Professor Rochester Institute of Technology Rochester, New York, United States
Introduction: Biomedical research and drug development increasingly depend on tissue barrier models that closely simulate physiological conditions for accurate preclinical testing. Traditional methods like animal testing and 2D cell cultures often inadequately replicate human responses. As a result, innovative approaches such as co-culture models and lab-on-chip devices have emerged, offering more relevant results and precise control over experimental parameters. These advancements are crucial in developing in vitro models that mimic endothelial tissues, essential for regulating blood pressure, coagulation, molecule transport, and immune responses. Such models depend on accurately replicating the basement membrane (BM), a complex component of the extracellular matrix that underlies epithelial and endothelial tissues. However, challenges remain in mimicking BM's structure due to its intricate composition and mechanical properties. Synthetic polymers and hydrogels provide some solutions with their flexibility and biocompatibility, but issues like thickness discrepancies still affect their physiological relevance. Our research focuses on developing ultrathin silicon nitride membranes for use in co-culture setups. These membranes support physiological conditions conducive to cell interaction and migration by providing tunable porosity, pore size, and thickness, yet challenges arise from their inherent stiffness compared to natural tissues. We aim to enhance their functionality by coating the surface with Poly(Ethylene Glycol) (PEG), which adjusts surface stiffness to better mimic the in vivo environment. We then explore the potential of softened surfaces to enhance cell adhesion, proliferation, and junctional protein expression, while maintaining the integrity of the membrane. Our objective is to deepen our understanding of how this modified surface impacts cell behavior.
Materials and
Methods: In this study, a microporous silicon nitride membrane with a 0.5 μm pore size was subjected to an oxygen plasma treatment to facilitate the electrostatic binding of PLL-g-PEG-Biotin on its surface. Subsequently, a 20 μL drop of 0.5 mg/mL PEG solution was applied to the treated surface and allowed to incubate at room temperature. The coated surface was then examined using Atomic Force Microscopy (AFM) to quantify the presence of the PLL-g-PEG-Biotin and to assess surface roughness. AFM imaging was conducted in tapping mode over a 20 μm x 20 μm area, calculating the root mean square roughness (Rq) from the obtained height profiles. To evaluate how cells interact with the modified surface, a cell adhesion experiment was conducted using streptavidin conjugated with fibronectin to modify PEG, a non-fouling polymer known to inhibit protein adsorption and cell adhesion. Streptavidin was chosen for its strong biotin-binding affinity, ensuring effective fibronectin anchoring. Three different sample types were prepared for testing: surfaces solely coated with PLL-PEG-biotin, those with an additional layer of streptavidin-fibronectin, and those directly treated with fibronectin. Human Umbilical Vein Endothelial Cells (HUVECs) were seeded on these samples with the density of 40,000 cell/cm2. After 24 hours, the cells were stained with DAPI and Phalloidin to visualize nuclei and cytoskeletal structures, respectively, assessing the adhesive properties of each surface treatment.
Results, Conclusions, and Discussions: Results and Discussions: Topographical imaging using AFM demonstrated clear differences in the surface profiles between coated and non-coated membranes (Figure 1A). Rq for the non-coated surface was measured at 1.325 nm, which contrasted sharply with the coated surface's roughness of 4.094 nm. This increase in roughness indicates the successful addition of PEG molecules to the surface. In the cell adhesion experiments, distinct variations in cell behavior were observed across different surface treatments (Figure 1B). The images featured a non-adhesive PEGylated surface, where minimal cell attachment occurred, highlighting the non-fouling nature of the PEG layer. However, subsequent treatment with Streptavidin-fibronectin markedly enhanced cell adhesion, validating the effectiveness of this surface modification in promoting cellular attachment. This finding underscores the potential of Streptavidin-fibronectin in modifying surface properties to favor cell adhesion. Further comparisons between surfaces treated with PEG-fibronectin and those directly fibronectin-coated showed significant differences in cell morphology. HUVECs on the PEGylated surfaces exhibited a more rounded shape and a reduced spreading area compared to those on directly fibronectin-coated surfaces. These observations suggest that the PEGylated surfaces provide a softer substrate, which may affect the mechanical interactions between the cells and the substrate, influencing how cells anchor and spread. This result points to the importance of surface softness in cellular behavior and provides insights into designing biomaterial surfaces for specific cellular interactions.
Conclusions: Preliminary data has confirmed that surface modifications were successful and that cells can distinguish differences in surface properties. Based on our observations, cells on PEG-coated surfaces exhibited less spreading compared to those on non-coated surfaces, consistent with previous studies on reduced cell spreading. Further research will focus on the effects of these softened substrates on HUVEC spreading, proliferation, and junction formation to better understand the implications of surface softness on cellular interactions and tissue barrier properties. This ongoing study will enhance our comprehension of cell-material interactions, crucial for optimizing biomaterial design in medical applications.
