(G-254) FSTL1-loaded 3D Bioprinted Vascular Cardiac Patch Regenerates the Myocardium after Myocardial Infarction (MI)

In recent years, methods to regenerate cardiac tissue following an ischemic injury have been of increasing focus to the tissue-engineering field. However, due to the low proliferative capacity of cardiomyocytes (CMs), along with the inadequate biological function of current patch devices, these methods have faced major setbacks. To address these shortcomings, we examined the use of a new generation of multifunctional cardiac patch devices using 3D bioprinting technology. These patches, containing perfusable vasculature, multi-cellular architecture, and pro-proliferative molecules, demonstrated improved therapeutic outcomes in mammalian myocardium following the ischemic heart injury. In this study, we present in vitro studies highlighting the pro-proliferative impact of follistatin-like-1 (FSTL1) protein on CMs, along with in vivo results confirming the regenerative effect of FSTL1-loaded 3D bioprinted cardiac patches to restore the heart function after myocardial infarction (MI).

The effect of FSTL1 on human induced pluripotent stem cell-derived CMs (hiPSC-CMs) were assessed by treating a monolayer of CMs with varying concentrations of FSTL1 in culture media. Immunofluorescence staining with alpha-actinin and Ki 67 was conducted to quantify the number of proliferating hiPSC-CMs in response to FSTL1. A vascular cardiac patch was bioprinted with a bioink containing human umbilical vein endothelial cells (HUVECs) and optimized amounts of gelatin methacrylate (GelMA), gelatin, and fibrinogen. FSTL1 was mixed with cellular bioink to fabricate the FSTL1-loaded vascular cardiac patch. The prepared cellular ink was cast in a custom designed mold, followed by 3D bioprinting of an intricate vascular tree geometry. The vasculature was printed between the two cast layers of cellular hydrogel using a sacrificial pluronic bioink. The pluronic channels were then dissolved to create a hollow vasculature, which was subsequently endothelialized by seeding HUVECs within the lumen. For in vivo studies using an athymic nude rat model, cardiac patches were surgically implanted on the site of injury (MI) immediately after left anterior descending artery (LAD) ligation. Experimental groups included: control (Sham), MI only, MI + HUVECs patch, and MI + HUVECs patch loaded with FSTL1. Echocardiography was performed longitudinally for the 4-week study duration. Masson’s Trichrome staining was conducted on harvested tissues for the quantification of scar tissue following the termination of the experiment.

FSTL1 treatment resulted in significant in-situ proliferation of hiPSC-CMs in vitro. Compared to the non-treated control group, hiPSC-CMs treated with varying concentrations of FSTL1 showed higher expression of Ki67. This effect was seen in all age groups of CMs, from day 11 to day 61. A vascular cardiac patch with FSTL1 was successfully bioprinted using HUVECs and GelMA/gelatin/fibrin bioink. These cellular patches were printed with high fidelity ( >90%) and supported 3D culture of embedded HUVECs within the patch. Our in vivo studies showed that the implantation of HUVECs patch loaded with FSTL1 results in reduced levels of myocardium damage compared to damages detected in the MI-only group. Trends seen in echocardiography data confirmed that ejection fraction showed improvement from initial injury in patch treatment groups, highlighting the healing capabilities of the patch. Additionally, trichrome staining at the end of the 4-week experiment demonstrated significant thinning and collagen accumulation in the left ventricle (LV) myocardium wall of rats without patch treatment, while treatment groups showed a trend of healthier cardiac structure and function. 

Altogether, this study demonstrates the enhanced regenerative capability of 3D bioprinted vascular cardiac patch loaded with FSTL1 following an ischemic injury in the myocardium. The synergistic effect of bioprinted patch with FSTL1 was evidenced by the higher ejection fraction and reduced fibrosis in the LV compared to those treated with HUVEC-only patch. The in vitro hiPSC-CM response to FSTL1 further sheds light on a potential mechanism of which the FSTL1 loaded cardiac patch results in mitigated myocardium damage and function after MI. The integration of 3D bioprinting with cell/tissue-therapy technologies introduces a novel regimen to treat diseased/damaged adult mammalian hearts.