Multicellular aligned bands disrupt global collective cell behavior.

Mechanical stress patterns emerging from collective cell behavior have been shown to play critical roles in morphogenesis, tissue repair, and cancer metastasis. In our previous work, we constrained valvular interstitial cell (VIC) monolayers on circular protein islands to study emergent behavior in a controlled manner and demonstrated that the general patterns of cell alignment, size, and apoptosis correlate with predicted mechanical stress fields if radially increasing stiffness or contractility are used in the computational models. However, these radially symmetric models did not predict the existence of local regions of dense aligned cells observed in seemingly random locations of individual aggregates. The goal of this study is to determine how the heterogeneities in cell behavior emerge over time and diverge from the predicted collective cell behavior. Cell-cell interactions in circular multicellular aggregates of VICs were studied with time-lapse imaging ranging from hours to days, and migration, proliferation, and traction stresses were measured. Our results indicate that elongated cells create strong local alignment within preconfluent cell populations on the microcontact printed protein islands. These cells influence the alignment of additional cells to create dense, locally aligned bands of cells which disrupt the predicted global behavior. Cells are highly elongated at the endpoints of the bands yet have decreased spread area in the middle and reduced mobility. Although traction stresses at the endpoints of bands are enhanced, even to the point of detaching aggregates from the culture surface, the cells in dense bands exhibit reduced proliferation, less nuclear YAP, and increased apoptotic rates indicating a low stress environment. These findings suggest that strong local cell-cell interactions between primary fibroblastic cells can disrupt the global collective cellular behavior leading to substantial heterogeneity of cell behaviors in constrained monolayers. This local emergent behavior within aggregated fibroblasts may play an important role in development and disease of connective tissues. STATEMENT OF SIGNIFICANCE: Mechanical stress patterns emerging from collective cell behavior play critical roles in morphogenesis, tissue repair, and cancer metastasis. Much has been learned of these collective behaviors by utilizing microcontact printing to constrain cell monolayers (aggregates) into specific shapes. Here we utilize these tools along with long-term video microscopy tracking of individual aggregates to determine how heterogeneous collective behaviors unique to primary fibroblastic cells emerge over time and diverge from computed stress fields. We find that dense multicellular bands form from local collective behavior and disrupt the global collective behavior resulting in heterogeneous patterns of migration, traction stresses, proliferation, and apoptosis. This local emergent behavior within aggregated fibroblasts may play an important role in development and disease of connective tissues.

The Role of Apoptosis and Oxidative Stress in a Cell Spheroid Model of Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is the most common heart valve disease among aging populations. There are two reported pathways of CAVD: osteogenic and dystrophic, the latter being more prevalent. Current two-dimensional (2D) in vitro CAVD models have shed light on the disease but lack three-dimensional (3D) cell-ECM interactions, and current 3D models require osteogenic media to induce calcification. The goal of this work is to develop a 3D dystrophic calcification model. We hypothesize that, as with 2D cell-based CAVD models, programmed cell death (apoptosis) is integral to calcification. We model the cell aggregation observed in CAVD by creating porcine valvular interstitial cell spheroids in agarose microwells. Upon culture in complete growth media (DMEM with serum), calcium nodules form in the spheroids within a few days. Inhibiting apoptosis with Z-VAD significantly reduced calcification, indicating that the calcification observed in this model is dystrophic rather than osteogenic. To determine the relative roles of oxidative stress and extracellular matrix (ECM) production in the induction of apoptosis and subsequent calcification, the media was supplemented with antioxidants with differing effects on ECM formation (ascorbic acid (AA), Trolox, or Methionine). All three antioxidants significantly reduced calcification as measured by Von Kossa staining, with the percentages of calcification per area of AA, Trolox, Methionine, and the non-antioxidant-treated control on day 7 equaling 0.17%, 2.5%, 6.0%, and 7.7%, respectively. As ZVAD and AA almost entirely inhibit calcification, apoptosis does not appear to be caused by a lack of diffusion of oxygen and metabolites within the small spheroids. Further, the observation that AA treatment reduces calcification significantly more than the other antioxidants indicates that the ECM stimulatory effect of AA plays a role inhibiting apoptosis and calcification in the spheroids. We conclude that, in this 3D in vitro model, both oxidative stress and ECM production play crucial roles in dystrophic calcification and may be viable therapeutic targets for preventing CAVD.