This study is the first to investigate whether integrin clustering mediates intracellular force transduction and subsequently regulates the phenotype 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 contraction 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). 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-C indicate when hBSMCs are cultured on hydrogel with smaller RGD spacing, both cell spreading area and proliferation rate increase, with 70 nm spacing appearing a threshold. Figure 2D-E further illustrates that as RGD spacing increases from 30 nm to 150 nm, hBSMCs tend to transition from a proliferative to contractive phenotype.
Figure 2F, 2G-K presents 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.

Taken together, these results indicate that hBSMCs have difficulty spreading and proliferating on adhesive interfaces where the spacing between adjacent ligands exceeds 70 nm, which notably enhances cell contraction. 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, spreading, contraction and proliferation, providing valuable insights for the surface and interface modification strategies of bladder repair materials[3].

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.
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.
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).

Continence 19S (2026) 102586
DOI: 10.1016/j.cont.2026.102586