Dynamic compression reulgates cell-matrix adhesion and cytoskeleton network of human mesemchymal stem cells in three dimensional collagen environments

Abstract

Comparing to two dimensional (2D) cultures, mechanoregulation study of cell in three dimensional (3D) cultures is more physiologically relevant. However, it remains largely unclear how cells sense and respond to mechanical loading in 3D system, particularly on changes of cell-matrix adhesion, cytoskeletal organization and signaling mechanism. Our lab has developed a 3D cell collagen microencapsulation culture system, and demonstrated alignment response of hMSCs in 3D collagen construct along loading direction under dynamic compression using a custom made micromanipulator based loading system. Understanding the mechanoregulation mechanism of hMSCs is important for rational loading regime degsin for tissue engineering purpose. Here we investigate mechanoregulation of cell matrix adhesion and cytoskeletal changes of hMSCs in 3D collagen construct under dynamic compression. In 5 hours, 7 days compression experiment, hMSCs respond to dynamics compression by formation of elongated ?5¬ integrin –containing -adhesions with colocalaziation with fibronectin. For shorter time point loading study, immunofluorescent study reveal that 10mins dynamics compression induce the colocalaziation of integrins ?v and FAKp397, while 30mins dyamics compression leads to concentration of FAKp397 into larger spots. In 9 hours compression, besides colocalization of integrins ?5 and fibronectin, diffues fibronectin was observed in collagen matrix. We have also further investigate changes of cytoskeletal network in 9 hours condition. We find that under dynamic compression, hMSCs respond by extending numerous filopodia liked actin strucure, colocalizing with Arp2/3, distrubuted around the whole cell together with the breakdown of microtubule network and disappearance of primary cilia labelled by acetylaed ?-tubulin, which were not observed in control, unloaded group. 2 hours after removal of compression, most of filpodia liked actin strcutre were disassembled, with numerous actin patches were observed instead and netowrk of microtubule started to re-establish. 24 hours after removal of compression, actin strcuture and microtubule network return to that simialr to control, unloaded group. We conclude that hMSCs respond to compression in 3D collagen environemnt by changing cell matrix adhesion components and reogranization cytoskeletal sturcutre. Delineating the mechanosensing mechanism will contribute to rationalize design of loading protocols for stem cell based functional tissue engineering.