Researchers from Virginia Tech and the University of Pittsburgh have collaborated to employ a novel nanoscale fibrous system that can measure the tiny forces exerted by and upon individual cells with extreme precision. The team hopes that this platform, which investigators call nanonet force microscopy (NFM), will provide new knowledge about smooth muscle cell biology that could have implications for treating cardiovascular disease, which is still a leading cause of death in the United States.

The results of investigations on cells using this platform appear in the “Forces” issue of the journal Molecular Biology of the Cell, in the article “Nanonet Force Microscopy for Measuring Forces in Single Smooth Muscle Cells of Human Aorta,” published July 7, 2017.

The main goal of this current study, said Julie Phillippi assistant professor at the University of Pittsburgh Department of Cardiothoracic Surgery whose laboratory provided healthy human smooth muscle cells for the study, was to quantify forces healthy cells experience in various conditions of stress. The fibrous nanonet itself was designed in the mechanical engineering laboratory of Amrinder Nain, associate professor at Virginia Tech University and member of the American Society for Cell Biology. Forces measured using NFM, Nain said, include forces exerted by the cells themselves and forces exerted by the environment on the cells.  “Everything in nature has a physical force,” said Nain. “This platform measures both simultaneously.”

Nanonet Force Microscropy (NFM) can measure the contractile inside-out forces of a single cell attached to multiple fibers. Shown here are f-actin (red), paxillin (green), and the nucleus (blue). Scale bar = 20 micron. Nanonet Force Microscropy (NFM) can measure the contractile inside-out forces of a single cell attached to multiple fibers. Shown here are f-actin (red), paxillin (green), and the nucleus (blue). Scale bar = 20 micron.

The basic design of the nanonet resembles the strings and frets of a guitar (minus the supporting fretboard). Thicker, non-flexible fibers approximately 1,000 nm in diameter run north and south and are widely spaced like frets. Perpendicular to the thicker fibers, thinner fibers approximately 250 nm in diameter run east and west and are more closely spaced (for this study ~15-20 microns apart), like guitar strings. The thinner fibers are fused to the thicker fibers at each intersection. In such an arrangement, cells are attached only to the suspended fibers that allow visualizing cell processes at high resolution.

A coating of adhesion factor fibronectin allows the single cells of human aortic smooth muscle to attach to the fibers. Several layers of nanonets were stacked loosely in a growth media chamber to allow for simultaneous studies on many individual cells. As the cells grow along and between the thin fibers, they tug with their intrinsic contractile forces. Phase contrast light microscope was used to collect images and video.

Inside-out forces were measured by the deflections of the thin fibers made by the contractions of cells growing along them. Outside-in forces were measured when thin fibers were stretched with a probe to simulate forces within the heart acting upon the cells. By adding hydrogen peroxide, the cells were placed under oxidative stress. Oxidative stress—when there are more reactive oxygen species in a biological system than the system can detoxify—results in the production of free radicals, which are associated with many diseases, such as heart disease, diabetes, and cancer. Not surprisingly, the study revealed that under stress from either chemical or physical assault, healthy cells exerted only about half as much force on their environment and could withstand only about half as much perturbation as normal.

Phillippi said that previous work tested the mechanical strength of whole aortic tissue and understanding the single cell biomechanics is vitally important. Single cell studies provide insight into the proteins involved in the fleeting so-called focal adhesions that most cells make as they move around their microenvironment. The NFM assembly aims to mimic, in as physiologically relevant a way as possible, what cells endure within the collagen fibers of the extra cellular matrix (ECM)—the matrix that supports cell growth in living things. Tweaking the artificial matrix by changing fiber diameter, density, and spacing in a controlled and repeatable manner, as well as using cells from diseased patients at different disease severities, will allow Phillippi and Nain to simulate conditions experienced by cells in many realistic situations.

“We have looked very closely at how the collagen and elastin fibers in the ECM are arranged and the micro-architecture and everything points to these microstructural defects in the ECM contributing to the weakening of the aortic walls and the ballooning of the vessel,” said Phillippi. “What we don’t know is, are these ECM proteins arranged that way from birth or is it something that happens over time? Or is it both? What role do the cells play? This engineered platform will allow us to answer some of those questions.”

Next steps for Phillippi and Nain include testing cells from the Pittsburgh team’s large repository of aortic specimens from patients, collected in collaboration with Thomas Gleason, Chief of the Division of Cardiac Surgery, University of Pittsburgh. Furthermore, Nain said, NFM could reveal the heterogeneity of cells taken from the same patient or from different patients with the same disease state down to the single-cell resolution.

“Gene expression profiles of selected cells could tell the story of why these differences might exist,” he said. “NFM makes it possible to conduct drug testing at the single-cell resolution to see if forces come back to normal phenotypic levels or if they are deviating in another way. Controlling the fiber diameter, spacing, and orientation brings a much needed new level of control and repeatability to interrogate matrix driven crosstalk within cells.”

Eventually, Nain and Phillippi said they hope to establish a database of baseline forces for many types of cells that researchers and clinicians can use to diagnose and treat disease.

“The platform gives us the ability to create in vitro disease models with multiple layers of sophistication,” said Phillippi.

In a broader context, Nain said he thinks the ability to achieve precise control on fiber diameter, spacing, and orientation to mimic native fibrous environments, will allow NFM to interrogate the push and pulls in a cell’s journey in developmental, disease, and repair biology. And, he said, “NFM is just another new ‘guitar’ whose true potential can be exploited only by those willing to who embrace multidisciplinary tuning to frequencies from sciences, philosophy, and engineering to listen to the songs of forces sung by cells.”

See original study here.

Watch a video about this research below or click here.

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Mary Spiro

Mary Spiro is ASCB's Science Writer and Social Media Manager. She has a master's degree in Biotechnology from Johns Hopkins University and bachelor's degrees in both Agronomy and Journalism from the University of Maryland, College Park. She can be reached at mspiro@ascb.org