Title

Hydrogels for central nervous system regeneration: Surface modulus and microtopographical effects on neuronal cell behavior

Date of Award

2008

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Chemical Engineering

Keywords

Mechanical properties, Topographical properties, Nerve regeneration, Cell behavior, Hydrogels, Substrates

Subject Categories

Biomedical Engineering and Bioengineering | Engineering

Abstract

This work investigated the effects of surface modulus and microtopography of hydrogel substrates on the behavior of neuronal cells in vitro . The dissertation focused on three polymers of varying moduli, poly(2-hydroxethyl methacrylate) (p(HEMA)), polyacrylamide and agarose, which were either flat or fabricated with a micropatterned topography. Pheochromocytoma (PC12) cells, Schwann cells (SC) and primary cortical neurons were cultured on each of the substrates and cell behavior was assessed to determine if effects of surface modulus and microtopography influenced cell behavior either individually or synergistically.

The mechanical properties of the substrates were determined using indentation-testing methods. A custom-built microindentation system was used to characterize the modulus of macroporous p(HEMA) hydrogels across a variety of hydrogel compositions. The microstructure of the substrates was characterized by scanning electron imaging in order to elucidate the relationships between the polymer chemistry and the effects on modulus. Improvements and modifications to the original microindentation system were undertaken, which permitted the testing of softer more compliant materials, including polyacrylamide and agarose. The modified system, termed the mesoindenter, was more sensitive, accurate and capable of testing substrates with moduli as low as 1.0 kPa.

The hydrogel substrates were synthesized to have moduli ranging from 22.4 kPa to 1412.4 kPa, as determined by indentation testing. The agarose substrates were the softest of the three hydrogels with a modulus ranging between 22.4-29.9 kPa, polyacrylamide substrates encompassed the intermediate modulus range with values between 206.7-210.8 kPa and the p(HEMA) substrates were the stiffest of the three hydrogels exhibiting a modulus of 1412.3 kPa.

Cell adhesion, neurite length and neurite alignment were characterized as a function of substrate surface modulus and microtopography on each of the different substrates. Adhesion of PC12 cells, SCs, primary neurons and co-cultures of SCs with primary neurons were examined on flat and micropatterned substrates of varying moduli. Furthermore, neurite length of PC12 cells and primary neurons, and cellular or neurite alignment across all three cell types were measured on flat and patterned p(HEMA), polyacrylamide and agarose to characterize neuronal cell behavior as a function of the substrate properties.

The effects of micropatterned surface topography of hydrogel substrates influenced Schwann cell, PC12 cell and primary neuron adhesion. Additionally, the micropatterned surface topography enhanced PC12 neurite and primary neurite outgrowth and alignment. The effects of surface modulus on neuronal adhesion and neurite outgrowth were not as clearly defined as the effects of surface topography; however, surface modulus had a noticeable effect on neurite outgrowth. The presence of SCs co-cultured with primary neurons had an effect on primary neurite outgrowth, but primary neuron adhesion and neurite alignment were not enhanced compared to primary cultures alone.

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