The research objective of this EArly-Concept Grant for Exploratory Research (EAGER) is to establish the link between the changing cadherin expression, or the cadherin switch model, and the evolving mechanical microenvironment that differentiating mesenchymal stem cells (MSCs) encounter during bone formation. MSCs experience both cell-matrix, or integrin-mediated, and cell-cell, or cadherin-mediated, forces during differentiation. However, the role of cell-cell forces through cadherins has been largely neglected despite that cadherin regulation is essential to normal bone formation. The "cadherin switch" specifies that as MSCs differentiate in to bone forming cells, one cadherin is up regulated and another is down regulated. The switch parallels the changing mechanical environment during bone formation: MSCs begin in soft marrow (~1kPa) and differentiate into bone matrix-producing cells in stiff, unmineralized matrix (10s of kPa). This work aims to relate the cadherin switch to the changing mechanical microenvironment for MSCs. The approach uses a combination of two tools: Electrohydrodynamic (E-Jet) and 3D, full-field traction force microscopy (3D TFM). E-Jet will pattern both proteins and stiffness on polyacrylamide substrates and enable manipulation cell-cell and cell-substrate forces. 3D TFM will measure the forces thus quantitatively establishing the relationship.
The influence of cell-cell forces in differentiation and bone formation has largely been neglected. Results from this work could lead to new treatments for bone diseases and a more complete understanding of tissue development both for bone and for other tissues. Through the work the relatively new techniques, 3D TFM and E-Jet patterning of cell culture substrates, will be made more accessible to a broader scientific community. The educational component focuses on graduate student training and research experiences through interactions between institutions and across disciplines. The research will also be used to highlight the breadth of the field of Mechanical Engineering (ME) specifically for the Campus Middle School (CMS) for Girls, with the goal of making ME more accessible to these and other K-12 students who otherwise may not be interested in ME based on preconceptions of the field.
This project had two research areas for which there were major outcomes. The first research focus was the development of a new technology for patterning soft gels with cell adhesion proteins in order to study the effect of substrate stiffness and chemistry and cell-cell interactions on cell behavior in vitro. The use of patterned gels to study cell behavior allows researchers to control how cells interact with each other and with the local environment. Thus, patterned gels allow for understanding how parameters like substrate stiffness and chemistry might influence tissue formation, like bone formation, and diseases like cancer, among others. Bone tissue formation, or osteogenesis, was of specific interest in this work. The publication by Poellmann et al  details the printing process and the ability to control of pattern feature size as well as the ability to print multiple materials of different stiffness onto a substrate of yet a different stiffness. In brief, a gel containing a fluorophore was printed on a silicon substrate and polymerized by UV exposure. Next, a second unpolymerized polymer solution was placed over the micropatterned droplets, exposed to the UV in order to cross-link, or solidify, the polymer solution, and finally the composite was peeled off the silicon substrate. The paper demonstrates a composite with two patterns of different stiffness, 44kPa and 2kPa, embedded in an 18kPa background. In a related paper, Poellmann et al  verified the chemistry used to pattern the substrates and how the chemistry influenced the protein attachment and mechanical properties of the soft gel substrates. The second research focus of the project was to measure with what force different cells pull on the substrate to which they are attached and relate this to cell shape measures and differentiation stage of the cells . In order for cells to be attached to a substrate, they must exert force on the substrate. This force is related to the cell behavior. Further, the cell shape is relevant both to the forces the cells exert and to their state of differentiation between a stem cell and a bone cell. The work showed increasing area and force as cells differentiated from the earliest stages to cells that resembled osteocytes, fully mature bone cells. Future work will utilize the techniques and technologies developed to study the interactions between cell-cell forces and cell-material forces to understand bone formation. Intellectual Merit and Broader Impact: This work contributes to the understanding of cell behavior in the context of bone formation. Specifically, the new patterning approach that was developed can be used to study the interaction of cells with their chemical and mechanical environment. This approach can be used across disciplines and cell systems to study cell interactions with their environment. Further, cell shape and force exerted on the substrate across stages of differentiation can help to design materials to enhance bone formation. Ultimately, this work can lead to a better understanding of bone formation and how to control cell behavior in this context, as well as in the context of other diseases in which the cellular mechanical and chemical environment is important. This is true with almost all cells and tissues, and for many diseases, as we understand them now. The project also contributed to advancing the careers of both graduate and undergraduate students through education and scientific mentoring, including a large fraction of which were from underrepresented groups.  Poellmann, M. J., & Wagoner Johnson, A. J. (2014). Multimaterial polyacrylamide: fabrication with electrohydrodynamic jet printing, applications, and modeling. 6. Biofabrication, 6, 035018. doi:10.1088/1758-5082/6/3/035018;  Poellmann, M. J., & Wagoner Johnson, A. J. (2013). Characterizing and Patterning Polyacrylamide Substrates Functionalized with N-Hydroxysuccinimide. Cellular and Molecular Bioengineering, 6(3), 299–309. doi:10.1007/s12195-013-0288-5;  Michael J. Poellmann, Jonathan B. Estrada, Thomas Boudou, Zachary Berent, Christian Franck, Amy J. Wagoner Johnson. Evolution of cells morphology and contractility during osteogenesis. in preparation.