Cyclic stretch inhibits cell invasion in 3D scaffolds.

Background: The development of clinically viable tissue-engineered heart valves (TEHVs) remains limited by inconsistent host cell infiltration. The dynamic hemodynamic environment may play a central role in driving or inhibiting cell invasion, yet the effects of cyclic stretch on cell migration and proliferation remain largely unexplored in 3D tissues and scaffolds. Given evidence that uniaxial constraint promotes directional invasion in 3D matrices, we hypothesized that uniaxial cyclic stretch would enhance cell invasion, particularly along the stretch direction. Methods: We embedded multicellular spheroids into collagen hydrogels and subjected them to uniaxial cyclic stretch (3-10%, 1 Hz) for two days and quantified invasion into the surrounding extracellular matrix using a custom image-processing program. Smooth muscle cells, valvular interstitial cells, and dermal fibroblasts were examined to represent cell populations relevant to TEHVs and for comparison across cell types with different contractility. To determine the mechanisms underlying changes in invasion with stretch, effects of cell tension were evaluated using gel compaction assays and inhibition of myosin IIA, and proliferation was assessed by Ki67 immunostaining. Results: Contrary to our hypothesis, cyclic stretch profoundly inhibited cell invasion into the matrix across all cell types and magnitudes of stretch. Invasion decreased by >50% in smooth muscle cells and fibroblasts and by up to 99% in valvular interstitial cells. Invasion suppression was inversely correlated with cell contractility, implicating a role for cell-generated tension. Inhibition of myosin IIA partially rescued invasion with stretch, though not to static levels. Stretched spheroids also exhibited reduced cell proliferation relative to static controls. Conclusions: These findings implicate actomyosin-mediated mechanotransduction in stretch-induced suppression of cell invasion and suggest that the dynamic valve environment may limit host-cell repopulation of TEHVs. More broadly, this work provides insight into how cyclic stretch regulates 3D cell invasion in mechanically active tissues with implications for wound healing and cancer metastasis.