S10 - Histological Characterization of Auxetic Cardiac Patches After Myocardial Infarction

Introduction: : Myocardial infarction (MI) causes permanent cardiomyocyte loss and fibrotic remodeling that impairs heart function. Engineered cardiac patches offer a potential strategy for repairing damaged tissue, and materials with auxetic properties — which expand in multiple directions under unidirectional strain — may better mimic native heart mechanics. This project focused on comparing the tissue response and cellular organization of auxetic versus control (non-auxetic) 3D-printed cardiac patches seeded with induced pluripotent stem cell-derived cardiomyocytes (iCMs). This work specifically examined fibrosis area of rodent hearts following patch implantation using brightfield microscopy, as well as iCM organization and structure within the patch using histological staining and confocal microscopy.

Materials and

Methods: : Cardiac patches were 3D printed using polycaprolactone (PCL) in both auxetic and control geometries, then coated with fibronectin and seeded with iCMs embedded in fibrin hydrogel. Patches were either cultured under dynamic bioreactor conditions or implanted onto infarcted rat myocardium and retrieved after several weeks for analysis. In vitro patches were fixed and stained for cardiac troponin T (cTnT), α-actinin, and the mitochondrial marker Tom20 to evaluate sarcomeric alignment and mitochondrial distribution. Explanted heart tissue was sectioned and stained with Picrosirius Red (PSR) to assess fibrotic collagen content at the patch-tissue interface and within the left ventricle. Confocal microscopy and Z-stack imaging were used to visualize cell distribution and structural organization within the patches.

Results, Conclusions, and Discussions:: Cardiomyocytes remained well-distributed within both auxetic and control patches following in vitro culture and in vivo implantation. Immunostaining confirmed cardiomyocyte identity through cTnT and α-actinin expression, while mitochondrial staining with Tom20 revealed dense perinuclear clustering and cytoplasmic organization. Cells cultured under mechanical stimulation exhibited more aligned sarcomeres and well-structured mitochondrial networks, suggesting enhanced intracellular maturation after bioreactor culture of 6 weeks compared to controls.
In the in vivo samples, Picrosirius Red staining revealed reduced collagen deposition in infarcted myocardium treated with either patch type compared to infarct-only controls. While both designs showed a decrease in fibrotic response, the collagen fibers surrounding the auxetic patches appeared more compact and linearly organized. These qualitative differences point to a possible role of patch mechanical architecture in guiding extracellular matrix remodeling, though further quantitative analysis is necessary to confirm this observation.
Taken together, these findings show that both auxetic and control 3D-printed cardiac patches can support cardiomyocyte retention and reduce post-infarct fibrosis. The auxetic design may offer subtle advantages in promoting organized tissue integration and intracellular structure. This work emphasizes the importance of evaluating not only mechanical properties, but also biological responses when designing scaffolds for cardiac repair. The staining and imaging techniques applied here provide a useful framework for assessing cell-material interactions and can inform future patch design and optimization.

Acknowledgements and/or References (Optional)::