Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells

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Entry by Max Darnell, AP 225, Fall 2011

Reference:

Title: Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells

Authors: Kristopher E. Kubow, Enrico Klotzsch, Michael L. Smith, Delphine Gourdon, William C. Little and Viola Vogel

Journal: Integrative Biology, Volume 1, Number 11-12, December 2009

Keywords: polymerization, extracellular matrix, crosslinking, polymer, FRET


Summary

A recent trend in biology and bioengineering has been the understanding of cellular microenvironments and how these properties determine cell fate and function. For instance, it has been shown that matrix rigidity influences stem cell fate, and that tumor-associated fibroblasts can excrete new extracellular matrix that promotes tumor migration. The real goal of this study was two-fold: 1) to examine how cells’ initial rigidity condition affects their placement of new extracellular matrix (ECM) and 2) to examine the effects of ECM crosslinking and whether the rigidity or conformation of crosslinked ECM dictates cellular response. From these results, the rigidity of the crosslinked tissue is the determining factor, not a more relaxed conformation. Also, new fibers bind to native ECM, not crosslinked ECM, due to an attempt to increase rigidity.


Methods/Results

Part one of the study examined whether it was rigidity or structure of Fn that regulated cell function. Decellularized cell-derived fibronectin (Fn) was labeled for Forster Resonance Energy Transfer (FRET) (by labeling different domains of the Fn) and reseeded with fibroblasts. The FRET essentially acted as a strain sensor, since the magnitude of the signal is tied to the distance between acceptor/donor labels. Intensification of FRET signal thus indicated increased ECM tension, while relaxation of the signal indicated ECM compression. FRET was then used in this manner to gauge the effects of crosslinking with 4% formaldehyde.

The figure below shows the FRET data as well as the setup for labeling the FRET donors and acceptors.

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The authors then tried to see how cross-linking the fibronectin affected the structure and isotropy of the ECM when cells were seeded. The figure below shows that for non cross-linked samples, the cells induced significant stretching of the fibers, from a folded to unfolded state, whereas for cross-linked samples, the fibers were already more isotropic. Thus, additional strains brought about by the addition of cells were much lower.

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Finally, the authors needed to confirm whether the effects of crosslinking described above were purely mechanical or whether they were having any effect on actual fibronectin polymerization during cell-traction-induced ECM remodeling. The figure below shows FRET data, which does not change in its intensity bands for crosslinked and non-crosslinked samples, meaning that fibronectin polymerization is not inhibited by crosslinking.

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Connection to Soft Matter

This emerging area of cell/matrix interaction is interesting from both a biology and soft matter perspective. If bioengineers plan to exploit the microenvironments of cells to dictate cellular function, the mechanics and behaviors of these substrates must be well-understood. ECM properties, such as the effect of different crosslinking must be understood on cellular length scales instead of using traditional rheological methods. On the biology side, several interesting questions arise. For example, increased crosslinking of ECM to enhance mechanical stability can result in inflammation, fibrosis, or change in cell fate. Why? Also, why is crosslinking an important aspect of wound healing, but can induce malignant phenotypes in other cases? How is this rigidity communicated and how does it translate into altered cell fate?