The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography–tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1_OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.
Objectives
This study aimed to evaluate the effect of betaine (BET) on immortalized human dental pulp stem cell (ihDP) osteogenic differentiation.
Materials and methods
hDPs were immortalized using SV40 T-antigen transfection. Characterization, multilineage differentiation, proliferation, cell cycle, colony-forming unit, and cellular senescence were evaluated (n = 4). The effect of BET on ihDP response was assessed (n = 4). Osteogenic differentiation was detected using ALP, ARS staining, and RT-qPCR (n = 4). To investigate the involvement of calcium signaling, the cells were pretreated with either 8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or thapsigargin before BET treatment (n = 6).
Results
ihDPs retained similar phenotypic characteristics presented in hDPs but exhibited an increase in cell proliferation and extended culture to passage 25. An increased proportion of cells in S and G2/M phases without senescence was observed in ihDPs. BET (50 mM) treatment significantly increased mineral deposition at 14 days and upregulated ALP, MSX2, BMP2, and RUNX2 expression. TMB-8 pretreatment reduced the effect of BET-induced ihDP osteogenic differentiation, whereas thapsigargin promoted osteogenic differentiation in ihDPs synergistically with BET.
Conclusion
ihDPs showed superior proliferation ability and a longer life span, which could serve as a promising cell for regenerative dentistry. BET promoted odonto/osteogenic differentiation via intracellular calcium regulation.
6-bromoindirubin-3′-oxime (BIO) is a candidate small molecule that effectively modulates Wnt signalling owing to its stable property. The present study investigated the influence of BIO on the odonto/osteogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were treated with 200, 400, or 800 nM BIO, and the effects on hDPSC responses and osteogenic differentiation were assessed. BIO-mediated Wnt activation was confirmed by β-catenin nuclear translocation detected by immunofluorescence staining. BIO attenuated colony formation and cell migration determined by in vitro wound-healing assay. BIO increased early apoptotic cell population evaluated using flow cytometry. For osteogenic induction, BIO promoted alkaline phosphatase (ALP) activity and mineralisation in a dose-dependent manner. ALP, RUNX2, OCN, OSX, ANKH, DMP1, and DSPP mRNA expression were significantly upregulated. The OPG/RANKL expression ratio was also increased. Further, BIO attenuated adipogenic differentiation as demonstrated by decreased lipid accumulation and adipogenic-related gene expression. Bioinformatic analysis of RNA sequencing data from the BIO-treated hDPSCs revealed that BIO modulated pathways related to autophagy and actin cytoskeleton regulation. These findings demonstrated that BIO treatment promoted hDPSC osteogenic differentiation. Therefore, this small molecule is a strong candidate as a bioactive molecule to enhance dentin repair.
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