The Effects of Melatonin on Proliferation, Osteogenic Differentiation and in Vivo Bone Regeneration of Dental Pulp-Derived Mesenchymal Stem Cells and Related Molecular Mechanisms

Mesenchymal stem-cell-based tissue engineering plays a significant role in bone defect repair and bone regenerative therapies. However, the efficiency of mesenchymal stem cells (MSCs) transplantation is questioned and the low survival of engrafted stem cells is still a major challenge in rent studies. Among the strategies of improving stem cell survival during the transplantation, small molecules (such as melatonin) can provide an efficient, rapid, safe, reversible and cost-effective way to differentiate stem cells when comparing to the growth factors, gene regulation and mechanical stimulation methods. Recently, melatonin has been regarded as a promising agent of regulating stem cell biology and protecting stem cells against oxidative stress and inflammatory mediators. Although previous studies have investigated the biological effects of melatonin on osteoblasts, the effects of melatonin on dental pulp-derived mesenchymal stem cells’ osteogenic differentiation, in vivo bone regeneration, and the molecular mechanisms involved in the process are still unknown. Thus, investigating the influences of melatonin on the proliferation, migration, osteogenic differentiation, mineralization, osteogenic gene expression, in vivo bone regeneration and the underlying signal transduction pathways of rabbit dental pulp-derived mesenchymal stem cells (rDPSCs) is great significance. The purposes of this research proposal are to (1) determine whether melatonin enhances rDPSCs’ proliferation, osteogenic differentiation and mineralization, (2) to elucidate potential signal transduction pathways that regulate this process and (3) to investigate the osteogenic effects of a combination of HA/TCP bone grafting materials and preconditioning MSCs with melatonin in rabbit calvarial defects. Based on these viewpoints, the present research proposal was designed to assess (1) the osteogenic effects and possible molecular mechanisms of melatonin on rDPSCs by using in vitro cell culture model, wound-healing migration analysis, biochemical methods, mineralization assay, real-time PCR, immunofluorescence staining and western blotting analysis (2) the in vivo bone healing capacity and molecular mechanisms of the combination of HA/TCP scaffold and preconditioning MSCs with melatonin in rabbit’s calvarial bone defect model by using micro-CT analysis, histological, histomorphometric, immunofluorescence staining (IHC), microarray and gene expression analysis. With the completion of the two-year research project, we not only demonstrated the biomechanical, molecular and histological evidences for the therapeutic abilities of MSCs with melatonin, but also provide scientific evidence for combining stem cell with small molecule-based regenerative therapy in the clinical application.