Reduced Graphene Oxide Doped Biodegradable Polyurethane Nanofibers for Cardiomyocyte Culture

Introduction: Myocardial infarction (MI) is a serious condition that can lead to adverse left ventricular (LV) remodeling, resulting in decreased heart function and even heart failure. One of the primary reasons for this is that cardiomyocytes lack the ability to regenerate, and the fibrotic response in the infarct region results in the formation of stiff scar tissue. To address this problem, tissue engineering has emerged as a promising approach, but there is a need to develop new and improved materials for use as scaffolding. One potential solution is to use conductive scaffolds, improving electrical signal transmittance and promoting stronger and more uniform contractions throughout the scaffold [1]. In this study, we developed a conductive scaffold by integrating an aligned nanofibrous patch made of biodegradable polyurethane (PU), a biocompatible and elastic polymer, with reduced graphene oxide (rGO).



Materials and Methods: We created PU-rGO membranes using rotating mandrel electrospinning with varying concentrations of rGO (0.5%, 1.0%, 2.0%, 4.0%, 10%) (w/w) in HFIP. We assessed the morphology, shrinkage, surface hydrophilicity, and conductivity of the nanofibers. Mechanical properties were evaluated using uniaxial and biaxial tensile testing. We conducted in vitro cell studies with C2C12 mouse myoblast cells, neonatal rat cardiomyocytes (NRCMs), and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) to verify cell compatibility, alignment, and functionality on the membranes.



Results and Discussions: The nanofibrous scaffolds fabricated in this study displayed a highly aligned microstructure, as confirmed by SEM images (Fig. 1A-B). They maintained their structure and alignment after immersion in PBS at 37°C for 24 hours. The mats' non-uniform shrinkage indicated anisotropic properties. The membranes' conductivity was 0.0013 ± 0.0002 S/m, 0.0080 ± 0.0022 S/m, and 0.0214 ± 0.0024 S/m for PU, PU-4%rGO, and PU-10%rGO, respectively. Increasing rGO concentrations did not significantly affect the membranes' hydrophilicity, as evidenced by contact angles of 120 ± 7° for pure PU and 132 ± 5° for 10% rGO. The scaffolds with higher rGO concentrations exhibited lower yield stress than those with lower concentrations but were stronger than left ventricle porcine heart tissue (Fig. 1C-1D). Biaxial mechanical testing revealed that the scaffolds had non-linear, anisotropic properties like native cardiac tissue. In vitro C2C12 cell studies showed proliferation in all groups. The NRCMs cultured on PU-10%rGO membranes demonstrated higher sarcomere and α-actinin lengths compared to the tissue culture polystyrene (TCP) and PU scaffolds. Immunofluorescent staining intensities of the NRCM indicated that the PU-10%rGO exhibited the highest expression of α-actinin, f-actin, troponin I, and connexin-43 (Fig. 1E). Furthermore, the results from the hiPSC-CMs were consistent with those from NRCMs, demonstrating that the PU-10%rGO group exhibited enhanced functional characteristics, including sarcomere and α-actinin lengths, CX-43 expression levels (Fig. 1F-1G), and more synchronized calcium transients compared to the PU and TCP groups (Fig. 1H).




Conclusion: Our study showed that PU-rGO displayed tunable physical properties and maintained cell viability and alignment, even at the highest rGO concentration. The rGO addition can improve cardiomyocyte function during in vitro culture, indicating the material has potential to be a good cardiomyocyte culture matrix in vitro.



Acknowledgements:

We greatly thank partial support from NSF CAREER #1554835 (YH), NIH R15 HL140503 (YH) and AHA EIA (YH). AT is supported by NIH T32 HL134613.


References: [1]. Dong R., et al. Biomaterials 2020;229:119584.