Injury to the central nervous system (CNS) can be extremely debilitating and the regenerative capacity of this tissue is notoriously poor. Intracranial pressure (ICP) levels after injury is known to be a predictor of patient outcome, however the reasons behind this remain unclear. It is known that neural stem cells, present in the CNS may be affected by the elastic modulus of their surrounding matrix however efforts to investigate this have also altered other physical parameters, most notably the pore size of the matrix. It was hypothesised that the activities of neural stem cells are affected by the elastic modulus of their 3D environment, altered independently of pore size; and by the hydrostatic pressure of their environment, altered within a biomimetic range of ICP after traumatic brain injury. The hypotheses were interrogated using an in vitro system, and with a hydrogel of tunable elastic modulus and a hydrostatic environment.
The effects of elastic modulus, independently of pore size, on the activities of neural stem cells was investigated using a chemically crosslinked hydrogel. Hydrogels variants were synthesised with two different crosslinker types, 1,3-phenylenediacetic acid (Ph) and tartaric acid (Ta), and shear storage modulus was found through rheometry to be significantly higher in the Ta hydrogel, although neither hydrogel fell into the target biomimetic range. Analysis of scanning electron microscopy (SEM) images, of a trinitrobenzenesulfonic acid (TNBS) assay and of T$_2$ relaxation by nuclear magnetic resonance (NMR) logging found no significant differences between the two hydrogels in pore diameter, degree of crosslinking or pore volume, respectively. The porosity of Ta hydrogels was found to be highly significantly larger by comparison of water content. The phenotype and cell spreading of the CB660 neural stem cell line was measured, finding no significant difference in protein expression, however cells cultured in the Ta hydrogel were significantly more spread. These findings indicated that it may be pore size and not elastic modulus that affects stem cell differentiation, however further research is required to improve the power of these stem cell studies.
The effects of hydrostatic pressure on the activities of the CB660 neural stem cell line was investigated using a pressurised modular incubator. The migration of cells was evaluated using a novel layered gel assay, a significant increase was observed in glial cell migration under increased pressure, while it was found that neural stem cell migration may be inhibited by increased pressure, with a 61.5% reduction in migrated cell number comparing 30mmHg to 10mmHg. Cell viability was assessed using successive CyQuant and ATPlite assays, finding that ATPlite readings were significantly lower at 50mmHg compared to 30mmHg, indicating that increased pressure may diminish cell viability. Cell phenotype and morphology was assessed in 2D, finding that increased pressure may encourage neuronal differentiation, however may inhibit the formation of projections. Again however, further research is required to improve the power of these stem cell studies due to low repeat numbers.
The results of the study of elastic modulus and pore size indicated that pore size may indeed be an important parameter to consider when studying stem cell differentiation in response to mechanosensitivity as it may be pore size and not elastic modulus that stem cells respond to, and this may be an important parameter for future researchers to include. The results of the study of hydrostatic pressure indicated that pressure does have a direct effect on both glial and neural stem cells, and that the combination of characterisitics of Lundberg A waves including high pressure (more than 40mmHg), short wavelength (less than 30 minutes) and a return to normal pressure levels (5-15mmHg) between waves may all be instrumental in the cellular repair processes and improved patient outcome. However, the power of the studies using the CB660 neuronal stem cell line were limited and should therefore be regarded with caution.
