Expression, purification and in vitro biological activity from human recombinant BMP-2 produced by a novel approach

Bone tissue engineering has been an increasing field of research during the last years. The ideal approach for a regenerative application would consist in the use of cells from the patient, scaffolding materials and differentiation growth factors. Bone morphogenetic protein-2 (BMP-2) is one such growth factors with a strong ability to induce new bone and cartilage formation and has been used as a powerful osteoinductive component of several late-stage tissue engineering products for bone grafting. In this work, we aimed at obtaining high yields of human recombinant BMP-2 in a stable, pure and biologically active form by use of a new bacteria expression system that circumvents the disadvantages of conventional recombinant protein preparation methods and to perform a study of the stability conditions and functionality of these peptides in vitro in human mesenchymal stem cells and C2C12 murine cell line.Portuguese Foundation for Science and Technology, FCT (PhD Grant to PC Bessa, to PC Bessa, SFRH/BD/17049/2004 SFRH/BD/17049/2004 ). This work was ). This work was also partially supported by the European STREP HIPPOCRATES (NMP3 also partially supported by the European STREP HIPPOCRATES (NMP3--CTCT--2003 2003--505758) and carried out under the scope of 505758) and carried out under the scope of European NoE EXPERTISSUES (NMP3 European NoE EXPERTISSUES (NMP3--CTCT- -2004 2004 --500283). 500283info:eu-repo/semantics/publishedVersio

A novel system for producing human recombinant BMP-2 and study of the growth factor stabilizing conditions

Bone tissue engineering has been an increasing field of research during the last years. The ideal approach for a regenerative application would consist in the use of cells from the patient, scaffolding materials and differentiation growth factors. Bone morphogenetic protein-2 (BMP-2) is one such growth factors with a strong ability to induce new bone and cartilage formation and has been used as a powerful osteoinductive component of several late-stage tissue engineering products for bone grafting. In this work, we aimed at obtaining high yields of human recombinant BMP-2 in a stable, pure and biologically active form by use of a new bacteria expression system that circumvents the disadvantages of conventional recombinant protein preparation methods and to perform a study of the stability conditions and the functionality of these peptides in vitro in human mesenchymal stem cells and C2C12 murine cell line.Portuguese Foundation for Science and Technology (PhD Grant to PC Bessa, SFRH/BD/17049/2004). This work was also partially supported by the European STREP HIPPOCRATES (NMP3-CT-2003-505758) and carried out under the scope of European NoE EXPERTISSUES (NMP3-CT-2004- 500283).info:eu-repo/semantics/publishedVersio

Enhanced reporter gene assay for the detection of osteogenic differentiation

Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner

The perceived impact of the National Health Service on personalised nutrition service delivery among the UK public

Personalised nutrition (PN) has the potential to reduce disease risk and optimise health and performance. Although previous research has shown good acceptance of the concept of PN in the UK, preferences regarding the delivery of a PN service (e.g. online v. face-to-face) are not fully understood. It is anticipated that the presence of a free at point of delivery healthcare system, the National Health Service (NHS), in the UK may have an impact on end-user preferences for deliverances. To determine this, supplementary analysis of qualitative data obtained from focus group discussions on PN service delivery, collected as part of the Food4Me project in the UK and Ireland, was undertaken. Irish data provided comparative analysis of a healthcare system that is not provided free of charge at the point of delivery to the entire population. Analyses were conducted using the 'framework approach' described by Rabiee (Focus-group interview and data analysis. Proc Nutr Soc 63, 655-660). There was a preference for services to be led by the government and delivered face-to-face, which was perceived to increase trust and transparency, and add value. Both countries associated paying for nutritional advice with increased commitment and motivation to follow guidelines. Contrary to Ireland, however, and despite the perceived benefit of paying, UK discussants still expected PN services to be delivered free of charge by the NHS. Consideration of this unique challenge of free healthcare that is embedded in the NHS culture will be crucial when introducing PN to the UK

The consumption of 12 Eggs per week for 1 year does not alter fasting serum markers of cardiovascular disease in older adults with early macular degeneration

Some studies suggest that eating more than one egg daily may increase risk of death from cardiovascular disease. The objective of this study was to determine the effects of consuming eggs on various serum markers associated with cardiovascular disease (CVD). Forty-five independently living adults diagnosed with early macular degeneration, but healthy otherwise were recruited into the study. Subjects were placed into the Intervention (n = 27) or Control group (n = 18) based on whether or not they would consume eggs. The Intervention group consumed 12 eggs per week while the Control group refrained from consuming any whole egg products for 1 year. Serum concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), glucose, apolipoprotein (apo) A-1 and apo B, lipoprotein (Lp)a and high-sensitive C reactive protein (hsCRP) were measured at baseline, 6, and 12 months. Serum low-density lipoprotein cholesterol (LDL-C) concentration was calculated via the Friedewald equation. Serum TC, TG, HDL-C, LDL-C, apo A-1, apo B, Lpa and hsCRP concentrations did not change at any time in both the Intervention and Control groups compared to baseline nor were there any differences between the two treatment. Serum glucose concentrations did increase significantly in the Intervention group at 6 months compared to baseline (23%, P < 0.05) but decreased back to baseline concentrations at 12 months. This study suggests that the consumption of 12 eggs per week for 1 year does not significantly alter fasting serum lipids, lipoprotein cholesterol, or other biomarkers of CVD in older adults diagnosed with early macular degeneration. Keywords: Apolipoprotein A-1, Apolipoprotein B, C-reactive protein (CRP), Cardiovascular disease (CVD), HDL cholesterol, LDL cholestero

Thermoresponsive self-assembled elastin-based nanoparticles for delivery of BMPs

Elastin-like polymers are a new type of protein-based polymers that display interesting properties in the biomaterial field. Bone morphogenetic proteins (BMPs) are cytokines with a strong ability to promote new bone formation. In this work, we explored the use of elastin-like nanoparticles (average size 237.5±3.0 nm), created by thermoresponsive self-assembly, for the combined release of bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protein-14 (BMP-14). These BMPs could be encapsulated at high efficiency into the elastin-like particles and delivered in a sustained way for 14 days. The activity of the growth factors was retained, as shown by the induction of ALP activity and osteogenic mineralization in C2C12 cells. Increased bioactivity was observed with a combined release of BMP-2 and BMP-14. This approach shows a significant potential for future tissue engineering applications in bone.This work was supported by Fundacao para a Ciencia e Tecnologia (PhD grants SFRH/BD/17049/2004 and SFRH/BD/36754/2007), project ElastM POCI/CTM/57177/2004 funded by FEDER and Fundacao para a Ciencia e Tecnologia; Marie Curie Alea Jacta EST short-term grant (MEST-CT-2004-8104) and European STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283). We acknowledge financial support from the MICINN (projects MAT 2007-66275-C02-01, NAN2004-08538, and PSE-300100-2006-1), the JCyL (projects VA034A09, VA016B08, and VA030A08), the CIBER-BBN (project CB06-01-0003), the JCyL and the Instituto de Salud Carlos III under the "Network Center of Regenerative Medicine and Cellular Therapy of Castilla and Leon"