A G-CSF functionalized scaffold for stem cells seeding: a differentiating device for cardiac purposes

Abstract

Myocardial infarction and its consequences represent one of the most demanding challenges in cell therapy and regenerative medicine. Transfer of skeletal myoblasts into decompensated hearts has been performed through intramyocardial injection. However, the achievements of both cardiomyocyte differentiation and precise integration of the injected cells into the myocardial wall, in order to augment synchronized contractility and avoid potentially life-threatening alterations in the electrical conduction of the heart, still remain a major target to be pursued. 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. Poly-(L-lactide) electrospun scaffolds were seeded with C2C12 murine skeletal myoblast for 48 hrs. 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. The possibility of fabricating extracellular matrix-mimicking scaffolds able to promote myoblast pre-commitment towards myocardiocyte lineage and mitigate the hazardous environment of the damaged myocardium represents an interesting strategy in cardiac tissue engineering.

fig 1

FE-SEM micrographs of PLLA/Ctrl (A) and PLLA/GCSF (B) electrospun scaffolds.

fig 2

Drug release curve from PLLA/GCSF scaffold, showing the cumulative release of G-CSF over time.

fig 3

Stress–strain profile for PLLA/GCSF computed starting from the recorded load-displacement data from longitudinal tensile tests.

fig 4

(A) PLLA/Ctrl seeded with C2C12 after 48 hrs culturing (100×); (B) PLLA/GCSF seeded with C2C12 after 48 hrs culturing (100×); (C) cells viability analysis in PLLA/GCSF and in PLLA/Ctrl scaffolds after 48 hrs of culture; (D) cell proliferation evaluated by measuring the total DNA content per construct; (E) percentage of Ki67 expression in both types of scaffolds. An increase in cell viability and proliferation could be detected in the PLLA/GCSF scaffold in comparison to PLLA/Ctrl scaffolds.

fig 5

Confocal microscopy. Immunofluorescence staining for Cx43, F-actin and nuclei. (A, B) PLLA/Ctrl seeded with C2C12 after 48 hrs (400×). (C–F) PLLA/GCSF seeded with C2C12 after 48 hrs (400×). Cx43 expression and a higher fibrillar distribution of cytoplasmic actin in the PLLA/GCSF scaffolds, with respect to the PLLA/Ctrl control could be seen (A–F). Cx43 antibody nicely stained membranes of cells (C, D, F) engrafted among polymeric fibres, but could also be detected in the perinuclear zone (E).

fig 6

(A) Cytofluorimetry analysis of Cx43 expression. (B) Western Blot for dystrophin, Cx43 and cTnI; MW: molecular weight marker. Lane 1: PLLA/Ctrl seeded with C2C12 for 48 hrs; lane 2: PLLA/GCSF scaffold seeded with C2C12 for 48 hrs. 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.

fig 7

Confocal microscopy. Immunofluorescence staining for cTnI, F-actin and nuclei. (A) PLLA/Ctrl seeded with C2C12 after 48 hrs (400×). (B) PLLA/GCSF seeded with C2C12 after 48 hrs (400×). cTnI expression was found only in the PLLA/GCSF sample, with a cytoplasmatic and granular pattern of expression, that indicates a non-mature organization of the protein inside the cells. Scale bar: 100 μm.

fig 8

TEM. (A, E) PLLA/Ctrl (B, C, D, F) PLLA/GCSF. Note cells on PLLA-GCSF polymer appeared elongated with more evidently represented RER and Golgi apparatus with respect to non-functionalized scaffolds (A–B), reliably indicating a higher metabolic activity. Elongated eccentric mitochondria typical of muscular cells could be detected on functionalized scaffolds (C). In the functionalized scaffold, several types of cell-to-cell interactions could be observed with cells acquiring a typical fuse-shape phenotype with elongated nuclei. Alignment of cells resembling myotube organization could be observed and electrondense fibrillar structures in the contact zone between cells could be seen (D). Membrane-to-membrane contact could be detected among cells seeded on bare PLLA scaffolds. In the functionalized scaffold a particular of a cell-to-cell junction characterized by a suggestive three-layered electron dense complex could be observed. Layers constituting the structure appeared highly rugous (E–F).