Fibroblast Morphology on Dynamic Softening of Hydrogels

Abstract

Despite cellular environments having dynamic characteristics, many laboratories utilized static polyacrylamide hydrogels to study the ECM-cell relationship. To attain a more in vivo like environment, we have developed a dynamic, DNA-crosslinked hydrogel (DNA gel). Through the controlled delivery of DNA, we can temporally decrease or increase gel stiffness while expanding or contracting the gel, respectively. These dual mechanical changes make DNA gels a cell-ECM model for studying dynamic mechanoregulated processes, such as wound healing. Here, we characterized DNA gels on a mechanical and cellular level. In contrast to our previous publication, in which we examined the increasing stiffness effects on fibroblast morphology, we examined the effects of decreased matrix stiffness on fibroblast morphology. In addition, we quantified the bulk and/or local stress and strain properties of dynamic gels. Gels generated about 0.5 Pa stress and about 6-11% strain upon softening to generate larger and more circular fibroblasts. These results complemented our previous study, where dynamic gels contracted upon stiffening to generate smaller and longer fibroblasts. In conclusion, we developed a biomaterial that increases and decreases in stiffness while contracting and expanding, respectively. We found that the dynamic deformation directionality of the matrix determined the fibroblast morphology and possibly influences function.

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

“…However kinetic information on DNA gels is limited, but we do know an additional 30% of crosslinking occurs two days after L2 delivery to 50% DNA gels 11 . Nevertheless, the generation of expansive and contractile forces from gel softening and stiffening, respectively, is unavoidable and cannot be decoupled 18,21 . 100→70% gels generate about 0.5 Pa of stress 21 , while 50→100% gels only generate about 0.04 Pa of stress since more energy is required to form bonds among the three stands of ssDNA 18 .…”

“…Second, standard cell and tissue culturing techniques are used for these experiments and can be found in numerous original articles, technical articles, and reference books. Techniques for specifically plating fibroblasts and neurons onto DNA gels can be found in previous publications [9][10][11][19][20][21] .…”

“…Plate cells. For protocols regarding dissection and/or culturing of neurons or fibroblasts mentioned in this article, refer to one of the following publications [9][10][11][19][20][21] . 2.…”

“…DNA-crosslinked polyacrylamide hydrogels (DNA gels) are dynamic twodimensional elastic substrates. DNA crosslinks allow for temporal, spatial, and reversible modulation of DNA gel stiffness by addition of singlestranded DNA (ssDNA) to media or buffer [9][10][11]13,14,18,21 . Unlike the aforementioned dynamic gels where stimuli are applied for modulation of elasticity, the DNA gels rely on the diffusion of applied ssDNA for the alteration of elasticity.…”

“…Image blurriness, as seen in Figure 3, was due to gel surface unevenness and is typical and unavoidable. Gel swelling and contraction, resulting from the addition of buffers and L2 18,21 , contributes to gel unevenness. Morphology was evaluated with ImageJ software (NIH, Bethesda, MD) and served as an indicator of function.…”