RESUMEN
Although the majority of current gene transfer techniques have focused on increasing the ability of the DNA to enter the cell, it is possible that changing the proliferative and migratory state of cells will influence the cells ability to take up and express plasmid DNA. This study was designed to test the hypothesis that growth factors (basic fibroblast growth factor (bFGF) and hepatocyte growth factor/scatter factor (HGF/SF)) used to alter the proliferative and migratory state of cells can alter plasmid DNA uptake and expression. In vitro studies indicate that enhancing cell proliferation with growth factor exposure enhances plasmid DNA uptake and expression. Furthermore, dual localized delivery of bFGF and plasmid DNA in vivo increases the expression, 3-6 times over control, as compared to plasmid delivery alone. Dual delivery of a factor promoting cell proliferation and a plasmid led to a further increase in the expression of the plasmid encoding bone morphogenetic protein-2 in a rat cranial defect by specific cell populations. The results of these studies suggest that increasing the proliferative state of target cell populations can enhance non-viral gene transfer.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Movimiento Celular/genética , Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Células 3T3 NIH , Plásmidos/genética , Ratas , Ratas Endogámicas Lew , Fracturas Craneales/genética , Fracturas Craneales/patología , Fracturas Craneales/terapia , TransfecciónRESUMEN
Tissue engineering approaches have been investigated as a strategy for hepatocyte transplantation; however the death of a majority of transplanted cells critically limits success of these approaches. In a previous study, a transient increase in hepatocyte survival was achieved through delivery of vascular endothelial growth factor (VEGF) from the porous polymer scaffold utilized for cell delivery. To enhance longer-term survival of the hepatocytes, this delivery system was modified to additionally deliver epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in a sustained manner. Hepatocytes were subcutaneously implanted in SCID mice on scaffolds containing EGF and/or HGF, in addition to VEGF, and survival was monitored for two weeks. A short-term enhancement of hepatocyte survival was observed after one week and is attributed to VEGF-enhanced vascularization, which was not altered by EGF or HGF. Surprisingly, long-term hepatocyte engraftment was not improved, as survival declined to the level of control conditions for all growth factor combinations after two weeks. This investigation indicates that the survival of hepatocytes transplanted into heterotopic locations is dependent on multiple signals. The delivery system developed for the current study may be useful in elucidating the specific factors controlling this process, and bring therapeutic transplantation of hepatocytes closer to implementation.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/trasplante , Factor A de Crecimiento Endotelial Vascular/farmacología , Animales , Materiales Biocompatibles , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/farmacocinética , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/farmacocinética , Humanos , Masculino , Ratones , Ratones SCID , Microesferas , Células 3T3 NIH , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/farmacología , Factores de Tiempo , Ingeniería de Tejidos/métodos , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/farmacocinéticaRESUMEN
There is no satisfactory conventional treatment for patients who experience irreversible salivary gland damage after therapeutic radiation for head and neck cancer or because of Sjögren's syndrome. Additionally, if most parenchyma is lost, these patients also are not candidates for evolving gene transfer strategies. To help such patients, several years ago we began to develop an artificial salivary gland. In the present study, we used a non-human primate tissue source, parotid glands from rhesus monkeys, to obtain potential autologous graft cells for development of a prototype device for in situ testing. Herein, we present 3 major findings. First, we show that primary cultures of rhesus parotid gland (RPG) cells are capable of attaining a polarized orientation, with Na(+)/K(+)-adenosine triphosphatase, zonula occludens-1, and claudin-1 distributed in specific domains appropriate for epithelial cells. Second, we show that RPG cells exhibit 2 essential epithelial functions required for graft cells in an artificial salivary gland device (i.e., an effective barrier to paracellular water flow and the generation of a moderate transepithelial electrical resistance). Third, we show that RPG cells can express functional water channels, capable of mediating directional fluid movement, after transduction by adenoviral and adeno-associated virus type 2 vectors. Together these results demonstrate that it is feasible to individually prepare RPG cells for eventual use in a prototype artificial salivary gland.
Asunto(s)
Bioprótesis , Células Epiteliales/citología , Técnicas de Cultivo de Órganos/métodos , Glándula Parótida/citología , Glándulas Salivales/citología , Glándulas Salivales/crecimiento & desarrollo , Ingeniería de Tejidos/métodos , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Epiteliales/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Macaca mulatta , Mucosa Bucal/citología , Mucosa Bucal/fisiología , Glándula Parótida/fisiologíaRESUMEN
Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.
Asunto(s)
ADN/administración & dosificación , Expresión Génica , Ácido Láctico/metabolismo , Polietileneimina/administración & dosificación , Ácido Poliglicólico/metabolismo , Polímeros/administración & dosificación , Animales , ADN/metabolismo , Estudios de Factibilidad , Liofilización , Terapia Genética , Vectores Genéticos , Cinética , Masculino , Plásmidos , Polietileneimina/metabolismo , Polímeros/metabolismo , Porosidad , Ratas , Ratas Endogámicas Lew , Ingeniería de Tejidos , Transfección , beta-Galactosidasa/análisis , beta-Galactosidasa/metabolismoRESUMEN
Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.
Asunto(s)
Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Hidrogeles/química , Plásmidos/administración & dosificación , Plásmidos/farmacocinética , Polietileneimina/química , Transfección/métodos , Células 3T3 , Animales , Elasticidad , Terapia Genética/métodos , Ratones , Vehículos Farmacéuticos/química , Plásmidos/química , Plásmidos/genéticaRESUMEN
Macroporous polymeric scaffolds are frequently used in tissue engineering to allow for cell seeding and host cell invasion of the scaffold following implantation. The process of gas foaming/particulate leaching (GF/PL) is one method to form porous three dimensional scaffolds from particulate poly(lactide-co-glycolide) (PLG). The current study was designed to test the hypothesis that the size of the polymer particles used in this process will control the properties of the scaffolds. Scaffolds were prepared from PLG particles of various sizes (less than 75 microm, 75-106 microm, 106-250 microm and 250-425 microm) and subsequently analyzed. Scaffolds formed from large particles (250-425 microm) displayed significantly decreased compressive moduli, as compared to scaffolds fabricated from smaller particles. In addition, these scaffolds have a pore structure that is less interconnected and contains closed pores. Analysis of tissue in-growth, utilizing a novel computer-aided method, demonstrated that scaffolds formed from smaller particle sizes (less than 106 microm) have significantly more tissue penetration than those formed from larger particle sizes (greater than 106 microm). These results indicate that using small PLG particles (less than 106 microm) leads to high elastic moduli, provides a more interconnected pore structure and promotes greater tissue penetration into the scaffolds in vivo.