Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (l-lactic acid) scaffolds for potential use in cardiac regeneration
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Diaphragmatic myoblasts (DMs) are precursors of type-1 muscle cells displaying high exhaustion threshold on account that they contract and relax 20 times/min over a lifespan, making them potentially useful in cardiac regeneration strategies. Besides, it has been shown that biomaterials for stem cell delivery improve cell retention and viability in the target organ. In the present study, we aimed at developing a novel approach based on the use of poly (L-lactic acid) (PLLA) scaffolds seeded with DMs overexpressing connexin-43 (cx43), a gap junction protein that promotes inter-cell connectivity. DMs isolated from ovine diaphragm biopsies were characterized by immunohistochemistry and ability to differentiate into myotubes (MTs) and transduced with a lentiviral vector encoding cx43. After confirming cx43 expression (RT-qPCR and Western blot) and its effect on inter-cell connectivity (fluorescence recovery after photobleaching), DMs were grown on fiber-aligned or random PLLA scaffolds. DMs were successfully isolated and characterized. Cx43 mRNA and protein were overexpressed and favored inter-cell connectivity. Alignment of the scaffold fibers not only aligned but also elongated the cells, increasing the contact surface between them. This novel approach is feasible and combines the advantages of bioresorbable scaffolds as delivery method and a cell type that on account of its features may be suitable for cardiac regeneration. Future studies on animal models of myocardial infarction are needed to establish its usefulness on scar reduction and cardiac function.
KeywordsDiaphragm Myoblasts Connexin-43 Absorbable implants Poly (L-lactic acid) Sheep Fluorescence recovery after photobleaching
We thank veterinarians María Inés Besansón and Pedro Iguaín for anesthetic management and animal house assistants Juan C. Mansilla, Osvaldo Sosa, and Juan Ocampo for care of the animals. We also thank Julio Martínez and Fabián Gauna for technical help.
CSG study design, data collection and/or assembly, data analysis and interpretation, manuscript writing, PL and MRB animal model preparation, LC and FDO data collection and interpretation, FMB and GAA provision of biomaterial, AO and EAA FRAP experiments, RAD and MP provision of lentiviral vector, AC study design, data analysis and interpretation, manuscript writing.
This work was supported by the National Agency for the Promotion of Science and Technology (ANPCyT) of Argentina (grants PICT 2011-1181 and 2012-0224) the René Barón Foundation of Argentina, the Florencio Fiorini Foundation of Argentina and the National Scientific and Technical Research Council (CONICET) of Argentina.
Compliance with ethical standards
Conflict of interest
The authors have no conflicts of interest to disclose.
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