A G-CSF functionalized scaffold for stem cells seeding: a differentiating device for cardiac purposes.
Bottom Line: Recently, granulocytes colony-stimulating factor (G-CSF) fuelled the interest of researchers for its direct effect on cardiomyocytes, inhibiting both apoptosis and remodelling in the failing heart and protecting from ventricular arrhythmias through the up-regulation of connexin 43 (Cx43).We propose a tissue engineering approach concerning the fabrication of an electrospun cardiac graft functionalized with G-CSF, in order to provide the correct signalling sequence to orientate myoblast differentiation and exert important systemic and local effects, positively modulating the infarction microenvironment.Biological assays demonstrated the induction of Cx43 expression along with morphostructural changes resulting in cell elongation and appearance of cellular junctions resembling the usual cardiomyocyte arrangement at the ultrastructural level.
Affiliation: CIR - Area of Cardiovascular Surgery, University Campus Bio-Medico of Rome, Rome, Italy.Show MeSH
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Mentions: Immunofluorescence analysis revealed Cx43 expression, along with a higher fibrillar distribution of cytoplasmic actin in the PLLA/GCSF scaffolds, with respect to the PLLA/Ctrl control (Fig. 5A–F). Cx43 antibody nicely stained membranes of cells engrafted among polymeric fibres, but could also be detected in the perinuclear zone. Flow cytometry confirmed and quantified Cx43 positivity, showing a statistical significant higher level of expression in the PLLA/GCSF scaffold. Interestingly, a significant increase in Cx43 positivity could be observed in 2D cell cultures exposed to PLLA/GCSF, in comparison with PLLA/Ctrl control (Fig. 6A). Immunofluorescence also showed appearance of cardiac-specific isoform of troponin I in the PLLA/GCSF group. Interestingly, antigen was localized in cytoplasm and presented a granular pattern of expression, clearly indicating a non-mature organization of the protein inside the cell (Fig. 7B). These results were confirmed with Western blotting analysis that showed co-expression of Cx43 and cTnI in the PLLA/GCSF patches, confirming a cardiac pre-commitment of myoblasts seeded in these scaffolds and a direct effect of G-CSF released from the polymer on cells in conventional culture (Fig. 6B). A non-cardiac specific dystrophin antibody recognizing a 60 kD fragment of the native protein was used as a general marker for myoblasts. The contemporary presence of both myoblast and cardiac-specific markers could reliably suggest different stages in the differentiation process. However, morphological and immunophenotypic changes achieved by myoblasts in this setting were not compatible with a complete differentiation in cardiomyocyte and cells did not acquire beating capability which is related to the appearance of a functional cardiac-specific calcium handling system.
Affiliation: CIR - Area of Cardiovascular Surgery, University Campus Bio-Medico of Rome, Rome, Italy.