الفهرس 
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Abstract
Introduction : Functional restoration of injured nerves is often hampered by lack of optimal extracellular conditions that are necessary to promote neuronal survival and regeneration. Dental pulp stem cells (DPSCs) secrete neurotrophic factors (NTFs) which may play an important therapeutic role in neural development, maintenance and repair. In this study the role of DPSCs-derived factors on rat phaeochromocytoma cell line (PC-12) and trigeminal ganglion neuronal cells (TGNC) survival, growth, migration and differentiation was investigated. Materials and methods. Conditioned medium (CM) was collected from 72h serum-free DPSCs cultures (DPSCs-CM), which were established from extracted rat incisors and NTFs (nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF)) were analysed by specific ELISAs. Neuronal cells (PC-12 and TGNC) were treated with 50 ng/ml NGF, 50% DPSCs-CM or live DPSCs were co-cultured with neuronal cells using Transwell inserts. The number of surviving cells with neurite outgrowths and the length of neurites were measured using ImageJ software. Transwell migration inserts were used to assess neuronal cell migration under the influence of DPSCs-CM. Immunocytochemical staining was used to evaluate the expression of neuronal markers; NeuN and MAP-2 and cytoskeletal marker βIII-tubulin. Gene expression levels of axonal growth-associated protein 43 (GAP-43) and synaptic protein Synapsin-I, NeuN, MAP-2, βIII-tubulin and thermo-sensitive transient receptor potential vanilloid channel 1 (TRPV1) were analysed by quantitative real time polymerase chain reaction (qRT-PCR). Specific neutralising antibodies against NGF, BDNF, GDNF, NT-3 and mixture of these antibodies were used to study the exact role of the NTFs detected in the DPSCs-CM on neuronal cells survival and neurite outgrowth length. Results Compared with the serum-free controls where most of the neuronal culture was showing cell death, DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth as assessed by NeuN, MAP-2 and βIII-tubulin immunostaining. DPSCs-CM was significantly more effective in stimulating neuronal cells neurite outgrowths than live DPSCs/neuronal cells co-cultures over the time studied in both PC-12 and TGNC cultures. The morphology of induced PC-12 cells in DPSCs-CM was similar to NGF positive controls; however, in TGNC culture, DPSCs-CM stimulation of neurite outgrowth was significantly higher than what was seen in NGF-treated cultures. DPSCs-CM also had a chemoattractant potential as was shown to increase migration of PC-12 and TGNC across the Transwell after 24h treatment. The number of surviving PC-12 neuronal cells were markedly reduced by the addition of anti-GDNF antibody while the neurite outgrowth was significantly attenuated by addition of anti-NGF, anti-GDNF and anti-BDNF antibodies. In TGNC cultures, blockage of NT-3 resulted in significant attenuation of neuronal survival while neurite outgrowth was significantly attenuated by the blockage of GDNF. Conclusion : This study demonstrated that DPSCs-derived factors promoted survival and regeneration of injured primary isolated TGNC as well as PC-12 neuronal cell line and appeared to be more effective in inducing neuronal survival and neurite outgrowth more than live DPSCs co-cultures. DPSCs-CM may be harnessed as a cell-free therapy for peripheral nerve repair.