Effect of cyclic compression on cytoskeleton remodeling and cell matrix interaction of hMSCs encapsulated in three dimensional type I collagen matrix

Abstract

The potential of determining stem cell fate through mechanoregulation has been demonstrated recently. However, the underlying mechanism remains largely unknown. Previously, we developed a novel microencapsulation technique to entrap cells in a nanofibrous collagen meshwork and use the cell-collagen model to study mechanoregulation of human mesenchymal stem cells (hMSCs). Initially, hMSCs were randomly distributed within the construct. Upon cyclic compression, hMSCs reoriented towards a direction along the loading axis. Cytoskeleton, being the major sub-cellular machinery supporting cell shape and motility, should play crucial role in sensing and responding mechanical signals. Therefore, a better understanding in the change of cytoskeleton and associated molecules upon mechanical loading is a prerequisite to rationalizing the loading regimes for stem cell-based functional tissue engineering.

In the current project, we hypothesize that hMSCs encapsulated in 3D collagen construct will respond to cyclic compression by remodeling the cytoskeleton structures and altering the interactions with collagen matrix. hMSCs collagen construct were cyclically compressed for 9 hours through micromanipulator based compression system. After compression, constructs were harvested either immediately after compression, 2 hours after compression and 24 hours compression, together with non-loading control group.

Here, we report compression-induced novel changes in cytoskeleton. Firstly, omnidirectional filopodia-like structures together with stress fibers bucking were observed immediately after 9hrs of cyclic compression. Secondly, actin patches were observed shortly after removal of 9hrs compression before the actin fibers resumed. Apart from exhibiting similar morphology with filopodia, the omnidirectional filopodia-like structures may share a similar function in interacting with ECM. Co-localization of the major membrane-bound matrix metalloproteinases MT1-MMP with actin staining was found along the length of the filopodia-like structures. A local collagen digestion zone, characterized by the presence of collagenase cleaved collage, was found co-localizing at least partially with the filopodia-like structures around the cell. Whether creating pericellular collagen digestion zone was mediated by MT1-MMP along the compression-induced filopodia like structures and what functions the digestion zone serves are interesting question to answer in the future.

Another interesting observation is the complete disassembly of pre-existing stress fibers followed by formation by numerous actin patches throughout the cell shortly after removal of the compression loading. Stress fibers reformed in 24 hours after removal of the loading. Quantitative measurement of F:G actin ratio agrees with such disassembly and reassembly dynamics. Colocalization of actin branching protein arp2/3 with the actin patches was found, suggesting that mechanically loaded hMSCs were re-establishing actin cytoskeleton network from these nucleation centers. Further studies are required to figure out the underlying significance of the loading-induced cytoskeleton dynamics in hMSCs and whether the actin patches Arp2/3 complex associates with endocytosis of cleaved collagen fragments.