Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration

Influence of iodine value on combustion and NOx emission characteristics of a DI diesel engine

Biodiesel is a renewable fuel that has been shown to reduce many exhaust emissions, except oxides of nitrogen (NOx), in diesel engine cars. This is of special concern in inner urban areas that are subject to strict environmental regulations, such as EURO norms. Also, the use of pure biodiesel (B100) is inhibited because of its higher NOx emissions compared to petroleum diesel fuel. The aim of this present work is to investigate the effect of the iodine value and cetane number of various biodiesel fuels obtained from different feed stocks on the combustion and NOx emission characteristics of a direct injection (DI) diesel engine. The biodiesel fuels were chosen from various feed stocks such as coconut, palm kernel, mahua (Madhuca indica), pongamia pinnata, jatropha curcas, rice bran, and sesame seed oils. The experimental results show an approximately linear relationship between iodine value and NOx emissions. The biodiesels obtained from coconut and palm kernel showed lower NOx levels than diesel, but other biodiesels showed an increase in NOx. It was observed that the nature of the fatty acids of the biodiesel fuels had a significant influence on the NOx emissions. Also, the cetane numbers of the biodiesel fuels are affected both premixed combustion and the combustion rate, which further affected the amount of NOx formation. It was concluded that NOx emissions are influenced by many parameters of biodiesel fuels, particularly the iodine value and cetane number.

Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.

Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2

This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods’ surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods’ surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 μg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 μg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 μg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points

Evaluation of a hybrid scaffold/cell construct in repair of high-load-bearing osteochondral defects in rabbits

The objective of this study was to evaluate the feasibility and potential of a hybrid scaffold system in large- and high-load-bearing osteochondral defects repair. The implants were made of medical-grade PCL (mPCL) for the bone compartment whereas fibrin glue was used for the cartilage part. Both matrices were seeded with allogenic bone marrow-derived mesenchymal cells (BMSC) and implanted in the defect (4 mm diameter×5 mm depth) on medial femoral condyle of adult New Zealand White rabbits. Empty scaffolds were used at the control side. Cell survival was tracked via fluorescent labeling. The regeneration process was evaluated by several techniques at 3 and 6 months post-implantation. Mature trabecular bone regularly formed in the mPCL scaffold at both 3 and 6 months post-operation. Micro-Computed Tomography showed progression of mineralization from the host–tissue interface towards the inner region of the grafts. At 3 months time point, the specimens showed good cartilage repair. In contrast, the majority of 6 months specimens revealed poor remodeling and fissured integration with host cartilage while other samples could maintain good cartilage appearance. In vivo viability of the transplanted cells was demonstrated for the duration of 5 weeks. The results demonstrated that mPCL scaffold is a potential matrix for osteochondral bone regeneration and that fibrin glue does not inherit the physical properties to allow for cartilage regeneration in a large and high-load-bearing defect site. Keywords: Osteochondral tissue engineering; Scaffold; Bone marrow-derived precursor cells; Fibrin glue

In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts

The ideal dermal matrix should be able to provide the right biological and physical environment to ensure homogenous cell and extracellular matrix (ECM) distribution, as well as the right size and morphology of the neo-tissue required. Four natural and synthetic 3D matrices were evaluated in vitro as dermal matrices, namely (1) equine collagen foam, TissuFleece®, (2) acellular dermal replacement, Alloderm®, (3) knitted poly(lactic-co-glycolic acid) (10:90)–poly(-caprolactone) (PLGA–PCL) mesh, (4) chitosan scaffold. Human dermal fibroblasts were cultured on the specimens over 3 weeks. Cell morphology, distribution and viability were assessed by electron microscopy, histology and confocal laser microscopy. Metabolic activity and DNA synthesis were analysed via MTS metabolic assay and [3H]-thymidine uptake, while ECM protein expression was determined by immunohistochemistry. TissuFleece®, Alloderm® and PLGA–PCL mesh supported cell attachment, proliferation and neo-tissue formation. However, TissuFleece® contracted to 10% of the original size while Alloderm® supported cell proliferation predominantly on the surface of the material. PLGA–PCL mesh promoted more homogenous cell distribution and tissue formation. Chitosan scaffolds did not support cell attachment and proliferation. These results demonstrated that physical characteristics including porosity and mechanical stability to withstand cell contraction forces are important in determining the success of a dermal matrix material.

Vitrification as a prospect for cryopreservation of tissue-engineered constructs

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

Sodium removal from Maramarua CSG waters using Ngakuru zeolites

Coal seam gas (CSG) waters are a by-product of natural gas extraction from un derground coal seams. The main issue with these waters is their elevated sodium content, which in conjunction with their low calcium and magnesium concentrations can generate soil infiltration problems in the long run , as well as short term toxicity effects in plants due to the sodium ion itself. Zeolites are minerals having a porous structure, crystalline characteristics, and an alumino-silicate configuration resulting in an overall negative charge which is balanced by loosely held cations. In New Zealand, Ngakuru zeolites have been mined for commercial use in wastewater treatment applications, cosmetics, and pet litter. This research focuses on assessing the capacity of Ngakuru zeolites to reduce sodium concentrations of CSG waters from Maramarua. Batch and column test (flow through) experiments revealed that Ngakuru zeolites are capable of sorbing sodium cations from concentrated solutions of sodium. In b atch tests, the sodium adsorption capacity ranged from 5.0 to 34.3meq/100g depending on the solution concentration and on the number of times the zeolite had been regenerated. Regeneration with CaCl2 was foun d to be effective. The calculated sodium adsorption capacity of Ngakuru zeolites under flow-through conditions ranged from 11 to 42meq/100g depending on the strength of the solution being treated and on w hether the zeolites had been previously regenerated. The slow kinetics and low cost of the zeolities, coupled with potentially remote sites for gas extraction, could make semi-batch operational processes without regeneration more favourable than in more industrial ion exchange situations.

Pressure filtration of Australian bagasse pulp

A one-dimensional pressure filtration model that can be used to predict the behaviour of bagasse pulp has been developed and verified in this study.The dynamic filtration model uses steady state compressibility parameters determined experimentally by uniaxial loading. The compressibility parameters M and N for depithed bagasse pulp were determined to be in the ranges 3000–8000kPa and 2.5–3.0 units, respectively. The model also incorporates experimentally determined steady state permeability data from separate experiments to predict the pulp concentration and fibre pressure throughout a pulp mat during dynamic filtration. Under steady state conditions, a variable Kozeny factor required different values for the permeability parameters when compared to a constant Kozeny factor. The specific surface area was 25–30% lower and the swelling factor was 20–25% higher when a variable Kozeny factor was used. Excellent agreement between experimental data and the dynamic filtration model was achieved when a variable Kozeny factor was used.

Effect of thermal treatment on adsorption-desorption of ammonia and sulfur dioxide on palygorskite : change of surface acid-alkali properties

Thermally activated Palygorskite (Pg) has been found to be a good adsorbent material for ammonia (NH3) and sulfur dioxide (SO2). This research investigated the effect of thermal treatment on pore structure and surface acid-alkali properties of Pg through the adsorption-desorption of NH3 and SO2. The results showed that, up to 200 °C, the adsorption of NH3 on Pg was significantly higher than SO2. This was due to NH3 being adsorbed in the internal surface of Pg and forming hydrogen bonds (H-bonds) with coordinated water. The increase in thermal treatment temp. from 150 to 550 °C, showed a gradual decrease in the no. of surface acid sites, while the no. of surface alk. sites increased from 200 to 400 °C. The change of surface acidity-alk. sites is due to the collapse of internal channels of Pg and desorption of different types of hydroxyls assocd. with the Pg structure.