Mechanisms and improvements of cell transplantation for nerve repair

Abstract: Trauma to the nervous system is a frequent clinical problem and new approaches to nerve repair are required. Autologous cell transplantation together with a suitable scaffold material could be used to create a bio-active artificial nerve graft to enhance regeneration. The work presented in this licentiate thesis attempts to improve both the biomaterial and cellular components of this repair strategy. In the first study, by using common biodegradable polyesters, namely poly-ε-caprolactone (PCL) and poly -L,D- lactic acid (PLA), a thin film scaffold prototype was fabricated by using a solvent-evaporation method. These scaffolds, with thicknesses of approximately 10-20 µm, exhibited a heterogenous but continuous surface topography decorated with pore/pits of regulated sizes. The sizes of the pore/pits ranged from 0.5 to 30 µm2and could be modulated by varying the ratios of PLA and PCL. Biocompatibility of these scaffolds was demonstrated by using adipose derived stem cells (ADSC) differentiated into a Schwann cell-like phenotype (dADSC), which showed attachment and proliferation on the films, maintenance of glial cell markers expression and enhancement of neurite outgrowth in co-culture with dorsal root ganglia (DRG) neurons. Transplantation of cells for nerve injuries remains sub-optimal due to their limited survival rates. In the second study, a chemical ischemia model (metabolically induced by sodium azide and 2-deoxyglucose) was established to investigate the differential effects of ischemia and serum deprivation on mesenchymal stem cells (MSCs). MSCs were more suseptible to combined than individual blockade of glycolysis and oxidative phosphorylation. Apoptotic and autophagy pathways were activated in the MSCs. Chemical ischemia or serum withdrawal alone induced a similar amount of cell death with significantly different intracellular ATP maintenance; but their effects were additive. The levels of various neurotrophin extracellular matrix and angiogenic factors expressed by the cells were shown to be differentially affected by ischemia but unaffected by changes in serum level. Stem cells isolated from both adipose tissue (ADSC) and bone marrow (BMSC) reacted similarly under these conditions. This chemical ischemia model will enable future screening of pharmacological agents to enhance the survival of MSCs under stress conditions. The mechanism underlying the neurotrophic potential of MSCs is unknown. In the third study in this thesis it is hypothesised that MSCs, upon stimulation with different growth factors, could produce brain derived neurotrophic factor (BNDF) with a similar molecular mechanism to that described in the nervous system. Within 24 hours of stimulation, ADSC and BMSC showed high secretion levels of BDNF, and these cells were able to enhance axonal outgrowth in DRG neurons at levels similar to long-term differentiated MSCs. Both the neuronal activity dependent promoterBDNFexon IV, along with full length protein encodingBDNFexon IX, were up-regulated upon stimulation.BDNFgating transcription factor, cyclic cAMP responsive element binding (CREB) protein, was also found to be activated but blocking of CREB phosphorylation with the small molecule inhibitor H89 did not suppress expression of BDNF protein suggesting compensatory pathways are involved. In summary, these studies indicate that MSCs are compatible with polyester based microporous scaffolds but the cells are highly susceptible to the stress conditions mimicking the hostile milieu at a nerve injury site. Preliminary studies hint at the molecular mechanism regulation BDNF expression in MSC and imply the interactions between MSCs and axons may play a role in the neurotrophic activity of the stem cells.