Meso-Scale Topological Cues for Endothelial Cell Growth on Fibrous Biomaterials

The long-term success of blood-contacting medical devices critically depends on the establishment of a stable, functional endothelial lining that can prevent thrombosis and inflammation. However, current vascular grafts and implantable materials often fail to reproduce the hierarchical microstructure and anisotropy of the native basement membrane, which governs endothelial organization and function. This work addresses this limitation by developing a novel hybrid lithography-electrospinning biofabrication approach to create biomimetic fibrous scaffolds that replicate the structural and topographical features of the vascular intima layer.Initially, native vascular tissues from human, porcine, and ovine sources were analyzed to establish morphological and structural benchmarks of the native tissue substrate. Scanning electron microscopy revealed a well-organized, anisotropic endothelial monolayer oriented along the circumferential direction of blood flow. Multiple decellularization strategies, mechanical, chemical, and sonication-based, were tested to expose the native basement membrane. None achieved complete endothelial removal without damaging the underlying structure, confirming the technical challenge of isolating intact basement membrane layers and motivating the design of synthetic analogues.To replicate the native vascular basement membrane topography, a hybrid lithography-electrospinning technique was developed, combining the geometric precision and resolution of lithography with the versatility of electrospinning. This method enabled the transfer of micro-scale patterns (square, honeycomb, and groove geometries) onto polyurethane-based fibrous scaffolds. Compared to bulk substrates, which are limited by low surface-to-volume ratio and poor permeability, electrospun scaffolds offer a dramatically higher surface for cell adhesion enhances cellular infiltration and nutrients diffusion. Moreover, while bulk technologies are typically restricted to small, localized devices, the electrospinning process is scalable, enabling the fabrication of large-area coatings or surfaces suitable for extended blood-contacting applications, such as extracorporeal oxygenators. Optical and scanning electron analyses confirmed successful pattern transfer, with correlation coefficients between 50-60%, indicating reproducible and high-fidelity replication. Notably, increasing the pattern depth from 1 μm to 4 μm improved fiber alignment and feature definition, highlighting the influence of topography depth on the electrospinning process.Biological assays using endothelial cells demonstrated excellent scaffold biocompatibility. Cell viability and proliferation analyses revealed robust viability and adhesion, particularlyivon functionalized substrates. Morphological quantification showed that 4 μm groove patterns induced strong cellular alignment and elongation, closely mimicking the anisotropic organization of native endothelium. In contrast, square and honeycomb patterns promoted isotropic growth. Over 48 hours, cells developed a continuous monolayer while progressively depositing collagen IV and fibronectin, demonstrating active extracellular matrix remodeling. Interestingly, extracellular matrix accumulation gradually masked the underlying microtopography, suggesting that substrate geometry plays a dominant role in early alignment but is later superseded by self-secreted matrix cues.Collectively, these results confirm that topographical guidance alone can drive endothelial morphogenesis, even in static culture conditions. Groove depth emerged as a critical design parameter, controlling both fiber organization during fabrication and cell alignment during culture. The hybrid lithography-electrospinning method thus establishes a reproducible and scalable platform for fabricating engineered basement membrane analogues with tunable anisotropy.From a translational perspective, the proposed approach provides a pathway toward hemocompatible coatings and vascular scaffolds capable of supporting endothelialization in a physiologically relevant manner. By coupling microstructural precision with fibrous biomimicry, this study bridges the gap between fundamental endothelial biology and clinical device engineering. The findings offer design principles for next-generation cardiovascular implants and large-scale blood-contacting devices, emphasizing the importance of early-stage topographical cues in achieving long-term endothelial stability and function, while demonstrating the power of electrospinning-based microfabrication to extend basement membrane-inspired topological cues beyond small-scale graft to large, clinically relevant surfaces.