Effects Of GPNMB On Odontoblastic Differentiation Of Human Dental Pulp Stem Cells In Vitro

Dental pulp plays a critical role in the reparative regeneration of tooth tissue. Dental pulp cells (DPCs) are composed of ectodermic and mesenchymal components containing neural crest-derived mesenchymal progenitors endowed with plasticity and multipotency. In response to dental injury and irreversible caries damage, dental pulp can produce new odontoblasts secreting areparative dentine matrix.Human dental pulp stem cells (hDPSCs) are easily obtained from human impacted third molars, possess high proliferative ability, and can be reprogrammed into induced pluripotent stem cells at relatively high rates. Cultured DPSCs have the ability to differentiate into odontoblast-like cells under certain culture conditions in vitro, with higher colony forming efficiency, proliferation ability, and the abilityof forming dense calcificationcolone even calcified nodule. Therefore, Studies showthat dental pulp stem cells can be used in the field of dental tissue regeneration.Dental tissue regenerationis a complex process, many cytokines involved in the regulation of this process.Glycoprotein (transmembrane) nonmetastatic melanoma protein b (GPNMB), also known as HGFIN, osteoactivin, and DC-HIL, is a type Ⅰ membrane glycoprotein involved in various biological processes, including inflammation, invasion and metastasis of malignant tumors, cell differentiation, and tissue regeneration.Previous studies showed that GPNMB acts as an osteogenic factor that stimulates osteoblast differentiation in vivo and in vitro.The odontogenic differentiation of DPCs is a biological process similar to hBMMSC osteogenic differentiation, which suggests that GPNMB may be involved in the odontogenic differentiation of hDPCs. However, the role of GPNMB in hDPCs is still unclear.In this study, after successfully isolated, expanded and identificatedhDPSCs from human impacted third molars, the expression of GPNMB was examined during the odontoblastic differentiation of hDPSCs in vitro, and then GPNMB was knocked-down in hDPSCs to determine its function on odontoblastic differentiation of hDPCs.Main results:1. Expression of GPNMB during the odontoblastic differentiation of hDPSCs in vitro.Human healthy impacted third molarswere collected. Theprocedure was approved by Institutional Review Board of School of Stomatology, Shandong University and performed with the informed consent of the patients. After isolation, expandation and identification of hDPSCs, the isolated cells showed the ability of osteo/odontogenic and adipogenic differentiation, when grew under defined culture conditions. The expression of the GPNMB in odontoblastic differentiation of hDPSCs,assessed by reverse transcriptase polymerase chain reaction and Western blot analysis,was significantly increased during odontoblastic differentiation of hDPCs.2. Effects of GPNMB on proliferation and odontoblastic differentiation of hDPSCsIn this part, gene knockdown of GPNMB in hDPSCs using liposome-mediated small interfering RNA (siRNA)-GPNMBwas performed.The proliferation of cells was measured by the MTT assay, and the differentiation of cells was detected with alkaline phosphatase (ALP) activity assay, qRT-PCR and Western blot were used to determine the expression levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). The study found the suppression of GPNMB expression by siRNA-GPNMB obviously promoted the proliferation of hDPSCs. Furthermore, siRNA-GPNMB significantly inhibited the activity of ALP and expression levels of DSPP and DMP-1 during odontoblastic differentiation of hDPSCs. The results show that GPNMB plays an important role in regulating the expression of key pluripotency genes in hDPSCs and modifying odontogenic differentiation.