Tissue engineering of co-cultured skin substitutes using biocomposite membranes based on collagen and polycaprolactone

The preparation and characterisation of collagen: PCL, gelatin: PCL and gelatin/collagen:PCL biocomposites for manufacture of tissue engineered skin substitutes are reported. Films of collagen: PLC, gelatin: PCL (1:4, 1:8 and 1:20 w/w) and gelatin/collagen:PCL (1:8 and 1:20 w/w) biocomposites were prepared by impregnation of lyophilised collagen and/or gelatin mats by PCL solutions followed by solvent evaporation. In vitro assays of total protein release of collagen:PCL and gelatin: PCL biocomposite films revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the biocomposite samples that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. Good compatibility of all biocomposite groups was proven by interaction with 3T3 fibroblasts, normal human epidermal keratinocytes (NHEK), and primary human epidermal keratinocytes (PHEK) and dermal fibroblasts (PHDF) in vitro respectively. The 1:20 collagen: PCL materials exhibiting good cell growth curves and mechanical characteristics were selected for engineering of skin substitutes in this work. The tissue-engineered skin model based on single-donor PHEK and PHDF with differentiated confluent epidermal layer and fibrous porous dermal layer was then developed successfully in vitro proven by SEM and immunohistochemistry assay. The following in vivo animal study on athymic mice revealed early complete wound healing in 10 days and good integration of co-cultured skin substitutes with adjacent mice skin structures. Thus the co-cultured skin substitutes based on 1:20 collagen: PCL biocomposite membranes was proven in principle. The approach to skin modelling reported here may find application in wound treatment, gene therapy and screening of new pharmaceuticals.

Evaluation of biodegradable polymer microspheres and an anionic dendrimer for the potential delivery of antisense oligonucleotides

Antisense oligonucleotides (AODNs) can selectively inhibit individual gene expression by binding specifically to rnRNA. The over-expression of the epidermal growth factor receptor (EGFR) has been observed in human breast and glioblastoma tumours and therefore AODNs designed to target the EGFR would be a logical approach to treat such tumours. However, poor pharmacokinetic/pharmacodynamic and cellular uptake properties of AODNs have limited their potential to become successful therapeutic agents. Biodegradable polymeric poly (lactide-co-glycolide) (P(LA-GA)) and dendrimer delivery systems may allow us to overcome these problems. The use of combination therapy of AODNs and cytotoxic agents such as 5-fluorouracil (5-FU) in biodegradable polymeric formulations may further improve therapeutic efficacy. AODN and 5-FU were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations (double emulsion method) and release profiles determined in vitro. The release rates (biphasic) of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Sustained release over 35 days was observed in both types of formulation. Naked and microsphere-loaded AODN and 5-FU (in separate formulations) were tested on an A431 vulval carcinoma cell line. Combining naked or encapsulated drugs produced a greater reduction in viable cell number as compared with either agent alone. However, controls and Western blotting indicated that non-sequence specific cytotoxic effects were responsible for the differences in viable cell number. The uptake properties of an anionic dendrimer based on a pentaerythritol structure covalently linked to AODNs (targeting the EGFR) have been characterised. The cellular uptake of AODN linked to the dendrimer was up to 3.5-fold higher in A431 cells as compared to naked AODN. Mechanistic studies suggested that receptor-mediated and adsorptive (binding protein-mediated) endocytosis were the predominant uptake mechanisms for the dendrimer-AODN. RNase H cleavage assay suggested that the dendrimer-AODN was able to bind and cleave the target site. A reduction of 20%, 28% and 45% in EGFR expression was observed with 0.05μM, 0.1μM and 0.5μM dendrimer-AODN treatments respectively with a reduction in viable cell number. These results indicated that the dendrimer delivery system may reduce viable cell number by an antisense specific mechanism.

Improving the function of islet and beta-cell grafts

Background: Human islet transplantation would offer a less invasive and more physiological alternative than whole pancreas transplantation and insulin injections respectively for the treatment of diabetes mellitus if islet graft survival can be improved. Initial recipient post-transplant insulin independence declines to <10% after 5 years. Factors contributing to graft failure include enzymatic disruption of the islet microenvironment during isolation, diabetogenic effects of immunosuppressants and metabolic stress resulting from slow revascularisation. Aims: To investigate the effect of co-culture in both static (SC) and rotational culture (RC) of BRINBDII beta-cells (Dl1) and human umbilical vein endothelial cells (HUVEC) on Dl1 insulin secretion; and the effect of a thiazolidinedione (TZD) on DII function and HUVEC proliferation. To assess the effect of culture media, SC, RC and a TZD on human islet morphology, insulin secretion and VEGF production. To initiate in vivo protocol development for assessment of revascularisation of human islet grafts. Methods: D11 cells were cultured +/-TZD and co-cultured with HUVEC +/-TZD in SC and RC. Dl1 insulin secretion was induced by static incubation with low glucose (1.67mM), high glucose (l6.7mM: and high glucose with 10mM theophylline (G+T) and determined by ELISA. HUVEC were cultured +/-TZD in SC and RC and proliferation was assessed by ATP luminescence assay and VEGF ELISA. D II and HUVEC morphology was determined by immunocytochemistry. Human islets were cultured in SC and RC in various media +/-TZD. Insulin secretion was determined as above and VEGF production by fluorescence immunocytochemistry (FI) and ELISA. Revascularisation of islet grafts was assessed by vascular corrosion cast and FI. Results: Dll cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and further improved by adding 10mM TZD. Untreated Dll/HUVEC co-cultures displayed significantly increased insulin secretion in response to 16.7mM and G+T over basal, again enhanced by RC and improved with 10mM TZD. 10mM TZD significantly increased HUVEC proliferation over control. Human islets maintained in medium 199 (mI99) in SC and RC exhibited comparable maintenance of morphology and insulin secretory profiles compared to islets maintained in RPMI, endothelial growth media and dedicated islet medium Miami# I. All cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and in certain instances further improved by adding 25mM TZD. TZD increased VEGF production and release as determined by ELISA. Post-implant vascular corrosion casts of mouse kidneys analysed by x-ray micro tomography indicates a possible TZD enhancement of microvessel growth via VEGF upregulation. Conclusions: D II /HUVEC co-culture in SC or RC does not alter the morphology of either cell type and supports D 11 function. TZD improves 0 I I and D I I/HUVEC SC and RC co-culture insulin secretion while increasing HUVEC proliferation. Human islet RC supports islet functional viability and structural integrity compared to SC while the addition of TZD occasionally further improves secretagogue induced insulin secretion. Expensive, 'dedicated' islet media showed no advantage over ml99 in terms of maintaining islet morphology or function. TZD upregulates VEGF in islets as shown by ELISA and suggested by x-ray micro tomography analysis of vascular corrosion casts. Maintenance of islets in RC and treatment with TZD prior to transplant may improve the functional viability and revascularisation rate of islet grafts.

New strategy to overcome the intrinsic difficulty of phospholipids solubilisation and delivery to the eye

Purpose: Lipids play a vital role at interfaces such as the tear film in the protection of the anterior eye. Their role is to act as lubricants and reduce surface and interfacial tension. Although there is a lack of appropriate methods to solubilize and dilute phospholipids to the tear film. Here, we report that styrene-maleic acid copolymers (PSMA), can form polymer–lipid complexes in the form of monodisperse nanometric particles, which can easily solubilise these phospholipid molecules by avoiding for example, the use of any kind of surfactant. Method: The interactions of PSMA with phospholipids have been studied by its adsorption from aqueous solutions into monolayers of dimyristoyl-phosphorylcholine (DMPC). The Langmuir trough (LT) technique is used to study this pH-dependant complex formation. The formed nanoparticles have been also analysed by 31P NMR, particle size distribution by light scattering (DLS) and morphology by electron microscopy (SEM). Results: The LT has been found to be a useful technique for in vitro simulation of in vivo lipid layer behaviour: The limiting surface pressure of unstable tear films ranges between 20 and 30 mN/m. More stable tear films show an increase in surface pressure, within the range of 35–45 mN/m. The DMPC monolayers have a limiting surface pressure of 38 mN/m (water), and 45 mN/m (pH 4 buffer), and the PSMA-DMPC complexes formed at pH 4 have a value of 42 mN/m, which resembles that of the stable tear film. The average particle size distribution is 53 ± 10 nm with a low polydispersity index (PDI) of 0.24 ± 0.03. Conclusions: New biocompatible and cheap lipid solubilising agents such as PSMA can be used for the study of the tear film composition and properties. These polymer–lipid complexes in the form of nanoparticles can be used to solubilise and release in a controlled way other hydrophobic molecules such as some drugs or proteins.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair. © 2003 Elsevier Ltd. All rights reserved.

Activation of proteinase-activated receptor 2 stimulates soluble vascular endothelial growth factor receptor 1 release via epidermal growth factor receptor transactivation in endothelial cells

The proteinase-activated receptor 2 (PAR-2) expression is increased in endothelial cells derived from women with preeclampsia, characterized by widespread maternal endothelial damage, which occurs as a consequence of elevated soluble vascular endothelial growth factor receptor-1 (sVEGFR-1; commonly known as sFlt-1) in the maternal circulation. Because PAR-2 is upregulated by proinflammatory cytokines and activated by blood coagulation serine proteinases, we investigated whether activation of PAR-2 contributed to sVEGFR-1 release. PAR-2–activating peptides (SLIGRL-NH2 and 2-furoyl-LIGRLO-NH2) and factor Xa increased the expression and release of sVEGFR-1 from human umbilical vein endothelial cells. Enzyme-specific, dominant-negative mutants and small interfering RNA were used to demonstrate that PAR-2–mediated sVEGFR-1 release depended on protein kinase C-ß1 and protein kinase C-e, which required intracellular transactivation of epidermal growth factor receptor 1, leading to mitogen-activated protein kinase activation. Overexpression of heme oxygenase 1 and its gaseous product, carbon monoxide, decreased PAR-2–stimulated sVEGFR-1 release from human umbilical vein endothelial cells. Simvastatin, which upregulates heme oxygenase 1, also suppressed PAR-2–mediated sVEGFR-1 release. These results show that endothelial PAR-2 activation leading to increased sVEGFR-1 release may contribute to the maternal vascular dysfunction observed in preeclampsia and highlights the PAR-2 pathway as a potential therapeutic target for the treatment of preeclampsia.

Trapping and unlimited delay of light pulses at microscale without distortion

A tunable bottle microresonator can trap an optical pulse of the given spectral width, hold it as long as the material losses permit, and release without distortion.

On arterial contractility, energetic requirements of the cardiovascular system, and the formation of arterial sclerosis

The aorta has been viewed as a passive distribution manifold for blood whose elasticity allows it to store blood during cardiac ejection (systole), and release it during relaxation (diastole). This capacitance, or compliance, lowers peak cardiac work input and maintains peripheral sanguine irrigation throughout the cardiac cycle. The compliance of the human and canine circulatory systems have been described either as constant throughout the cycle (Toy et al. 1985) or as some inverse function of pressure (Li et al. 1990, Cappelo et al. 1995). This work shows that a compliance value that is higher during systole than diastole (equivalent to a direct function of pressure) leads to a reduction in the energetic input to the cardiovascular system (CV), even when accounting for the energy required to change compliance. This conclusion is obtained numerically, based on a 3-element lumped-parameter model of the CV, then demonstrated in a physical model built for the purpose. It is then shown, based on the numerical and physical models, on analytical considerations of elastic tubes, and on the analysis of arterial volume as a function of pressure measured in vivo (Armentano et al. 1995), that the mechanical effects of a presupposed arterial contraction are consistent with those of energetically beneficial changes in compliance during the cardiac cycle. Although the amount of energy potentially saved with rhythmically contracting arteries is small (mean 0.55% for the cases studied) the importance of the phenomenon lies in its possible relation to another function of the arterial smooth muscle (ASM): synthesis of wall matrix macromolecules. It is speculated that a reduction in the rate of collagen synthesis by the ASM is implicated in the formation of arteriosclerosis. ^

Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Benthic Exchange of C, N, and P Along the Estuarine Ecotone of Lower Taylor Slough, Florida (USA): Effect of Seasonal Flows and Phosphorus Availability

The southern Everglades and Florida Bay have experienced a nearly 50 % reduction in freshwater flow resulting in increased salinity and landward expansion of mangrove forest. Given the marine end-member is a natural source of P to this region, it is necessary to understand the interactions between inflows and P availability in controlling the exchange of materials across the mangrove ecotone. From 2007 to 2008, we used sediment core incubations to quantify fluxes of dissolved inorganic N and P and dissolved organic carbon (DOC) in three ecotone areas (dwarf mangrove, pond, and bay). Experiments were repeated seasonally over 2 years involving P-enriched surface water as a factor. We saw consistent uptake of soluble reactive P (SRP), DOC, and nitrate + nitrite (N+N) by the soils/sediments and release of ammonium (NH4 +) from soils/sediments to the water column across all sites and seasons. P enrichment had no discernible effect on DIN or DOC flux, suggesting that rapid P uptake may have been more geochemically mediated. However, uptake of added P occurred across all sites and seasons, reflecting high uptake capacity in this carbonate system and the potential of the mangrove ecotone to sequester P as it becomes more available.

(Table 1) Dissolved Hafnium and Neodymium in the South Atlantic measured on water bottle samples during POLARSTERN cruise ANT-XXIV/3

We present the first combined dissolved hafnium (Hf) and neodymium (Nd) concentrations and isotope compositions of deep water masses from the Atlantic sector of the Southern Ocean. Eight full depth profiles were analyzed for Hf and twelve for Nd. Hafnium concentrations are generally depleted in the upper few hundred meters ranging between 0.2 pmol/kg and 0.4 pmol/kg and increase to relatively constant values of around 0.6 pmol/kg in the deeper water column. At the stations north of the Polar Front (PF), Nd concentrations increase linearly from about 10 pmol/kg at depths of ~ 200 m to up to 31 pmol/kg close to the bottom indicating particle scavenging and release. Within the Weddell Gyre (WG), however, Nd concentrations are essentially constant at 25 pmol/kg at depths greater than ~ 1000 m. The distributions of both elements show a positive correlation with dissolved silicon implying a close linkage to diatom biogeochemistry. Hafnium essentially shows invariant isotope compositions with values averaging at epsilon-Hf = +4.6, whereas Nd isotopes mark distinct differences between water masses, such as modified North Atlantic Deep Water (NADW, epsilon-Nd = -11 to -10) and Antarctic Bottom Water (AABW, epsilon-Nd = -8.6 to -9.6), but also waters locally advected via the Agulhas Current can be identified by their unradiogenic Nd isotope compositions. Mixing calculations suggest that a small fraction of Nd is removed by particle scavenging during mixing of water masses north of the PF. Nevertheless, the Nd isotope composition has apparently not been significantly affected by uptake and release of Nd from particles, as indicated by mixing calculations. A mixing envelope of an approximated North Pacific and a North Atlantic end-member shows that Nd isotope and concentration patterns in the Lower Circumpolar Deep Water (LCDW) can be fully explained by ~ 30:70 percentage contributions of these respective end-members.

Distribution of neodymium isotopes and concentrations during M77/3 and M77/4

The radiogenic isotope composition of the Rare Earth Element (REE) neodymium (Nd) is a powerful water mass proxy for present and past ocean circulation. The processes controlling the Nd budget of the global ocean are not quantitatively understood and in particular source and sink mechanisms are still under debate. In this study we present the first full water column data set of dissolved Nd isotope compositions and Nd concentrations for the Eastern Equatorial Pacific (EEP), where one of the globally largest Oxygen Minimum Zones (OMZ) is located. This region is of particular interest for understanding the biogeochemical cycling of REEs because anoxic conditions may lead to release of REEs from the shelf, whereas high particle densities and fluxes potentially remove the REEs from the water column. Data were obtained between 11400N and 161S along a nearshore and an offshore transect. Near surface zonal current bands, such as the Equatorial Undercurrent (EUC) and the Subsurface Countercurrent (SSCC), which are supplying oxygen-rich water to the OMZ are characterized by radiogenic Nd isotope signatures (eNd=-2). Surface waters in the northernmost part of the study area are even more radiogenic (eNd = +3), most likely due to release of Nd from volcanogenic material. Deep and bottom waters at the southernmost offshore station (141S) are clearly controlled by advection of water masses with less radiogenic signatures (eNd=- 7) originating from the Southern Ocean. Towards the equator, however, the deep waters show a clear trend towards more radiogenic values of up to eNd=-2. The northernmost station located in the Panama basin shows highly radiogenic Nd isotope signatures in the entire water column, which indicates that particle scavenging, downward transport and release processes play an important role. This is supported by relatively low Nd concentrations in deep waters (3000-6000 m) in the EEP (20 pmol/kg) compared to locations in the Northern and Central Pacific (40-60 pmol/kg), which suggests enhanced removal of Nd in the EEP.

Geochemical data and radiocarbon ages of organic matter fractions and bacteriohopanepolyol data of sediment core GeoB6518-1 from the Congo River basin

The age of organic material discharged by rivers provides information about its sources and carbon cycling processes within watersheds. While elevated ages in fluvially-transported organic matter are usually explained by erosion of soils and sediments, it is commonly assumed that mainly young organic material is discharged from flat tropical watersheds due to their extensive plant cover and high carbon turnover. Here we present compound-specific radiocarbon data of terrigenous organic fractions from a sedimentary archive offshore the Congo River in conjunction with molecular markers for methane-producing land cover reflecting wetland extent in the watershed. We find that the Congo River has been discharging aged organic matter for several thousand years with increasing ages from the mid- to the Late Holocene. This suggests that aged organic matter in modern samples is concealed by radiocarbon from nuclear weapons testing. By comparison to indicators for past rainfall changes we detect a systematic control of organic matter sequestration and release by continental hydrology mediating temporary carbon storage in wetlands. As aridification also leads to exposure and rapid remineralization of large amounts of previously stored labile organic matter we infer that this process may cause a profound direct climate feedback currently underestimated in carbon cycle assessments.

Spatially explicit estimates of stock size, structure and biomass of North Atlantic albacore tuna (Thunnus alalunga) in the North Atlantic for the period 1956-2010, compiled from the International Commission for the Conservation of Atlantic Tunas

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.

Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay.

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.