Quantifying species and age-dependent fibroblast morphologies on biomaterial surfaces via optical diffraction tomography

Fibroblasts are essential for maintaining tissue structure by producing the extracellular matrix (ECM) and collagen fibers. Their potential in regenerative medicine and personalized therapy has recently attracted attention, particularly for reprogramming into neuronal cells. Despite ongoing methodological advances aimed at fully exploiting the potential of fibroblasts, variability in their responses, driven by donor-specific factors such as species and age, remains poorly understood. In this study, we investigate fibroblasts differentiation in vitro , focusing on how biomaterial surfaces can guide cell growth, proliferation, and morphology through mechanical cues. We compare fibroblasts from two species (rodent and human) and, within the human donor group, across two distinct age ranges. By combining conventional morphological and cytoskeletal analyses with advanced three-dimensional (3D) label-free imaging techniques such as optical diffraction tomography (ODT), we characterize the morphological transformations of these cells cultured on two biomaterial surfaces, silk fibroin (SF) and Zinc-Aluminum hydrotalcite (HTlc), by measuring cell dry mass and projected area. Our findings provide insights into how substrate characteristics shape fibroblast morphology and introduce an effective strategy to identify substrates that preferentially promote neuron-like morphological shapes, an important feature for inducing neuronal reprogramming.