Biophysical Characterization and Theoretical Analysis of Molecular Mechanisms Underlying Cell Interactions with Poly(N-isopropylacrylamide) Hydrogels

• Main Author: Cross, Michael C.
• Format: Others
• Published: Scholar Commons 2016
• Subjects: atomic force microscopy; isopropylacrylamide; tissue engineering; flory rehner theory; bioprinting; Biomedical Engineering and Bioengineering; Biophysics
• Online Access: http://scholarcommons.usf.edu/etd/6213; http://scholarcommons.usf.edu/cgi/viewcontent.cgi?article=7409&context=etd

So-called, “Dynamic biomaterials” comprised of stimuli-responsive hydrogels are useful in a wide variety of biomedical applications including tissue engineering, drug delivery, and biomedical implants. More than 150,000 peer-reviewed articles (as of 2016) have been published on these materials, and more specifically, over 100,000 of these are on the most widely studied, poly(N-isopropylacrylamide). This thermoresponsive polymer in a crosslinked hydrogel network undergoes a large volume phase transition (𝑉/𝑉0 ~ 10 − 100) within a small temperature range (𝑇 ~ 1 − 3𝐾) making it particularly useful for tissue engineering applications because of the ability to control the topographical configuration of cells into tissue modules which can be applied in multiple layers to form three dimensional constructs. Nevertheless, applications with poly(N-isopropylacrylamide) hydrogels are hindered by two key obstacles: 1. there is presently no quantitative prediction of mechanical properties over the volume phase transition and 2. the mechanisms of cell attachment and detachment remain controversial and unclear. Current polymer-solution theory, first postulated by Paul Flory and Maurice Huggins in 1942, successfully predicts hydrogel swelling for non-stimuli-responsive polymers based on an empirically derived interaction parameter. However, for stimuli-responsive polymer hydrogels, this theoretical framework fails to quantitatively predict swelling and mechanical properties of the polymer. Currently, only qualitative agreement with experiment has been shown. Cell-cell and cell-matrix interactions are mediated through proteins collectively known as cell adhesion molecules. For cell-matrix interactions, these are generally the transmembrane protein, integrin, and the serum protein, fibronectin. It is widely accepted that nearly all molecular mechanisms of cell-matrix interactions are dependent on recognition of the peptide sequence Arg-Gly-Asp. However, much less is known about mechanical mechanisms involved in cell-cell and cell-matrix interactions. Obstacles to the advancement of these applications are 1) unclear mechanisms of cell release and 2) extended exposure of cells to hypothermic conditions. The author, in collaboration with others, has published work demonstrating reduced cell exposure to hypothermic conditions during tissue module release and elucidated a mechanism of tissue module release: mechanical strain. The central hypothesis of work in this proposal is that tissue module release occurs due to a mechanical strain-rate coinciding with critical force needed overcome the dynamic bond strength of cell adhesion molecules. Advances in this area could improve biomaterial design and accelerate the field of regenerative medicine by reducing or eliminating the need for allograft transplants. This dissertation project, then, seeks to address these two obstacles through biophysical characterization methods and analysis including: atomic force microscopy, scanning electron microscopy, laser-scanning confocal micrscopy, phase-contrast microscopy, and mass-balance analysis. It is hypothesized that, (1) mechanical properties of PNIPAAm hydrogels are quantitatively predicted based on crosslinker ratio in the water-rich phase, (2) release of cells from micropatterned PNIPAAm hydrogels occurs when the lateral strain in the surface exceeds ϵ > 0.25, and (3) the molecular mechanism of rapid cell release from micro-patterned PNIPAAm hydrogels is mediated by the transmembrance protein integrin and its extracellular matrix receptor, fibronectin. Results from these studies could be useful for improving the design of biomaterials based on PNIPAAm hydrogels for applications in tissue engineering.