Researchers created a library of micromolded, hexagonally spaced elastomeric micropost arrays, one to a few microns high, on which they cultivated cells.
The micropost system allowed engineers to modulate the rigidity and flexibility of the substrate surface without changing the adhesive or other material surface properties that could affect cell growth. Post height determined the degree to which a post would bend in response to a cell's horizontal traction force. The system enabled researchers to map cell traction forces to individ¬ual focal adhesions and spatially quantify sub-cellular distributions of focal-adhesion area, traction force and focal-adhesion stress.
The research, published in the current issue of the journal Nature Methods, demonstrated that the height of the posts determined the flexibility of the surface substrate, which in turn impacted the cell's morphology, leading to differences in focal adhesions, cytoskeletal contractility and stem-cell differentiation. Furthermore, early changes in cytoskeletal contractility measured by the devices predicted lineage fate decisions made days later by the stem cells.
"The library of micro¬post arrays spanned a more than 1,000-fold range of rigidity from 1.31 nN ìm−1 up to 1,556 nN ìm−1," said Chris Chen, lead author and the Skirkanich Professor of Innovation in Bioengineering in the School of Engineering and Applied Science at Penn. "Furthermore, the micropost array library will be made available to researchers in other laboratories."
Using current methods, it was not possible to change surface rigidity without also affecting other cellular properties such as the amount of active ligand molecules presented to cells, making it difficult to tease out the precise contributions of rigidity to cellular behavior.
Prior techniques employed the culture of cells on hydrogels derived from natural extracellular matrix proteins at different densities; however, changing densities of the gels impacted not only mechanical rigidity but also the amount of the binding or signaling ligand, leaving uncertainty as to the relevant contribution of these two matrix properties on the observed cellular response. Other synthetic hydrogels have been used that can vary rigidity without altering ligand density, but such systems cannot separate whether cells are sensing flexibility of individual molecules or of the macroscale mechanics.
"Although hydrogels will continue to be important in characterizing and controlling cell-material interactions, alternative approaches are necessary to understand how cells sense changes in substrate rigidity," Chen said.
In the body, cells do not exist in isolation but are in constant contact with other cells and with the extracellular matrix, providing structural support as well as both molecular and mechanical signals. In prior research, Chen's team has demonstrated that the push and pull of cellular forces drives the buckling, extension and contraction of cells during tissue development. These processes ultimately shape the architecture of tissues and play an important role in coordinating cell signaling, gene expression and behavior, and they are essential for wound healing and tissue homeostasis in adult organisms.
This study was conducted by Chen, Jianping Fu, Yang-Kao Wang, Michael T. Yang, Ravi A. Desai, Xiang Yu and Zhijun Liu of the Department of Bioengineering at Penn. Fu and Wang are now faculty members at the University of Michigan and the Skeletal-Joint Research Center of the National Cheng-Kung University Medical School.
The research was funded by grants from the National Institutes of Health, the National Science Foundation, the Army Research Office Multidisciplinary University Research Initiative, the Material Research Science and Engineering Center, the Institute for Regenerative Medicine, Penn's Nano/Bio Interface Center, the Center for Musculoskeletal Disorders of the University of Pennsylvania, the New Jersey Center for Biomaterials and the American Heart Association.
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