Cells getting stiffed: cellular response to material properties
Cells on gels
How cells sense their mechanical environments and how they integrate mechanical signaling cues into their behavior has become one of the fundamental questions of biomedical engineering and remains a focus of my own studies. One important mechanical property is stiffness. About fifteen years ago, Robert Pelham and Yu-li Wang described an elegant system for producing surfaces of the same material but with different stiffnesses, by combining polyacrylamide (PAAm) and its crosslinker bis-acrylamide in different ratios but at the same total concentration, and then conjugating the gels they produced to type I collagen to permit cell adhesion. They showed marked differences in the behavior of two different cell types on flexible vs. stiff materials. Their method became quite popular and inspired a number of studies considering different cell types and processes.
One such study was conducted by Adam Engler in Dennis Discher's lab at U Penn. Engler, using the PAAm system, showed that human mesenchymal stem cells (MSCs) can be directed towards neural, muscle, or bone-like lineages in the same media on gels of different stiffnesses. His results were published in Cell.
Trouble lurks
A challenge to the PAAm system was published in Nature Materials last year by a group of 13 authors sprawling across England, Germany, Switzerland, and the Netherlands. Trappmann (now a postdoc in Chris Chen's group at Penn) and colleagues, working with human epidermal stem cells, found that their cells were profoundly affected by their PAAm gels of different stiffnesses. But when they made silicone rubber (PDMS) substrates that had the same stiffnesses as their PAAm gels, their cells behaved identically independent of stiffness. This happened whether the PAAm gels had collagen or fibronectin, another adhesion molecule, tethered to the surface. Having noticed the Engler study, they decided to try with human mesenchymal stem cells. They reproduced Engler's findings on PAAm but MSCs differentiated into osteoblasts on all of the PDMS surfaces.
If materials of the same stiffness are affecting cells in two different ways, there has to be another factor at play. Trappmann & co. looked at SEM images of their gels and discovered big changes in porosity as the stiffness of polyacrylamide gels varied:
In order to assess whether porosity was a factor affecting the cells, they freeze-dried polyacrylamide gels of different stiffnesses and coated them with a thin layer of gold, so that they would each display the same rigidity but different porosities to the cells. As before, even though the surfaces now had the same stiffness, cells on "soft" gels with large pores detached; cells on "stiff" gels with small pores spread.
Anchors aweigh
So what makes porosity matter? Trappmann & colleagues decided to take a look at how the different surfaces affected how collagen was anchored to the surface. Like many polymers, collagen acts a little bit like a bicycle chain: over short distances, like the individual links in a chain, collagen is very hard to bend. The "persistence length" of a polymer describes the largest distance over which it acts like a rigid chain link. Over long distances, like a well-oiled chain of many links, collagen is quite flexible. If you tied a bike chain down to a board at every link, it would be very rigid. If you tied it down less often and left some slack in the chain, the exposed parts of the chain would be more flexible.
Which is exactly the experiment Trappmann performed: by embedding gold nanoparticles (which bind to collagen) at fixed distances in a polyethylene glycol (PEG) gel (which doesn't), they tied down collagen either every 60 nm (about once per persistence length), or every 190 nm. Then, they exposed the surfaces to epidermal stem cells. Cells that saw tightly bound collagen behaved like cells on stiff substrates, spreading without differentiating. Cells that saw the loosely bound collagen behaved like cells on very soft substrates, differentiating and detaching.
Therefore, they conclude, the difference between the PAAm gels that the MSCs were responding to wasn't stiffness of the gels at all! It was the microscopic porosity, which controlled how tightly bound collagen was to the surface. Big pores offered fewer binding sites, which meant cells found it easy to pull on the collagen they were attached to. Small pores offered more tethering sites, so the collagen was much harder to pull on. Local stiffness still matters, but not in the way we thought.
I found the Trappmann study convincing. I'm going to have to look into whether there are other experimental systems that allow the collagen tethering to be modulated independent of surface stiffness, or whether it's possible to fabricate the gold-in-PEG system in our lab. Next, I'll talk about an analysis of another system which I found a little less impressive…
Reference: Trappmann, B., Gautrot, J., Connelly, J., Strange, D., Li, Y., Oyen, M., Cohen Stuart, M., Boehm, H., Li, B., Vogel, V., Spatz, J., Watt, F., & Huck, W. (2012). Extracellular-matrix tethering regulates stem-cell fate Nature Materials, 11 (7), 642-649 DOI: 10.1038/nmat3339