Design and quantitative mechanical characterization of stiffness gradient cell culture substrates

Polymer channels have been proposed as an alternative to autologous nerve grafting to treat severed and damaged peripheral nerves. Most nerve guidance channels are constructed of a single, mechanically uniform material. However, nerve regeneration and neurite extension have been shown to be dependent on mechanical stimuli in rat models and in vitro cultures. In an effort to improve the function of nerve guidance channels, the effect of mechanical gradients on nerve cell function was studied using phase contrast microscopy and instrumented indentation. Instrumented indentation techniques are well developed for hard materials, but a consensus has yet to form on appropriate mechanical models and testing protocols for analyzing experimental data from hydrated biomaterials.;First, a method for synthesizing non-toxic polyHEMA (poly-2-hydroxyethyl methacrylate) hydrogels using Irgacure 2959 as the photoinitiator was developed. The polyHEMA hydrogels were then UV-polymerized in thermoplastic molds under a circular, gradient photomask to produce stiffness gradient hydrogels. The gels were coated with solubilized rat tail collagen to promote neuron adhesion.;Second, nanoindentation testing and analysis methods were selected to accommodate the unique surface mechanical behavior of soft, hydrated hydrogels. Several mechanical models were evaluated including Oliver-Pharr, Hertz Contact Theory, Standard Linear Solid, and Johnson-Kendall-Roberts adhesion models. A Maxwell-Wiechert model provided the best correlation between results from nanoindentation and unconfined compression on uniform polyHEMA hydrogels.;Third, the cellular response of undifferentiated and differentiated PC-12 cells to the gradient gels was determined using phase contrast microscopy. PC-12 cells were seeded on the stiffness gradient pHEMA gels and Corning cell bind controls. Cell location, cell area, and neurite extension were monitored as a function of a gel's mechanical properties. Both undifferentiated and differentiated PC-12 neurons preferentially attached to the stiffest region of the gel. The differentiated PC-12 neurons attached to the stiffest regions of the gel also extended more neurites than other neurons. This research demonstrates that a complex mechanical microenvironment has the potential to improve nerve guidance channel design and peripheral nerve recovery.