RESUMEN
Collagen-glycosaminoglycan scaffolds that have been used clinically for skin regeneration have also shown significant promise for other applications in tissue engineering. However, regeneration of thicker tissues with the aid of implanted biomaterials is likely to depend on, or be accelerated by, the ability to establish rapid vascularisation of the implant. The present study aims to establish a nascent vascular network in vitro within a CG scaffold as a first step towards that goal. Mesenchymal stem cells (MSCs) were chosen as primary vasculogenic candidate cells and a culture medium that promoted maximal network formation on Matrigel by these cells was selected. MSCs seeded in the CG scaffold formed networks of cord-like structures after one to two weeks in the presence of the vasculogenic medium; similar structures were formed by aortic endothelial cells (ECs) cultured for comparison. Gene expression analysis suggested that the MSCs began to adopt an endothelial phenotype, with RNA for PECAM and VCAM rising while that for alpha-smooth muscle actin fell. However there was no increase in Tie-2 and vWF expression. Addition of smooth muscle cells (SMCs) as a potential perivascular stabilising component did not have a noticeable effect on MSC-derived networks, although it enhanced EC-derived structures.
Asunto(s)
Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Andamios del Tejido , Actinas/genética , Animales , Aorta/citología , Huesos/irrigación sanguínea , Técnicas de Cultivo de Célula , Células Cultivadas , Células Endoteliales , Matriz Extracelular/química , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Células Madre Mesenquimatosas/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Fenotipo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Ratas , Ratas Wistar , Receptor TIE-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula 1 de Adhesión Celular Vascular/genética , Factor de von Willebrand/genéticaRESUMEN
BACKGROUND: Cardiovascular diseases (CVD) account for 36% of deaths in Europe and the United States. Gene therapy can act as a therapeutic modality for the treatment of CVD. The use of microRNA mimetics may be advantageous as they regulate important processes in health and pathology. A major hurdle for using miRNA therapies relates to site specific delivery and sufficient cellular uptake of material to achieve efficacy OBJECTIVE: To assess the feasibility of ultrasound responsive microbubble mediated delivery of miR mimics to cardiomyocytes. METHODS: Liposome/microbubble formulations were added to HL-1 cardiomyocytes in the presence/absence of ultrasound (US). Transfection efficacy and functionality was assessed using epifluorescent microscopy, flow cytometry and qRT-PCR. DNA Quantification post-ultrasound mediated transfection of HL-1s using microbubbles was quantified. The capability of miR-133 microbubble formulations to suppress hypertrophy were measured by quantifying changes in cell size. RESULTS: Ultrasound mediated microbubble formulations enhanced intracellular delivery of miR mimics in cardiomyocytes. Both complexed/encapsulated miR-microbubble formulations delivered functional miR mimics and showed no adverse effect on cardiomyocyte viability. Furthermore, ultrasound mediated microbubble transfection of miR-133 mimics reversed cardiomyocyte hypertrophy in an in-vitro model. CONCLUSIONS: This novel delivery method has the potential for further development as a targeted delivery strategy for miR therapeutics to the heart.