This study is the first to investigate whether integrin clustering mediates intracellular force transduction and subsequently regulates the behavior of human bladder smooth muscle cells (hBSMCs). Previous studies have shown that specific integrin subtypes form focal adhesions (FA) by binding to extracellular RGD sequences, thereby transmitting mechanical signals to cells and activating downstream pathways that regulate various cellular behaviors[1]. Building on this foundation, we aimed to explore how different integrin ligand spacings influence hBSMC spreading and proliferation.

Hydrogels with RGD spacings of 30 nm, 70 nm, and 150 nm were fabricated (Figure 1C). Briefly, PS(x)-b-P2VP(y) was stirred overnight in dry o-xylene. HAuCl3·3H2O precursor was added, and the solution was stirred to obtain Au-loaded micelles. Glass slides were spin-coated with the micellar solution and plasma-etched using a microwave plasma system (Figure 1A). After that, gold nanoarray-decorated slides were immersed in N,N′-bis(acryloyl)cystamine solution. The polymerization precursor was prepared by dissolving poly(N-acryloyl glycinamide) (NAGA) monomer, ammonium persulfate, and N,N,N′,N′-tetramethylethylenediamine in Milli-Q water[2]. This solution was applied to the AuNP-patterned slides and polymerized under UV light. The resulting hydrogels were detached from the slides and stored in Milli-Q water for later use. For hBSMC adhesion, hydrogels were incubated in a 25 μM cyclic RGDfK solution with an OEG spacer (Figure 1B). The hydrogel storage modulus was approximately 23 kPa (Figure 1D, 1E). Finally, hBSMCs were cultured on the UV-sterilized nanopatterned hydrogels for 12 h, 24 h, and 48 h, followed by immunofluorescence staining for paxillin, p-myosin II, and laminA/C.

Figure 2A demonstrates fluorescent immunostaining of cells adhering to substrates with varying RGDfK spacings, suggesting differential integrin clustering and focal adhesion formation. Notably, cells on 30nm-spaced substrates likely exhibit denser integrin aggregation (indicated by paxillin) and stronger intracellular force (indicated by p-myosin II and laminA/C) compared to larger spacings. Figure 2B and 2C quantify cell spreading areas and aspect ratios, revealing that smaller RGDfK spacings (30nm and 70nm) promote greater cell spreading (larger than 3000 μm2 on average) and lower aspect ratios (indicating a more circular morphology) compared to 150nm spacing. Figure 2D tracks cell proliferation over 48 hours. At 12 hours, non-adherent cells were removed, leaving adherent populations that proliferate over time. We found that cell number increased across all groups, with cells on the 30 nm ligand-spacing substrate exhibiting the highest proliferation rates.

Taken together, these results indicate that hBSMCs have difficulty adhering and spreading on adhesive interfaces where the spacing between adjacent ligands exceeds 70 nm, which also significantly impairs cell proliferation. This suggests that intracellular force transduction mediated by FA formation plays a critical role in regulating cell behavior in response to different ligand spacings.

The design of artificial bladder repair materials remains a challenge, with surface morphology often overlooked. This study demonstrates that ligand spacing significantly impacts hBSMC adhesion, spreading, and proliferation, providing valuable insights for the surface and interface modification strategies of bladder repair materials[3].

[1]	Miroshnikova YA, Rozenberg GI, Cassereau L, et al. a5ß1-Integrin promotes tension-dependent mammary epithelial cell invasion by engaging the fibronectin synergy site. Drubin DG, ed. Molecular Biology of the Cell. 2017;28(22):2958-2977.
[2]	Ye K, Wang X, Cao L, et al. Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate. Nano Letters. 2015;15(7):4720-4729.
[3]	Peng L, Jin X, He Q, et al. Remodelling landscape of tissue-engineered bladder with porcine small intestine submucosa using single-cell RNA sequencing. Cell proliferation. 2022;56(1)