In-vitro model of young and aged PU-based scaffold for cardiac aging studies

Background-aim Aging is associated with a progressive decline in numerous physiological processes, leading to an increased risk of health complications and disease.In vitro cardiac tissue engineering, through the use of scaffolds able to favour cell adhesion and survival, is a promising tool for identification of aging-related molecular mecha- nisms. Aim was to show a new approach focused on tissue-specific architecture and mechanical properties mimicking of young and aged tissue (scaffold) integrated with mechanical stimuli (loading) to generate an in-vitro pathophysiological model of cardiac aging. Methods Young and aged artificial tissues were produced by polyurethane (PUR) and polyurethane-polycaprolactone blend, respectively. The poly- mer blends were studied to simulate the aged muscle, which is stiffer compare to the young one. Polymer scaffolds were produced by Thermal Induce Phase Separation to obtain oriented fibres texture like cardiac tissues. Scaffolds surface was functionalized with fibronectin. Sprague- Dawley primary neonatal rats cardiomyocytes were seeded on young and aged scaffold and cultured for 7 days. For mechanical tests, scaffolds were placed in SQPR bioreactor and subjected to a cyclic loading stimulus (1 Hz) for 24 h. To mimic ischemic pathology, a hypoxia/reperfusion protocol was applied. Cell viability with CellTiter Blue assay was evaluated. Natriuretic Peptides (NPs) and Endothelin (ET-1) system mRNA expression, to evaluate cardiac phenotype, and Connexin (CX)-43, to confirm cellular interaction by gap junction formation, were measured by Real time-PCR. Results Results showed a good viability in static and after mechanical loading stimulation in SQPR. An increased expression of ANP/BNP in parallel to a reduction of CNP mRNA levels in young scaffold with respect to old ones were observed in static condition. An activation of NPR-A and NPR-B was also found. After mechanical stimulation, ANP and BNP trend significantly decreased in old scaffold with respect to young ones (p b .0001/p = .0008, respectively) and, on the contrary, CNP was significantly higher (p = .011) with a counter-regulation of NPR-B. At the end of hypoxia/reperfusion protocol, an acceptable reduction of 30% in cell viability was observed. During I/R, only CNP was up regulated in SQPR bioreactor scaffold. ET-1 mRNA was higher in old scaffold while CX43 mRNA decreased. During I/R CX43 mRNA levels resulted significantly higher in SQPR bioreactor scaffold with respect to static conditions (p = .0028) and plastic surface (p = .014). Conclusions Our engineered model, thanks to integration of structural proper- ties and mechanical stimuli, furnishes a new approach to study in-vitro cardiac aging.