(3) Mastication mimicking mechanical stretch of fibroblast induces active neurotropic factors

Introduction: Evidence suggests that brain-derived neurotrophic factor (BDNF) enhances periodontal regeneration. Mechanosensory nerve with ruffini endings are immunoreactive to tyrosine kinase B, a high-affinity receptor for BDNF, and are concentrated in the area where periodontal fibers are being stretched the most when teeth are in function during mastication. This study investigates a possible molecular mechanism of how stimulated periodontal ligament cells induced sensory nerve cells attachment at a cellular level. Our testing hypothesis is that masticatory tension stimulates fibroblasts to secrete nerve chemotactic, BDNF, and the activated form of BDNF initiates the migration of neuronal cells.

Methods: Masticatory tension was mimicked by a mechanical cyclic stretch of a type I collagen (PureCol) coated round silicone sheet (40mm diameter) on which NIH-3T3 fibroblasts (120 x 10^4 cells) were cultured in with the growth medium (DMEM). Cyclic stretch was generated by a biaxial stretching machine (CellScale-MCB1) at 0.2Hz. Stretching frequencies were done in a 30-minute stretch cycle (repeats of 30s stretch and 30s rest) for two rounds, 3 to 4 hours apart for each round of stretching for 3 consecutive days. The culture medium was collected on day 0 as the pre-stretch sample, and a fresh growth medium was replaced for the stretching experiment. Post-stretch media was collected on days 1, 2, and 3 after the stretching experiment which was performed on days 0, 1, and 2, respectively. The total BDNF concentration was measured by ELISA. Differentiation of immature proBDNF and the pro-peptide cleaved mature BDNF (mBDNF) were identified by immunoprecipitation using Western blot. The effect of experimental media on Neuro-2a cells was tested using migration assay and migrated cells were counted by CCK-8 assay. All measurements were triplicates for statistical analysis.

Results: The total BDNF released in the stretched medium (3 days average: 346.99 pg/ml) was doubled compared to the pre-stretch medium (171,73 pg/ml) with a significant difference between the pre-stretch and each stretched sample (Tukey Post Hoc Test; p< 0.0001). There is no significant difference between each stretched sample. A pronounced presence of proBDNF and mBDNF were identified in the post-stretch medium than in the control sample with no stretch. Post-stretch medium significantly attracted more Neuro-2a cells migration compared to the pre-stretch medium. When the stretched medium was neutralized with anti-BDNF antibodies, the migrated cells decreased similar to the pre-stretch medium.

Conclusions: Mastication mimicking cyclic stretch of fibroblasts induced secretion of both pro-and mature BDNF. The increased activated mBDNF concentrations in this mechanically induced extracellular matrix provided an environment favorable to neuronal cells migrations.

Lead Author’s Contributions to the Research Project: The Lead Author was responsible for performing all experiments, data collection, data analysis and the composition of the abstract.