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Affording high-through put cardiogenesis in human mesenchymal stem cells
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Abstracts / Biomedicine & Pharmacotherapy 60 (2006) 468–479
appropriate fibre manufacturing. The transfer of mechanical stress to the biopolymer scaffold is engineered through a device using a linear motion form without need to be reduced and/or transformed through kinematic pairs. High performances result in terms of precision and reproducibility of the movement. Moreover the inherent speed and bandwidth limits are suitable to generate a motion compatible with the rat heartbeat. An additional useful feature of this device is its relevant flexibility, allowing the setting and the control of kinetic shape and speed through a personal computer. Indeed, during this step it is needed that all the parameters within the bioreactor are easily and quickly controlled and monitored by the user, to understand the relative importance of each single variable. We envisage to set 5 Hz stretch cycles, extending the biopolymer scaffold up to 20% of its initial dimension, throughout a defined time span. During the test the scaffold bearing the cells will be housed into a culture mini-chamber with all the controls of a standard cell culture incubator (i.e. sterility, temperature, pH and relative humidity) and access ports for media supply and cell manipulation. 6 Affording high-through put cardiogenesis in human mesenchymal stem cells C. Ventura Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems, Institute of Cardiology, University of Bologna, 40138 Bologna, Italy Loss of cardiomyocytes due to myocardial infarction or hereditary cardiomyopathies, combined with the absence of regenerative capacity in myocardial cells, represent causative factors in the progression toward heart failure. Establishing ex vivo models of cardiogenesis in suitable populations of human adult stem cells is therefore a major assignment in the perspective of a cell therapy for damaged hearts. We have developed mixed esters of hyaluronan with butyric and retinoic (HBR) acid and provide evidence that they acted as novel powerful cardiogenic agents in human mesenchymal stem cells (hMSCs) isolated from bone marrow (BMhMSCs), fetal membranes (FMhMSCs), and dental pulp (DPhMSCs). HBR dramatically increased the transcription of GATA-4 and Nkx-2.5, acting as cardiac lineage-promoting genes in different animal species. Exposure to HBR ultimately ensued in a high throughput yield of cardiomyocytes expressing sarcomeric α-myosin heavy chain and α-sarcomeric actinin. The HBR effect was selective in nature, since the mixed ester failed to affect the expression of genes involved in skeletal myogenesis or neuronal specification. HBR also remarkably enhanced the expression of the KDR gene, encoding for a VEGF receptor involved in angiogenesis, and led to the formation of vWF-expressing cells. These differentiating responses were remarkably more pronounced in FMhMSCs than in DPhMSCs or BMhMSCs. The current findings demonstrate the potential for chemically manipulating a gene program of human stem cell cardiogenesis
without the need of gene transfer technologies and may set the basis for unprecedented approaches of tissue engineering and myocardial regeneration. 7 Multifunctional drug carriers by biochemical surface modification of drug-eluting stents: surface characterization and 28 days in vivo results in a pig model M. Morra Nobil Bio Ricerche, Street S. Rocco 36, Villafranca d’Asti, Italy Introduction. – Sustained release of drug from drug-elutingstents (DES) requires tight control of the polymeric carrier chemistry and properties. Synthetic biostable polymeric carriers are commonly used in commercially available DES, but, according to several research reports, they promote severe inflammation in the vessel wall, inducing significant sideeffects and hypersensitivity. This often requires an increase of the drug dosage, to overcome carrier-related inflammation, leading however to unwanted drug-related side effects and unpaired endothelialization. After initial studies mostly concerned with DES efficacy, today’s works are strongly aiming at assessing DES safety, and at reducing the required carrier and drug amount. An alternative strategy is to increase the biocompatibility of drug carriers by surface modification techniques. Modifications should involve just the first few nanometers, in order to leave the bulk properties of the carrier, and hence drug release kinetic, unaffected. Immobilization of naturally occurring biological molecules to drug carrier surfaces improves the biological acceptance of synthetic polymers, by the presentation of a more natural interface to the surrounding tissue. In this work we present our results on the surface modification of DES by immobilization of hyaluronan (HA). HA is a glicosaminoglycan that plays a key role in cell motility and has been suggested as a possible natural aid against restenosis. The aim of the work is to present results of surface characterization of HA coupled DES and in vivo data in the coronary pig model. Results and discussion. – Surface chemical analysis by XPS confirms the successful linking of HA to the carrier surface. Importantly, the HA surface layer is not damaged by the steps involved is stent preparation (heat setting, crimping, packaging, sterilization), as confirmed by XPS analysis throughout these steps. AFM images of the carrier surface show that surface topography is not affected by the process and force-distance curves indicate that surface-linked HA molecules swell at the aqueous interface, keeping, albeit terminally constrained, conformational freedom required for biological activity. Concerning in vivo studies, histopathology evaluation shows low luminal stenosis with low injury and inflammation score and good intimal maturation and endothelialization. Despite the 600 μg acrylate carrier loaded on the DES, HA coated stents do not show any significant worsening of the evaluated parameters over the unloaded bare stents. Thus, this
Abstracts / Biomedicine & Pharmacotherapy 60 (2006) 468–479
appropriate fibre manufacturing. The transfer of mechanical stress to the biopolymer scaffold is engineered through a device using a linear motion form without need to be reduced and/or transformed through kinematic pairs. High performances result in terms of precision and reproducibility of the movement. Moreover the inherent speed and bandwidth limits are suitable to generate a motion compatible with the rat heartbeat. An additional useful feature of this device is its relevant flexibility, allowing the setting and the control of kinetic shape and speed through a personal computer. Indeed, during this step it is needed that all the parameters within the bioreactor are easily and quickly controlled and monitored by the user, to understand the relative importance of each single variable. We envisage to set 5 Hz stretch cycles, extending the biopolymer scaffold up to 20% of its initial dimension, throughout a defined time span. During the test the scaffold bearing the cells will be housed into a culture mini-chamber with all the controls of a standard cell culture incubator (i.e. sterility, temperature, pH and relative humidity) and access ports for media supply and cell manipulation. 6 Affording high-through put cardiogenesis in human mesenchymal stem cells C. Ventura Laboratory of Molecular Biology and Stem Cell Engineering, National Institute of Biostructures and Biosystems, Institute of Cardiology, University of Bologna, 40138 Bologna, Italy Loss of cardiomyocytes due to myocardial infarction or hereditary cardiomyopathies, combined with the absence of regenerative capacity in myocardial cells, represent causative factors in the progression toward heart failure. Establishing ex vivo models of cardiogenesis in suitable populations of human adult stem cells is therefore a major assignment in the perspective of a cell therapy for damaged hearts. We have developed mixed esters of hyaluronan with butyric and retinoic (HBR) acid and provide evidence that they acted as novel powerful cardiogenic agents in human mesenchymal stem cells (hMSCs) isolated from bone marrow (BMhMSCs), fetal membranes (FMhMSCs), and dental pulp (DPhMSCs). HBR dramatically increased the transcription of GATA-4 and Nkx-2.5, acting as cardiac lineage-promoting genes in different animal species. Exposure to HBR ultimately ensued in a high throughput yield of cardiomyocytes expressing sarcomeric α-myosin heavy chain and α-sarcomeric actinin. The HBR effect was selective in nature, since the mixed ester failed to affect the expression of genes involved in skeletal myogenesis or neuronal specification. HBR also remarkably enhanced the expression of the KDR gene, encoding for a VEGF receptor involved in angiogenesis, and led to the formation of vWF-expressing cells. These differentiating responses were remarkably more pronounced in FMhMSCs than in DPhMSCs or BMhMSCs. The current findings demonstrate the potential for chemically manipulating a gene program of human stem cell cardiogenesis
without the need of gene transfer technologies and may set the basis for unprecedented approaches of tissue engineering and myocardial regeneration. 7 Multifunctional drug carriers by biochemical surface modification of drug-eluting stents: surface characterization and 28 days in vivo results in a pig model M. Morra Nobil Bio Ricerche, Street S. Rocco 36, Villafranca d’Asti, Italy Introduction. – Sustained release of drug from drug-elutingstents (DES) requires tight control of the polymeric carrier chemistry and properties. Synthetic biostable polymeric carriers are commonly used in commercially available DES, but, according to several research reports, they promote severe inflammation in the vessel wall, inducing significant sideeffects and hypersensitivity. This often requires an increase of the drug dosage, to overcome carrier-related inflammation, leading however to unwanted drug-related side effects and unpaired endothelialization. After initial studies mostly concerned with DES efficacy, today’s works are strongly aiming at assessing DES safety, and at reducing the required carrier and drug amount. An alternative strategy is to increase the biocompatibility of drug carriers by surface modification techniques. Modifications should involve just the first few nanometers, in order to leave the bulk properties of the carrier, and hence drug release kinetic, unaffected. Immobilization of naturally occurring biological molecules to drug carrier surfaces improves the biological acceptance of synthetic polymers, by the presentation of a more natural interface to the surrounding tissue. In this work we present our results on the surface modification of DES by immobilization of hyaluronan (HA). HA is a glicosaminoglycan that plays a key role in cell motility and has been suggested as a possible natural aid against restenosis. The aim of the work is to present results of surface characterization of HA coupled DES and in vivo data in the coronary pig model. Results and discussion. – Surface chemical analysis by XPS confirms the successful linking of HA to the carrier surface. Importantly, the HA surface layer is not damaged by the steps involved is stent preparation (heat setting, crimping, packaging, sterilization), as confirmed by XPS analysis throughout these steps. AFM images of the carrier surface show that surface topography is not affected by the process and force-distance curves indicate that surface-linked HA molecules swell at the aqueous interface, keeping, albeit terminally constrained, conformational freedom required for biological activity. Concerning in vivo studies, histopathology evaluation shows low luminal stenosis with low injury and inflammation score and good intimal maturation and endothelialization. Despite the 600 μg acrylate carrier loaded on the DES, HA coated stents do not show any significant worsening of the evaluated parameters over the unloaded bare stents. Thus, this