Regeneration of crush-injured mental nerve using Schwann-like cells derived from dental pulp stem cells combined with pulsed electromagnetic field

Regeneration of crush-injured mental nerve using Schwann-like cells derived from dental pulp stem cells combined with pulsed electromagnetic field Wei-Hong Hei (Directed by Professor: Jong-Ho Lee, DDS, MSD, PhD) Department of Oral and Maxillofacial Surgery, Graduate School, Seoul National University Background Peripheral nerve injury is relatively common in clinical situation which may result in serious disturbance such as dysesthesia, persistent paresthesia and pain. Various techniques have been investigated to enhance peripheral nerve regeneration including the application of pulsed electromagnetic field (PEMF) and administration of Schwann cells from mesenchymal stem cells as the potential sources. Purpose of study The purpose of study was firstly to clarify the influence of frequency and duration of PEMF and find the suitable condition of PEMF on the peripheral nerve regeneration; and secondly to investigate the effects of combining PEMF and local injection of Schwann-like cells, in comparison with each single modality, on peripheral nerve regeneration in a rat model with crush-injured mental nerve. Materials & Methods The experiments were divided into two parts, part I and part II. Part I: Immortalized rat Schwann cells (iSCs) (1x10/well) were exposed at 4 different conditions (50 Hz 1 Hr/day, 50 Hz 12 Hr/day, 150 Hz 1 Hr/day and 150 Hz 12 Hr/day). Cell proliferation and mRNA expression of S100 and BDNF (brain-derived neurotrophic factor) were analyzed. Sprague-Dawley rats (200–250 g) were divided into 6 groups (n=10 each): Control, Sham, 50 Hz 1 Hr/day, 50 Hz 12 Hr/day, 150 Hz 1 Hr/day and 150 Hz 12 Hr/day groups. Mental nerve was crush-injured and exposed at condition of PEMF for 3 weeks. Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labeling of trigeminal ganglion (TG). Part II: Schwann-like cells derived from human dental pulp stem cells (hDPSCs) (1 ×10 cells per well) were used. Integrin β4 (CD104), S100, glial fibrillary acidic protein (GFAP), laminin, P75 were detected with Immunocytochemistry. P75 and S100 mRNA were analyzed using Quantitative Real-time polymerase chain reaction (qRT-PCR); Male Sprague-Dawley rats (200250 g, six-week-old) were divided into 7 groups (n=10 each): Control, Sham, PEMF, hDPSCs, hDPSCs + PEMF, Schwann-like cells, Schwann-like cells + PEMF group. Cells were transplanted (106 cells/6μl) after crush injury with or without PEMF exposure. Outcomes of nerve regeneration were evaluated with functional test, histomorphometry and retrograde labeled neurons. Statistical analyses were performed using SPSS program version 21 (SPSS Inc., Chicago, IL, USA). All data were presented as mean values with standard error means (SEM). The normal distribution of the measurements was verified by One-Sample Kolmogorov-Smirnov normality test. One-Way ANOVA test (axon counts, labeled neurons result) and Repeated Measure ANOVA test (sensory test, MTT and qRT-PCR results) followed by post-hoc Tukey test was used to compare data between different groups. P value ≤. 05 was considered statistically significant. Results Part I: iSCs proliferation with 50 Hz, 1 Hr/day was increased from 4th to 7th day; mRNA expression of S100 and BDNF was significantly increased at the same condition from first week to third week (p<.05, vs. control); Difference score was increased at the second and third week, and Gap score was improved at the third week under 50 Hz 1 Hr/day PEMF compared with control while other conditions showed no statistical significance. Axon counts and the number of retrograde labeled neurons were significantly increased under PEMF of four different conditions compared with control. Although there was no statistical difference, 50 Hz, 1 Hr/day PEMF showed highest regeneration ability than other conditions. Part II: Schwann-like cells differentiated from hDPSCs expressed CD104, S100, GFAP (glial fibrillary acidic protein), laminin, P75 (P75 neurotrophin receptor). P75 and S100 mRNA expression was the highest in Schwann-like cells exposed with PEMF. Difference and Gap scores were increased significantly at the first and third week than control while other treatment groups showed no significance. Axon and retrograde labeled neuron counts were obviously increased in all interference groups. Schwann-like cells + PEMF group showed the highest ability and significances were found when compared with PEMF (P=.033, axon count; P=.035, retrograde labeling); hDPSCs (P=.042, axon count) and Schwann-like cells group (P=.038, axon count). Conclusion 1. PEMF enhanced peripheral nerve regeneration, and that it may be due to cell proliferation and increase in BDNF and S100 gene expression. Among the various conditions, 50 Hz, 1 Hr was revealed to be the most suitable inferred from the data of in Vitro and in Vivo study. 2. Schwann-like cells and hDPSCs with or without PEMF in addition to PEMF alone improved peripheral nerve regeneration after crush injury. Among these, the Schwann-like cells combined with PEMF showed the highest regeneration ability. PEMF had a synergic effect on the ability for hDPSCs and Schwann-like cells to regenerate nerves in Vitro and, after transplantation, in Vivo.