrochemical studies and emission quenching experiments involving guanosine monophosphate are consistent with an excited state located on a TAP moiety. This hypothesis is further supported by detailed DFT calculations, which take into account solvent effects on excited state dynamics. Cell-free steady-state and time-resolved optical studies on the interaction of the new complex with duplex and quadruplex DNA show that the complex binds with high affinity to both structures and indicate that its photoexcited state is also quenched by DNA, a process that is accompanied by the generation of the guanine radical cation sites as photo-oxidization products. Like the parent complex, this new compound is taken up by live cells where it primarily localizes within the nucleus and displays low cytotoxicity in the absence of light. However, in complete contrast to [{RuII(phen)2}2(tpphz)]4+, the new complex is therapeutically activated by light to become highly phototoxic toward malignant human melanoma cell line showing that it is a promising lead for the treatment of this recalcitrant cancer.EPSRC grant EP/M015572/1 Unviersity of Sheffield/EPSRC Doctoral Fellowship Prize EPSRC Capital Equipment Award ERASMUS