RGD constructs with physical anchor groups as polymer co-electrospinnable cell adhesives

The tissue integration of synthetic polymers can be promoted by displaying RGD peptides at the biointerface with the objective of enhancing colonization of the material by endogenous cells. A firm but flexible attachment of the peptide to the polymer matrix, still allowing interaction with receptors, is therefore of interest. Here, the covalent coupling of flexible physical anchor groups, allowing for temporary immobilization on polymeric surfaces via hydrophobic or dipole–dipole interactions, to a RGD peptide was investigated. For this purpose, a stearate or an oligo(ethylene glycol) (OEG) was attached to GRGDS in 51–69% yield. The obtained RGD linker constructs were characterized by NMR, IR and MALDI-ToF mass spectrometry, revealing that the commercially available OEG and stearate linkers are in fact mixtures of similar compounds. The RGD linker constructs were co-electrospun with poly(p-dioxanone) (PPDO). After electrospinning, nitrogen could be detected on the surface of the PPDO fibers by X-ray photoelectron spectroscopy. The nitrogen content exceeded the calculated value for the homogeneous material mixture suggesting a pronounced presentation of the peptide on the fiber surface. Increasing amounts of RGD linker constructs in the electrospinning solution did not lead to a detection of an increased amount of peptide on the scaffold surface, suggesting inhomogeneous distribution of the peptide on the PPDO fiber surface. Human adipose-derived stem cells cultured on the patches showed similar viability as when cultured on PPDO containing pristine RGD. The fully characterized RGD linker constructs could serve as valuable tools for the further development of tissue-integrating polymeric scaffolds

The influence of electrospinning parameters on polydioxanone scaffold properties

Conduits currently used to reconstruct the right ventricular outflow tract (RVOT) have no growth potential and require reoperations, resulting in an increased level of morbidity and mortality. This work investigates the effect of electrospinning parameters on the mechanical properties and biocompatibility of bioresorbable tubular scaffolds, as part of a program to develop a tissue-engineered valved tube for RVOT replacement. Electrospinning was used to develop tubular scaffolds of polydioxanone, with the experimental parameters systematically varied. Three electrospinning parameters (volume of liquid, flow rate, and speed of mandrel rotation) were investigated, and their effects on the mechanical properties and cellular response of the scaffolds were analysed using scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, gas chromatography, uniaxial tensile tests, cell viability and cytotoxicity assays. Mechanical properties were compared to those of the native RVOT reported in the literature. Increasing the mandrel rotation speed tended to increase fibre alignment slightly, and led to more profound rises in the stress at failure and Young's modulus. An increase in flow rate also increased the rigidity of the tubes. Cell viability and cytotoxicity assays showed all the tubes produced to have excellent biocompatibility. Through variation of the processing parameters, it is possible to tune mechanical properties of medical-grade polymer tubes over a wide range. By using a mandrel rotation speed of 50 rpm and a flow rate of 20 mL/h or higher we can prepare materials with Young's modulus, strain at failure, and tensile stress close to the native tissue. Electrospinning therefore offers great promise in the development of scaffolds to match the properties of the native RVOT, paving the way to a future bioresorbable device to replace the RVOT in children

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.We tested the hypothesis that Wnt/beta-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/beta-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for beta-myosin heavy chain (beta-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/beta-catenin signaling is required for Smad1 activation by BMP4.Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/beta-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications

Effects of autologous bone marrow stem cell transplantation on beta-adrenoceptor density and electrical activation pattern in a rabbit model of non-ischemic heart failure

BACKGROUND: Since only little is known on stem cell therapy in non-ischemic heart failure we wanted to know whether a long-term improvement of cardiac function in non-ischemic heart failure can be achieved by stem cell transplantation. METHODS: White male New Zealand rabbits were treated with doxorubicine (3 mg/kg/week; 6 weeks) to induce dilative non-ischemic cardiomyopathy. Thereafter, we obtained autologous bone marrow stem cells (BMSC) and injected 1.5–2.0 Mio cells in 1 ml medium by infiltrating the myocardium via a left anterolateral thoracotomy in comparison to sham-operated rabbits. 4 weeks later intracardiac contractility was determined in-vivo using a Millar catheter. Thereafter, the heart was excised and processed for radioligand binding assays to detect β(1)- and β(2)-adrenoceptor density. In addition, catecholamine plasma levels were determined via HPLC. In a subgroup we investigated cardiac electrophysiology by use of 256 channel mapping. RESULTS: In doxorubicine-treated animals β-adrenoceptor density was significantly down-regulated in left ventricle and septum, but not in right ventricle, thereby indicating a typical left ventricular heart failure. Sham-operated rabbits exhibited the same down-regulation. In contrast, BMSC transplantation led to significantly less β-adrenoceptor down-regulation in septum and left ventricle. Cardiac contractility was significantly decreased in heart failure and sham-operated rabbits, but was significantly higher in BMSC-transplanted hearts. Norepinephrine and epinephrine plasma levels were enhanced in heart failure and sham-operated animals, while these were not different from normal in BMSC-transplanted animals. Electrophysiological mapping revealed unaltered electrophysiology and did not show signs of arrhythmogeneity. CONCLUSION: BMSC transplantation improves sympathoadrenal dysregualtion in non-ischemic heart failure

Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections

<p>Abstract</p> <p>Background</p> <p>Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.</p> <p>Results</p> <p>Our model revealed that adding normal MSCs to the ischemic cell population significantly decreased the ratio of dead H9c2 cells (H9c2 only: 0.85 ± 0.086 vs. H9c2+MSCs: 0.16 ± 0.035). This effect was dependent on direct cell-to-cell contact since co-cultivation with MSCs cultured in cell inserts did not exert the same beneficial effect (ratio of dead H9c2 cells: 0.90 ± 0.055). Confocal microscopy revealed that cardiomyoblasts and MSCs frequently formed 200-500 nm wide intercellular connections and cell fusion rarely occurred between these cells.</p> <p>Conclusion</p> <p>Based on these results we hypothesize that mesenchymal stem cells may reduce the number of dead cardiomyoblasts after ischemic damage via direct cell-to-cell interactions and intercellular tubular connections may play an important role in these processes.</p

Plantando, colhendo, vendendo, mas não comendo: práticas alimentares e de trabalho associadas à obesidade em agricultores familiares do Bonfim, Petrópolis, RJ.

Objetivo: verificar a prevalência de obesidade entre adultos das 86 famílias agricultoras de um bairro de Petrópolis, RJ, e analisar seus determinantes socioculturais. Métodos: estudo quantitativo e qualitativo sobre nutrição, práticas alimentares e de trabalho realizado em 2008. Dados antropométricos foram coletados por inquérito nutricional domiciliar e o material qualitativo por observação participante e entrevistas. Resultados: a prevalência de obesidade foi baixa (9,3%) entre os homens, mas bastante elevada entre as mulheres (29,9%). A prática agrícola local implica em atividade física leve para mulheres e intensa para homens. Essa diferença não é acompanhada na dieta, semelhante para homens e mulheres, com predomínio de alimentos de alto valor calórico. A produção familiar objetiva essencialmente a venda. A agricultura mercantil e a decorrente especialização dos cultivos favorecem comprar alimentos no mercado em vez de produzir para autoconsumo. Conclusão: os aspectos socioculturais e ocupacionais estudados podem ter contribuído para elevar a prevalência de obesidade nas mulheres e podem ser úteis no estudo de outros grupos com características semelhantes. Esta pesquisa ratifica a importância de estudar a obesidade em nível local, integrando abordagens quantitativas e qualitativas para identificar possíveis limitações e portas de entrada para ações de intervenção localmente relevantes