Expression of the candidate fat taste receptors in human fungiform papillae and the association with fat taste function.

Significant experimental evidence supports fat as a taste modality; however, the associated peripheral mechanisms are not well established. Several candidate taste receptors have been identified, but their expression pattern and potential functions in human fungiform papillae remain unknown. The aim of this study is to identify the fat taste candidate receptors and ion channels that were expressed in human fungiform taste buds and their association with oral sensory of fatty acids. For the expression analysis, quantitative RT-PCR (qRT-PCR) from RNA extracted from human fungiform papillae samples was used to determine the expression of candidate fatty acid receptors and ion channels. Western blotting analysis was used to confirm the presence of the proteins in fungiform papillae. Immunohistochemistry analysis was used to localise the expressed receptors or ion channels in the taste buds of fungiform papillae. The correlation study was analysed between the expression level of the expressed fat taste receptors or ion channels indicated by qRT-PCR and fat taste threshold, liking of fatty food and fat intake. As a result, qRT-PCR and western blotting indicated that mRNA and protein of CD36, FFAR4, FFAR2, GPR84 and delayed rectifying K+ channels are expressed in human fungiform taste buds. The expression level of CD36 was associated with the liking difference score (R -0·567, ß=-0·04, P=0·04) between high-fat and low-fat food and FFAR2 was associated with total fat intake (ρ=-0·535, ß=-0·01, P=0·003) and saturated fat intake (ρ=-0·641, ß=-0·02, P=0·008).

Biomineralization of osteoblasts on DLC coated surfaces for bone implants.

Diamond like carbon (DLC) films were deposited onto Ti6Al4V and Si wafer substrates by RF plasma enhanced chemical vapor deposition. The influence of dopants such as fluorine (F), silicon (Si), and nitrogen (N) on composition, structure, and biocompatibility was investigated. Ion scattering spectroscopy analysis revealed the presence of dopant atoms in the outer-most layers of the films. Raman studies showed that the position of the G-band shifts to higher frequencies with the fluorine and nitrogen content in the DLC film, whereas the incorporation of Si into DLC induces a decrease of the position of the G peak. The corrosion behavior was studied in simulated body fluid. A higher charge transfer resistance (Rct) was observed for the doped DLC films. The indirect cytotoxicity was performed using L929 fibroblast cells. The coated surfaces were hemocompatible when tested with red blood cells. DLC films were noncytotoxic to L929 cells over a 24 h exposure. Saos-2 osteoblast cell response to the doped and undoped DLC coated surfaces was studied in adhesion, proliferation, differentiation, and mineralization assays. The production of calcium and phosphate by cells on doped DLC, particularly, nitrogen doped DLC, was higher than that on undoped DLC.

Grafting of Bioactive Polymers with Various Architectures: A Versatile Tool for Preparing Antibacterial Infection and Biocompatible Surfaces.

The aim of this Research Article is to present three different techniques of poly(sodium styrene sulfonate) (polyNaSS) covalent grafting onto titanium (Ti) surfaces and study the influence of their architecture on biological response. Two of them are "grafting from" techniques requiring an activation step either by thermal or UV irradiation. The third method is a "grafting to" technique involving an anchorage molecule onto which polyNaSS synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization is clicked. The advantage of the "grafting to" technique when compared to the "grafting from" technique is the ability to control the architecture and length of the grafted polymers on the Ti surface and their influence on the biological responses. This investigation compares the effect of the three different grafting processes on the in vitro biological responses of bacteria and osteoblasts. Overall outcomes of this investigation confirmed the significance of the sulfonate functional groups on the biological responses, regardless of the grafting method. In addition, results showed that the architecture and distribution of grafted polyNaSS on Ti surfaces alter the intensity of the bacteria response mediated by fibronectin.

Co-encapsulation and co-transplantation of mesenchymal stem cells reduces pericapsular fibrosis and improves encapsulated islet survival and function when allografted.

Pericapsular fibrotic overgrowth (PFO) is associated with poor survival of encapsulated islets. A strategy to combat PFO is the use of mesenchymal stem cells (MSC). MSC have anti-inflammatory properties and their potential can be enhanced by stimulation with proinflammatory cytokines. This study investigated whether co-encapsulation or co-transplantation of MSC with encapsulated islets would reduce PFO and improve graft survival. Stimulating MSC with a cytokine cocktail of IFN-γ and TNF-α enhanced their immunosuppressive potential by increasing nitric oxide production and secreting higher levels of immunomodulatory cytokines. In vitro, co-encapsulation with MSC did not affect islet viability but significantly enhanced glucose-induced insulin secretion. In vivo, normoglycemia was achieved in 100% mice receiving islets co-encapsulated with stimulated MSC as opposed to 71.4% receiving unstimulated MSC and only 9.1% receiving encapsulated islets alone. Microcapsules retrieved from both unstimulated and stimulated MSC groups had significantly less PFO with improved islet viability and function compared to encapsulated islets alone. Levels of peritoneal immunomodulatory cytokines IL-4, IL-6, IL-10 and G-CSF were significantly higher in MSC co-encapsulated groups. Similar results were obtained when encapsulated islets and MSC were co-transplanted. In summary, co-encapsulation or co-transplantation of MSC with encapsulated islets reduced PFO and improved the functional outcome of allotransplants.

Grafting of architecture controlled poly(styrene sodium sulfonate) onto titanium surfaces using bio-adhesive molecules: Surface characterization and biological properties.

This contribution reports on grafting of bioactive polymers such as poly(sodium styrene sulfonate) (polyNaSS) onto titanium (Ti) surfaces. This grafting process uses a modified dopamine as an anchor molecule to link polyNaSS to the Ti surface. The grafting process combines reversible addition-fragmentation chain transfer polymerization, postpolymerization modification, and thiol-ene chemistry. The first step in the process is to synthetize architecture controlled polyNaSS with a thiol end group. The second step is the adhesion of the dopamine acrylamide (DA) anchor onto the Ti surfaces. The last step is grafting polyNaSS to the DA-modified Ti surfaces. The modified dopamine anchor group with its bioadhesive properties is essential to link bioactive polymers to the Ti surface. The polymers are characterized by conventional methods (nuclear magnetic resonance, size exclusion chromatography, and attenuated total reflection-Fourier-transformed infrared), and the grafting is characterized by x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, and quartz crystal microbalance with dissipation monitoring. To illustrate the biocompatibility of the grafted Ti-DA-polyNaSS surfaces, their interactions with proteins (albumin and fibronectin) and cells are investigated. Both albumin and fibronectin are readily adsorbed onto Ti-DA-polyNaSS surfaces. The biocompatibility of modified Ti-DA-polyNaSS and control ungrafted Ti surfaces is tested using human bone cells (Saos-2) in cell culture for cell adhesion, proliferation, differentiation, and mineralization. This study presents a new, simple way to graft bioactive polymers onto Ti surfaces using a catechol intermediary with the aim of demonstrating the biocompatibility of these size controlled polyNaSS grafted surfaces.

Biomineralisation with Saos-2 bone cells on TiSiN sputtered Ti alloys.

Surface modifications of metallic implants are important in order to protect the underlying metals from the harsh corrosive environment inside the human body and to minimize the losses caused by wear. Recently, researches are carried out in developing bioactive surfaces on metallic implants, which supports the growth and proliferation of cells on to these surfaces. Titanium silicon nitride (TiSiN) hard nanocomposites thin films were fabricated on Ti alloys (Ti-6Al-4V) by pulsed direct current (DC) reactive magnetron sputtering. The films were characterized for its microstructural and electrochemical behavior. The higher charge transfer resistance (Rct) and positive shift in Ecorr value of TiSiN/Ti alloys than the bare Ti-alloys indicates a better corrosion resistance offered by the TiSiN thin films to the underlying substrates. The biological response to TiSiN/Ti alloys and control bare Ti-alloys was measured in vitro using cell-based assays with two main outcomes. Firstly, neither the Ti alloy nor the TiSiN thin film was cytotoxic to cells. Secondly, the TiSiN thin film promoted differentiation of human bone cells above the bare control Ti alloy as measured by alkaline phosphatase and calcium production. TiSiN thin films provide better corrosion resistance and protect the underlying metal from the corrosive environment. The thin film surface is both biocompatible and bioactive as indicated from the cytotoxicity and biomineralization studies.

Bone tissue response induced by bioactive polymer functionalized Ti6Al4V surfaces: In vitro and in vivo study.

Ti6Al4V is commonly used for orthopedic applications. This study was designed to test the potentially added benefit of Ti6Al4V functionalized with a bioactive polymer poly(sodium styrene sulfonate) both in vitro and in vivo. Cell-based assays with MC3T3-E1 osteoblast-like cells were used to measure the cell adhesion strength, cell spreading, focal contact formation, cell differentiation and the mineralization of extracellular matrix on grafted and ungrafted Ti6Al4V discs in combination with FBS and collagen type I. Bone morphogenetic protein-2 (BMP-2) was also included in the cell differentiation assay. Results showed that the grafted surface combined with collagen I gave superior levels in every parameter tested with cell-based assays and was almost equivalent to BMP-2 for cell differentiation. In vivo testing was conducted in rabbits (n=42) with cylinders of grafted and ungrafted Ti6Al4V implanted in defects made to the femoral and lateral condyles and animals that were maintained to 1, 3 and 12months. Hydroxyapatite coated Ti6Al4V cylinders were included as a clinical reference control. Osseointegration was assessed post-mortem using histomorphometric analysis conducted on resin sections of explanted undecalcified bone. Two histomorphometric parameters, that of bone-to-implant contact and the bone area, were analyzed by a trained observer blinded to sample identity. Results showed osseointegration on grafted Ti6Al4V was marginally better than both ungrafted and hydroxyapatite coated Ti6Al4V. Overall, the study found that the grafted Ti6Al4V significantly promoted all aspects of osteogenesis tested in vitro and, although in vivo outcomes were less compelling, histomorphometry showed osseointegration of grafted Ti6Al4V implants was equivalent or better than controls.

Coencapsulation of Target Effector Cells With Mesenchymal Stem Cells Reduces Pericapsular Fibrosis and Improves Graft Survival in a Xenotransplanted Animal Model.

Pericapsular fibrotic overgrowth (PFO) is a problem that thwarts full implementation of cellular replacement therapies involving encapsulation in an immunoprotective material, such as for the treatment of diabetes. Mesenchymal stem cells (MSCs) have inherent anti-inflammatory properties. We postulated that coencapsulation of MSCs with the target cells would reduce PFO. A hepatoinsulinoma cell line (HUH7) was used to model human target cells and was coencapsulated with either human or mouse MSCs at different ratios in alginate microcapsules. Viability of encapsulated cells was assessed in vitro and xenografted either intraperitoneally or subcutaneously into C57BL/6 mice. Graft retrieval was performed at 3 weeks posttransplantation and assessed for PFO. Coencapsulation of human MSCs (hMSCs) or mouse MSCs (mMSCs) with HUH7 at different ratios did not alter cell viability in vitro. In vivo data from intraperitoneal infusions showed that PFO for HUH7 cells coencapsulated with hMSCs and mMSCs in a ratio of 1:1 was significantly reduced by ∼30% and ∼35%, respectively, compared to HUH7 encapsulated alone. PFO for HUH7 cells was reduced by ∼51% when the ratio of mMSC/HUH7 was increased to 2:1. Implanting the microcapsules subcutaneously rather than intraperitoneally substantially reduced PFO in all treatment groups, which was most significant in the mMSC/HUH7 2:1 group with a ∼53% reduction in PFO compared with HUH7 alone. Despite the reduced PFO reaction to the individual microcapsules implanted subcutaneously, all microcapsule treatment groups were contained in a vascularized fibrotic pouch at 3 weeks. The presence of MSCs in microcapsules retrieved from these fibrotic pouches improved graft survival with significantly higher cell viabilities of 83.1 ± 0.6% and 79.1 ± 0.8% seen with microcapsules containing mMSC/HUH7 at 2:1 and 1:1 ratios, respectively, compared to HUH7 alone (51.5 ± 0.7%) transplanted subcutaneously. This study showed that coencapsulation of MSCs with target cells has a dose-dependent effect on reducing PFO and improving graft survival when implanted either intraperitoneally or subcutaneously in a stringent xenotransplantation setting.

Contributions of adhesive proteins to the cellular and bacterial response to surfaces treated with bioactive polymers: case of poly(sodium styrene sulfonate) grafted titanium surfaces.

The research developed on functionalized model or prosthetic surfaces with bioactive polymers has raised the possibility to modulate and/or control the biological in vitro and in vivo responses to synthetic biomaterials. The mechanisms underlying the bioactivity exhibited by sulfonated groups on surfaces involves both selective adsorption and conformational changes of adsorbed proteins. Indeed, surfaces functionalized by grafting poly(sodium styrene sulfonate) [poly(NaSS)] modulate the cellular and bacterial response by inducing specific interactions with fibronectin (Fn). Once implanted, a biomaterial surface is exposed to a milieu of many proteins that compete for the surface which dictates the subsequent biological response. Once understood, this can be controlled by dictating exposure of active binding sites. In this in vitro study, we report the influence of binary mixtures of proteins [albumin (BSA), Fn and collagen type I (Col I)] adsorbed on poly(NaSS) grafted Ti6Al4V on the adhesion and differentiation of MC3T3-E1 osteoblast-like cells and the adhesion and proliferation of Staphylococcus aureus (S. aureus). Outcomes showed that poly(NaSS) stimulated cell spreading, attachment strength, differentiation and mineralization, whatever the nature of protein provided at the interface compared with ungrafted Ti6Al4V (control). While in competition, Fn and Col I were capable of prevailing over BSA. Fn played an important role in the early interactions of the cells with the surface, while Col I was responsible for increased alkaline phosphatase, calcium and phosphate productions associated with differentiation. Poly(NaSS) grafted surfaces decreased the adhesion of S. aureus and the presence of Fn on these chemically altered surfaces increased bacterial resistance ≈70% compared to the ungrafted Ti6Al4V. Overall, our study showed that poly(NaSS) grafted Ti6Al4V selectively adsorbed proteins (particularly Fn) promoting the adhesion and differentiation of osteoblast-like cells while reducing bacterial adhesion to create a bioactive surface with potential for orthopaedic applications.

Contribution of fibronectin and vitronectin to the adhesion and morphology of MC3T3-E1 osteoblastic cells to poly(NaSS) grafted Ti6Al4V.

This study is focused on understanding the underlying mechanisms involved in the improved in vitro and in vivo responses of osteoblasts on poly(sodium styrene sulfonate) (poly(NaSS)) functionalized Ti6Al4V surfaces. We probed the contribution of cell-adhesive glycoproteins fibronectin (Fn) and vitronectin (Vn) in the initial adhesion of MC3T3-E1 osteoblastic cells to poly(NaSS) functionalized and control Ti6Al4V surfaces. Firstly, culture media containing serum depleted of Fn and Vn (DD) were used to establish the contribution of Fn and Vn in the adhesion and spreading of cells on poly(NaSS) grafted and control surfaces. Compared to ungrafted surfaces, poly(NaSS) grafted surfaces enhanced the levels of cell adhesion, cell spreading and the formation of intracellular actin cytoskeleton and focal contacts in serum treatments where Fn or Vn were present (FBS, DD+Fn, DD+Vn). Cell responses to Fn were more significant than to Vn. Secondly, blocking Fn and Vn integrin receptors using antibodies to α5ß1 (Fn) and αvß1 (Vn) showed that adhesion of cells to poly(NaSS) grafted surfaces principally involved the Fn integrin receptor α5ß1. Thirdly, blocking of the heparin and cell-binding regions of Fn molecule (RGD, C-HB, N-HB) showed that grafting with poly(NaSS) altered the conformation of Fn. Together these outcomes explained why the presence of sulfonate (SO3(-)) groups grafted on the Ti6Al4V surface enhanced the early cell adhesion and spreading processes which determine clinical success for applications that require osseointegration. STATEMENT OF SIGNIFICANCE: This study is devoted to the basic analysis of the mechanism at the origin of the improved in vitro and in vivo osteoblast cell responses exhibited by poly(sodium styrene sulfonate) (poly(NaSS)) functionalized Ti6Al4V surfaces. The aim was to probe the contribution of cell adhesive glycoproteins fibronectin and vitronectin in the initial adhesion of MC3T3-E1 osteoblastic cells to poly(NaSS) functionalized Ti6Al4V surfaces. The outcomes of this research explained why the presence of SO3(-) (sulfonate) groups grafted on the Ti6Al4V surface enhanced the early cell adhesion and spreading processes which determine clinical success for applications that require osseointegration. This work is a step further in the research of poly(NaSS), a very promising bioactive polymer with potential to the orthopedic and dental fields.

Self-assembled gold coating enhances X-ray imaging of alginate microcapsules.

Therapeutic biomolecules produced from cells encapsulated within alginate microcapsules (MCs) offer a potential treatment for a number of diseases. However the fate of such MCs once implanted into the body is difficult to establish. Labelling the MCs with medical imaging contrast agents may aid their detection and give researchers the ability to track them over time thus aiding the development of such cellular therapies. Here we report the preparation of MCs with a self-assembled gold nanoparticle (AuNPs) coating which results in distinctive contrast and enables them to be readily identified using a conventional small animal X-ray micro-CT scanner. Cationic Reversible Addition-Fragmentation chain Transfer (RAFT) homopolymer modified AuNPs (PAuNPs) were coated onto the surface of negatively charged alginate MCs resulting in hybrids which possessed low cytotoxicity and high mechanical stability in vitro. As a result of their high localized Au concentration, the hybrid MCs exhibited a distinctive bright circular ring even with a low X-ray dose and rapid scanning in post-mortem imaging experiments facilitating their positive identification and potentially enabling them to be used for in vivo tracking experiments over multiple time-points.

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system.

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Atmospheric gas plasma-induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis.

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PURPOSE: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. METHODS: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. RESULTS: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. CONCLUSIONS: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal.

OBJECTIVE: To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears. ANIMAL STUDIED: Domestic short-haired cats (7-17 months; 2.6-5.2 kg) were used. PROCEDURES: Eye-flush tears were collected periodically for up to 18 weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n = 4) or with nictitating membranes intact (n = 4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix-metalloproteinase (MMP)-9 measurements using sandwich enzyme-linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP-2 and -9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18 weeks were also investigated in histological sections using immunoperoxidase for visualization. RESULTS: Nictitating membrane removal did not significantly change TPC and MMP-9 in tears within the first 4 weeks. MMP-9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP-2 and -9 activity in tears from eyes without nictitating membranes at week 1 and a decrease following week 2 post-surgery. MMP-2 and -9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18 weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes. CONCLUSIONS: Based on this preliminary study, nictitating membrane removal appeared to cause long-term changes in expression of tear proteins, including reduced MMP-9 expression.

Encapsulated pancreatic progenitors derived from human embryonic stem cells as a therapy for insulin-dependent diabetes.

BACKGROUND: Cellular-based therapies for insulin-dependent diabetes are potential means of achieving and maintaining normal blood glucose levels (BGL) without the need for insulin administration. Islets isolated from donor pancreases have been the most common tissue used to date, but supply is a limiting factor. The use of human embryonic stem cells (hESC) as a therapy became a possibility with the report that these cells could be differentiated to pancreatic progenitors (PP) over 12 days in vitro. Conversion of PP to glucose-responsive insulin-secreting cells can be achieved by transplanting the progenitors in vivo where cell maturation occurs. To date this step has not been shown under in vitro conditions. METHODS: Prior to transplanting, cells are encapsulated in alginate to prevent the immune cells of recipient attacking the graft. The alginate capsules have pores with a molecular weight cut-off of 250 kDa. These are too small to allow entry of immune cells, but large enough for passage of nutrients and insulin. RESULTS: Encapsulated insulin-producing cells survive and function when transplanted, and have been shown to normalize BGL when allografted into diabetic mice. As few as 750 encapsulated human islets are sufficient to normalize BGL of diabetic non-obese diabetic severe combined immunodeficient (NOD/SCID) recipient mice for at least 2 months. The safety of transplanting encapsulated human islets as demonstrated by the lack of major adverse events and infection was recently shown in a first-in-human clinical trial. Finally, fetal porcine islet-like cell clusters, which are akin to PP derived from ESC, mature and normalize BGL of diabetic recipient mice with the same efficiency as non-encapsulated clusters placed under the kidney capsule. CONCLUSION: Transplanting encapsulated PP, derived from hESCs, into diabetic recipients is the strategy that is now being explored in the Australia Diabetes Therapy Project.

The influence of surface topography of a porous perfluoropolyether polymer on corneal epithelial tissue growth and adhesion.

Design principles for corneal implants are challenging and include permeability which inherently involves pore openings on the polymer surface. These topographical cues can be significant to a successful clinical outcome where a stratified epithelium is needed over the device surface, such as with a corneal onlay or corneal repair material. The impact of polymer surface topography on the growth and adhesion of corneal epithelial tissue was assessed using porous perfluoropolyether membranes with a range of surface topography. Surfaces were characterised by AFM and XPS, and the permeability and water content of membranes was measured. Biological testing of membranes involved a 21-day in vitro tissue assay to evaluate migration, stratification and adhesion of corneal epithelium. Similar parameters were monitored in vivo by surgically implanting membranes into feline corneas for up to 5 months. Data showed optimal growth and adhesion of epithelial tissue in vitro when polymer surface features were below a 150 nm RMS value. Normal processes of tissue growth and adhesion were disrupted when RMS values approached 300 nm. Data from the in vivo study confirmed these findings. Together, outcomes demonstrated the importance of surface topography in the design of implantable devices that depend on functional epithelial cover.

A perfluoropolyether corneal inlay for the correction of refractive error.

This study assessed the long-term biological response of a perfluoropolyether-based polymer developed as a corneal inlay to correct refractive error. The polymer formulation met chemical and physical specifications and was non-cytotoxic when tested using standard in vitro techniques. It was cast into small microporous membranes that were implanted as inlays into corneas of rabbits (n = 5) and unsighted humans (n = 5 + 1 surgical control) which were monitored for up to 23 and 48 months respectively. Overall, the inlays were well tolerated during study period with the corneas remaining clear and holding a normal tear film and with no increased vascularisation or redness recorded. Inlays in three human corneas continued past 48 months without sequelae. Inlays in two human corneas were removed early due to small, focal erosions developing 5 and 24 months post-implantation. Polymer inlays maintained their integrity and corneal position for the study duration although the optical clarity of the inlays reduced slowly with time. Inlays induced corneal curvature changes in human subjects that showed stability with time and the refractive effect was reversed when the inlay was removed. Outcomes showed the potential of a perfluoropolyether inlay as a biologically acceptable corneal implant with which to provide stable correction of refractive error.

A comparison of basal and eye-flush tears for the analysis of cat tear proteins.

PURPOSE: To identify a rapid and effective tear collection method providing sufficient tear volume and total protein content (TPC) for analysis of individual proteins in cats. METHODS: Domestic adult short-haired cats (12-37 months; 2.7-6.6 kg) were used in the study. Basal tears without stimulation and eye-flush tears after instillation of saline (10 µl) were collected using microcapillary tubes from animal eyes either unwounded control or wounded with 9-mm central epithelial debridement giving four groups with n = 3. Tear comparisons were based on total time and rate for tear collection, TPC using micro bicinchoninic acid (BCA), tear immunoglobulin A (IgA), total matrix-metalloproteinase (MMP)-9 concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity. RESULTS: Eye-flush tears were collected significantly faster than basal tears in wounded eyes with higher rates for tear collection in unwounded control and wounded eyes. TPC was significantly lower in eye-flush tears compared to basal tears. The relative proportion of tear IgA normalized to TPC (% IgA of TPC) was not significantly different between basal and eye-flush tears. In unwounded control eyes, MMP-9 was slightly higher in eye-flush than in basal tears; activity of MMP-9 in both tear types was similar. In wounded eyes, eye-flush tears showed highest MMP-9 levels and activity on Day 1, which subsequently decreased to Day 7. MMP-9 activity in basal tears from wounded eyes did not display changes in expression. CONCLUSIONS: Eye-flush tears can be collected rapidly providing sufficient tear volume and TPC. This study also indicates that eye-flush tears may be more suitable than basal tears for the analysis of MMPs following corneal wounding.

Biocompatibility and modification of the protein-based adhesive secreted by the Australian frog Notaden bennetti.

When provoked, Notaden bennetti frogs secrete a proteinaceous exudate, which rapidly forms a tacky and elastic glue. This material has potential in biomedical applications. Cultured cells attached and proliferated well on glue-coated tissue culture polystyrene, but migrated somewhat slower than on uncoated surfaces. In organ culture, dissolved glue successfully adhered collagen-coated perfluoropolyether lenses to debrided bovine corneas and supported epithelial regrowth. Small pellets of glue implanted subcutaneously into mice were resorbed by surrounding tissues, and all of the animals made a full recovery. An initial but transient skin necrosis at the implant site was probably caused by some of the potentially toxic metabolites present in the frog secretion; these include sterols and carotenoids, as well as fatty alcohols, aldehydes, ketones, acids, and aromatic compounds. Removal of the carotenoid pigments did not significantly alter the glue's material properties. In contrast, peroxidase treatment of dissolved glue introduced unnatural crosslinks between molecules of the major protein (Nb-1R) and resulted in the formation of a soft hydrogel, which was very different to the original material.