The research objective of this award is to use microfabrication, live cell imaging, and genetic and molecular tools to investigate the physical effects of the cell microenvironment on the osteogenic (or bone) differentiation of human mesenchymal stem cells (hMSCs). The osteogenic differentiation of hMSCs is sensitive to the mechanical properties (such as matrix stiffness) of the surrounding cellular microenvironment. In this project, we will use microfabrication to establish a novel library of elastomeric micropost arrays, where by modulating the geometrical factors of these micropost arrays, we can precisely change the stiffness of the micropost array and thus the innate contractile or mechanical state of hMSCs lying on the microposts. We propose to use these elastomeric micropost arrays coupled with different genetic and molecular tools to investigate the functional crosstalk between substrate stiffness and intracellular cytoskeletal contractility of hMSCs and their crucial involvements in the mechanoresponsive hMSC osteogenesis. hMSCs are adult stem cells that form and heal nearly all of the mechanical tissues in humans, including bone. Thus, understanding the mechanical regulation of hMSC osteogenesis may provide insights into hMSC biology and further improve hMSC osteogenesis in vitro for regenerative therapeutics. The educational activities of this award will have broad impacts on students from different educational levels and genders and ethnicities. The technologies developed in the context of this award will be used as vehicles for outreach activities to K-12 students and other underrepresented female and minority students in the Michigan Ann Arbor and Ypsilanti school districts. The outreach activities will reveal to K-12 students the exciting challenges in science and engineering and their close relevance to our society, thus motivating them to pursue science and engineering curricula.
This research is to leverage recent advances in microfabrication to develop novel functional microscale tools for mechanistic investigation of mechanosensitive properties of human mesenchymal stem cells (hMSCs). Specifically, we have proposed to use such micromechanical tools to investigate effects of substrate rigidity and external mechanical forces on osteogenic (bone) differentiation of hMSCs. hMSCs are adult stem cells that form and heal nearly all of the mechanical tissues in humans, including bone. Thus, understanding mechanoregulation of hMSC osteogenesis may provide insights into hMSC biology and further improve hMSC osteogenesis in vitro for regenerative therapeutics. Intellectual Merit Outcome: The mechanoresponsive hMSC osteogenesis is thought to be regulated by molecular mechanisms centering on the intracellular actin cytoskeleton (CSK)-focal adhesion (FA)-extracellular matrix (ECM) linkage for the reciprocal transmission of mechanical signals. Despite the clear need to elucidate the relationships between matrix mechanics, external forces, CSK tension (intracellular force), and FA stress/signaling, very few tools currently exist to precisely modulate substrate rigidity and external forces while simultaneously measuring live-cell subcellular responses of CSK tension and FA dynamics. To address this critical need, in this research we have successfully developed a standardized library of microfabricated elastomeric micropost arrays to precisely regulate substrate rigidity. These micropost arrays have a uniform surface geometry and different post heights to modulate substrate rigidity independent of effects on adhesion and other material surface properties. These microposts can also serve simultaneously as cantilevers to report traction forces exerted by adherent cells. More recently, we have developed another unique micromechanical system, termed micropost array membrane (mPAM), in which the elastomeric microposts are integrated onto a stretchable membrane, such that when the base membrane is stretched, stretching forces can be transmitted through the posts to adherent cells seeded on top of the posts. Using the mPAM, we have developed a novel strategy for whole-cell cell stiffness measurement with a subcellular spatial resolution. We have further studied the functional role of actin CSK architecture in regulating cell shape-dependent mechano-sensitivity to directional cell stretch. In our most recent effort, we have applied the mPAM system to perform live-cell subcellular studies of force-mediated FA morphogenesis, to examine the spatiotemporal evolution and coordination of FA dynamics and CSK contractile force. We have further extended our research to study mechanosensitive behaviors of human pluripotent stem cells (hPSCs) and how such knowledge can be applied to promote their self-renewal and enhance their directed differentiation efficiency. Together, our research has developed novel technology platforms and methodologies for mechanobiology research. Our mechanistic studies have also provided quantitative information about the mechanotransductive events centering on the cytoskeleton-focal adhesion-extracellular matrix signaling axis. Broader Impact Outcome: Owing to its multi-disciplinary nature, our research has seamlessly integrated knowledge from different fields including micro/nanoengineering, mechanobiology, and stem cell science. To achieve our research, we have developed different integrated micromechanical tools that can be extremely useful for other researchers working in the general field of mechanobiology. Our research has also helped establish a novel mechanistic framework for understanding the mechanosensitive stem cell-biomaterial interactions, which is important and critically needed for future developments of functional biomaterials for large-scale stem cell culture. In this research, we have conducted educational activities to target students from different educational levels and genders and ethnicities. Some of the technologies developed in our research have been used as vehicles for our outreach activities to K-12 students and other underrepresented female and minority students. Through established educational programs at UM (including the UROP, SURE/SURE, and NNIN REU programs), we have recruited many undergrads to participate in our research. Many of these undergraduate students are female and belong to minority groups. By exposing the students to exciting challenges in research, these students become more motivated to pursue a career in science and engineering. Lastly, we have developed a completely new interdisciplinary course in biomechanics for the College of Engineering at the University of Michigan. This course can prepare engineering students to pursue research in a variety of multi-disciplinary areas such as tissue engineering and regenerative medicine that are of great importance to our society today.
