Effects Of High-mobility Group Box1on The Proliferation, Migration And Odontoblastic Differentiation Of Human Dental Pulp Cells

Background and Objective:Human dental pulp cells (hDPCs) can proliferate and differentiate into odontoblasts thus creating reparative dentine in response to the appropriate stimuli. The response to stimuli and formation of reparative dentine of pulp tissue is a complex biological process, which involves the migration, proliferation and differentiation of hDPCs into odontoblast/odontoblasts-like cells. However, the precise mechanisms underlying the proliferation and differentiation of hDPCs remain unclear.High-mobility group box1protein (HMGB1) belongs to a family of high-mobility group nuclear proteins, which is a DNA-binding protein that has nuclear effects, including proper transcriptional regulation and DNA recombination and repair. In addition to its nuclear role, HMGB1functions as an extracellular signaling molecule that regulates both inflammation and regeneration. According to previous studies, HMGB1induced cardiac progenitor cell proliferation and differentiation in myocardial regeneration; HMGB1promoted the wound healing through inducing fibroblast cell proliferation and migration; HMGB1took part in the skeletal muscle regeneration, modulating in an autocrine or paracrine manner; and HMGB1played an important role in the proliferation and neuronal differentiation of mouse neuronal progenitor cells by binding with receptor for advance glycation end products (RAGE). A recent study showed that the expression of HMGB1was particularly high in ameloblasts and odontoblasts at regions of ongoing mineralization, which indicated that HMGB1participates in tooth mineralization. Therefore, it is meaningful to research the biological effects of HMGB1on hDPCs. The aims of this study were to investigate the expression of high-mobility group box1(HMGB1) in human dental pulp and the biological effects of HMGB1on proliferation, migration and odontoblastic differentiation of hDPCs.Methodology:Human dental pulp cells were culticated from pulp connective tissue explants. Cells between the third and sixth passages were used in this study. Immunohistochemical assay, immunoflurescence staining and flow cytometric analysis were used to detect the expression of HMGB1in the human dental pulp and hDPCs, respectively. After culturing human primary hDPCs in the presence of HMGBl with different doses, the proliferation of hDPCs was examined by CCK-8and the migration of hDPCs was evaluated using Scratch Test. Odontoblastic differentiation of hDPCs was determined using alkaline phosphatase (ALP) activity assay and mineralized nodule formation. Important mineralization-related genes such as ALP, dentin sialophosphoprotein (DSPP) and dentin matrix protein-1(DMP-1) were determined by real-time polymerase Chain reaction and Western Blot. Messenger RNA and protein levels of HMGB1and RAGE were also detected. immunoflurescence staining and Western Blot were used to detect the expression of HMGB1on the nuclei and the cytoplast. Meanwhile, the protein level of HMGB1in the supernatants was quantified using ELISA analysis on day0,3,7,11and14. Real-time polymerase Chain reaction and Western blot analysis were performed to determine the difference of expressions of DMP-1, DSP and ALP with or without the presence of exogenous HMGB1. Mineralized nodule formation, messenger RNA and protein levels of RAGE were also detected.Results:hDPCs culticated from pulp connective tissue explants was available. HMGB1was found in human dental pulp and in the nuclei of hDPCs, and was expressed on about98%of hDPCs. The appropriate concentration of HMGB1promotes the proliferation and migration of hDPCs. During hDPC odontoblastic differentiation, the mRNA level of HMGB1was up-regulated and translocated from the nuclei to the cytoplasm and then secreted out from hDPCs. Exogenous HMGB1promoted hDPC mineralized nodule formation. It up-regulated the activity of ALP and the mRNA and protein levels of DMP-1, ALP, DSPP, and RAGE of hDPCs.Conclusion:Taken together, these results suggest that hDPCs can be cultivated preferably from tissue explants in vitro. HMGB1was found in human dental pulp and in the nuclei of hDPCs, and was expressed on about98%of hDPCs. The appropriate concentration of HMGB1promotes the proliferation and migration of hDPCs. During hDPC odontoblastic differentiation, HMGB1was translocated from the nuclei to the cytoplasm and then actively secreted out from hDPCs. The appropriate concentration of HMGB1promotes the proliferation, migration and odontoblastic differentiation of hDPCs which maybe depend on RAGE.