Journal Club 20/01/2016

1. Journal Club20/01/2016

2. Background • Looking in to biocompatibility of hydrogels • Grow neuronal cells on the surface • Ultimately, I want a material that is biologically inert, and one which encourages the attachment and growth of neurons.

3. Introduction • The stiffness of a cells environment impacts cell adhesion, migration, differentiation and phenotype. • In 1997 Wang first demonstrated fibroblast migration is greater on stiffer substrates. • This dependence is less clear for neuronal cells and cells of softer tissues. • 2 relationships proposed for neurite behavior and matrix stiffness: • Neurite outgrowth varies inversely with stiffness • Neurite outgrowth occurs maximally at intermediate stiffness

4. Materials and MethodsSubstrate preparation • Polyacrylamide substrates • Varied crosslink density • Constant covalently bonded fibronectin throughout

5. Materials and MethodsShear modulus characterization • Prepolymer solution cast as 5ml hydrogels in 6cm culture dishes • Washed 2x5 mins distilled water, then allowed to swell overnight • Cut into 4cm diameter discs • Shear moduli at 20˚C TA Instruments AR100 rheometer • Parallel plate geometry set to constant strain of <2% • Stress sweep 0.1-20Pa for softer gels, 1-500Pa for stiffer

6. Materials and MethodsCell Culture • PC12 cells induced by neurite growth factor into neuronal phenotype • Received at passage 1, used at 5-6

7. Materials and MethodsAnalysis of neurite outgrowth • Plates warmed to room temperature and imaged using an Axiovert S25 microscope with a CCD camera and Matrox Intellicam imaging software or a Zeiss Axiovert S100 microscope equipped with a digital camera and Metamorph imaging software • Images were captured under phase-contrast microscopy at 5–16 random locations. • Neurite length, branching, the number of neurites per cell and per cent cells expressing neurites were analyzed with ImageJ • Criteria: • only PC12 cells responsive to NGF were included • Only neurites >5 μm in length and that did not contact other cells or neurites were included • 5 μm bin size was used in the neurite length frequency analysis. • Branching was calculated from counting the total number of occurrences per sample where one neurite split into two and dividing the branch count by the total neurite length in the sample.

8. Statistical Analysis • ANOVA tests (for normally distributed data) • Kolmogorov–Smirnov tests (for data that were not normally distributed) • Significance level of p < 0.05.

9. Results

15. Discussion • Found that PC12 neurons display a threshold response to substrates varying in shear modulus from ∼101–104 Pa • On hydrogels of the lowest shear modulus examined (7 Pa), the few neurites that extended were relatively short and unbranched. • On hydrogels of shear modulus of 190 Pa and above, more neurites were initiated per cell, and in general, the neurites per cell were longer and more branched. • For these stiffer materials (>190 Pa), a correlation was not found between the increased modulus and neurite length, branching, neurites per cell, or the percentage of cells expressing neurites. • PC12 cells are limited in their ability to mimic fully the behaviour of primary neurons, thus possibly explaining differences in behaviour • By using K-S test, we are able to detect the significant differences in neurite outgrowth on materials of differing compliance without distortions of significance levels due to tests based on normal distribution assumptions.

16. Discussion • On very soft substrates (e.g., <103 Pa), PC12 neurons are able to remain viable and extend neurites, whereas other cell types are only loosely adherent or unspread • Because neural tissues are much softer than dermal, orthopedic and cardiovascular tissues, neurons are able to grow on much softer substrates. • Increasing stiffness beyond those found in neural tissues (i.e., modulus∼102–103 Pa), however, does not seem to impact the extent of PC12 neurite outgrowth, branching, neurites per cell or percentage of cells expressing neurites. • In contrast to other published studies of neural response to substrate stiffness, PC12 cells extend neurites that are more numerous, longer and more branched on stiffer substrates • Further stress the importance of quantifying ligand density, analyzing large sample sets and applying appropriate statistical tests.