Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Erratum to: The role of lysyl oxidase-like 2 in the odontogenic differentiation of human dental pulp stem cells

Molecules and Cells -     

The emerging role of extracellular Ca2+ in osteo/odontogenic differentiation and the involvement of intracellular Ca2+ signaling: From osteoblastic cells to dental pulp cells and odontoblasts

Calcium ions (Ca²⁺) is the main element of dental pulp capping materials. Ca ²⁺ signaling plays a crucial role in a myriad of cell activities. An overwhelming array of studies have already reported the experimental and clinical benefits of Ca²⁺-enriched materials in the treatment of teeth with accidental vital pulp exposure and incomplete root formation. Thus, Ca²⁺ signaling has always been an excellent target for the design of various novel biomaterials for use in revitalizing or regenerative endodontic procedures. However, the molecular mechanisms that enable dental pulp cells (DPCs) to detect and respond to extracellular Ca²⁺ have not been characterized in detail before. In this review, we mainly outline the pathways by which the cell detects and responds to extracellular Ca²⁺, as well as the relevant regulatory paths in DPCs and odontoblasts, and discuss the potential role of Ca²⁺ as a therapeutic tool. Moreover, because DPCs share many of the same functional properties that are found in osteoblasts, some comparisons with bone cells were additionally incorporated into this text.     

4-Methylumbelliferone promotes the migration and odontogenetic differentiation of human dental pulp stem cells exposed to lipopolysaccharide in vitro

Hyaluronic acid (HA), a major component of the extracellular matrix, is essential to inflammatory regulation. 4-Methylumbelliferone (4-mu), as the specific inhibitor of HA synthesis, is an anti-inflammatory in multiple systems. However, there have been no studies, to our knowledge, regarding 4-mu treatment in pulp inflammation. Therefore, the purpose of this study was to investigate the effects of 4-mu on biological behaviors in human dental pulp stem cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro. hDPSCs were exposed to LPS to construct the inflammation model in vitro. Immunocytochemistry, quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, scratch/Transwell assay, and alizarin red staining/alkaline phosphatase staining were selected to explore the effect of 4-mu on the expression of inflammatory factors, cell proliferation, cell migration, and the odontogenic differentiation ability of hDPSCs. LPS stimulated hDPSCs to highly express the related inflammatory factors and CD44 (the major HA receptor), which were all inhibited by 0.1 mM of 4-mu. In addition, the cell proliferation ability of hDPSCs was suppressed by 4-mu, while cell migration and odontogenic differentiation abilities were significantly improved under inflammation. In conclusion, 4-mu suppressed inflammatory cytokines in inflamed hDPSCs and had a positive effect on the migration and odontogenic differentiation of hDPSCs.     

IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells

Dental stem cells for dental pulp regeneration have become a new strategy for pulpitis treatment. Angiogenesis and neurogenesis play a vital role in the pulp-dentin complex regeneration, and appropriate growth factors will promote the process of angiogenesis and neurogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5) is involved in the regulation of tooth growth and development. A previous study showed that IGFBP5 enhanced osteo/odontogenic differentiation of dental stem cells. Our research intends to reveal the function of IGFBP5 in the angiogenic and neurogenic differentiation of human dental stem cells. Human dental pulp stem cells (DPSCs) were used in the present study. Lentiviral IGFBP5 shRNA was used to silence the IGFBP5. Retroviruses expressing Wild-type IGFBP5 were used to over-express IGFBP5. Angiogenic and neurogenic differentiation were carried out by in vitro study. Real-time RT-PCR and western blot results showed that over-expression of IGFBP5 upregulated the expressions of angiogenic markers, including VEGF, PDGFA and ANG-1, and neurogenic markers, including NCAM, TH, Nestin, βIII-tubulin, and TH, in DPSCs. Moreover, microscope observation confirmed that over-expression of IGFBP5 enhanced neurosphere formation in DPSCs in size and amount. Immunofluorescence staining results showed that over-expression of IGFBP5 also prompted the percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. While depletion of IGFBP5 downregulated the expressions of VEGF, PDGFA, ANG-1, NCAM, TH, Nestin, βIII-tubulin, and TH, it decreased the neurosphere formation and percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. In conclusion, our results revealed that IGFBP5 promoted angiogenic and neurogenic differentiation potential of DPSCs in vitro and provided the possible potential target for enhancing directed differentiation of dental stem cells and dental pulp-dentin functional regeneration.     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, ΔNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.     

Increased strength in the Col-Tgel induces apoptosis in the human dental pulp stem cells: 3D culturing of human dental pulp stem cells at different strengths of collagen

Human dental pulp stem cells (HDPSCs) have great potential to be used in regenerative medicine. To use these stem cells effectively for this purpose, they should be grown in a 3D cell culture that mimics their natural niches instead of a 2D conventional cell culture. The aim of this study was to grow the HDPSCs in the 3D cell culture created by Transglutaminase-crosslinked collagen hydrogels (Col-Tgel) in two different strengths to find a suitable 3D cell culture environment for these stem cells. Two stiffness of the 3D Col-Tgel were used to grow the HDPSCs: soft and medium matrix with strength of 0.9–1.5 kPa and 14–20 kPa, respectively. HDPSCs express markers similar to MSCs, therefore seven such markers were analyzed in the HDPSCs during their growth in the 2D and in the 3D soft and medium Col-Tgel. The CD105 and CD90 markers were significantly (p < 0.05) downregulated in HDPSCs cultured in both 3D cell culture conditions compared with HDPSCs in 2D cell culture. Furthermore, CD34 marker, a negative marker, expressed by a few cells in HDPSCs culture was upregulated (p < 0.05) in HDPSCs cultured in medium 3D Col-Tgel, indicating cells that expressing the marker grow better in medium 3D Col-Tgel. The apoptosis results revealed that HDPSCs in medium 3D Col-Tgel had the least number of live cells and a significantly (p < 0.05) higher early apoptosis rate compared to HDPSCs in 2D and 3D Col-Tgel medium. MTT analysis also showed a significant difference among the three cell culture conditions. We conclude that HDPSCs cultured on 3D soft Col-Tgel showed better proliferation than cells cultured in 3D medium gel. These results demonstrate that the ideal environment to grow HDPSCs in 3D is the soft Col-Tgel not medium Col-Tgel.     

Lipopolysaccharide upregulates the proliferation,migration, and odontoblastic differentiation of NG2+ cells from human dental pulp in vitro

NG2⁺ cells have been proven to differentiate into odontoblasts in vivo, and their contribution to odontoblasts is significantly increased, especially after tooth injury. However, their characteristics in vitro, especially under an inflammatory environment, are still not fully understood. Therefore, this study aimed to explore their proliferation, migration, and odontoblastic differentiation ability after treatment with lipopolysaccharide (LPS) in vitro. In our study, NG2 ⁺ cells were isolated from the human dental pulp by magnetic‐activated cell sorting, and these isolated cells were proven to be NG2 ⁺ by immunostaining. When compared with human dental pulp cells (hDPCs), the NG2 ⁺ cells showed no significant differences in cell migration with or without LPS incubation, but their proliferative ability was weaker. When treated with LPS, NG2 ⁺ cells expressed elevated levels of pro‐inflammatory cytokines including interleukin‐1β (IL‐1β), IL‐6, IL‐8, and tumor necrosis factor‐α, and among these, the expression of IL‐1β and IL‐6 were higher than that of hDPCs. Their multipotent differentiation potential was confirmed by the induction of odontoblastic and adipogenic differentiation, and LPS increased their odontoblastic differentiation capacity. In the odontoblastic differentiation process, Wnt5a, BMP2, and BMP7 mRNA were increased, while the canonical Wnt‐related genes were decreased. In conclusion, the LPS stimulation promotes the migration, proliferative, and odontoblastic differentiation ability of NG2 ⁺ cells from the human dental pulp in vitro, and bone morphogenetic protein and the noncanonical Wnt pathway may be involved in their odontoblastic differentiation. These results indicated their special roles in tooth injury repair and potential application in pulp regeneration.     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

Wnt signaling reprograms metabolism in dental pulp stem cells

Human dental pulp stem cells (DPSCs) can differentiate to a wide range of different cell lineages, and share some gene expression and functional similarities with pluripotent stem cells. The stemness of DPSCs can also be pharmacologically enhanced by the activation of canonical Wnt signaling. Here, we examined the metabolic profile of DPSCs during reprogramming linked to Wnt activation, by a short (48 hr) exposure to either the GSK3-β inhibitor BIO (6-bromoindirubin-3´-oxine) or human recombinant protein WNT-3A. Both treatments largely increased glucose consumption, and induced a gene overexpression of pyruvate and mitochondrial acetyl-coA producing enzymes, thus activating mitochondrial tricarboxylic acid cycle (TCA) metabolism in DPSCs. This ultimately led to an accumulation of reducing power and a mitochondrial hyperpolarization in DPSCs. Interestingly, Nile Red staining showed that lipid fuel reserves were being stored in Wnt-activated DPSCs. We associate this metabolic reprogramming with an energy-priming state allowing DPSCs to better respond to subsequent high demands of energy and biosynthesis metabolites for cellular growth. These results show that enhancement of the stemness of DPSCs by Wnt activation comes along with a profound metabolic remodeling, which is distinctly characterized by a crucial participation of mitochondrial metabolism.     

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.     

Distal C‐terminus of Cav1.2 is indispensable for the chondrogenic differentiation of rat dental pulp stem cells

The α1 subunit (Cav1.2) of the L‐type calcium channel (LTCC), which is presently existing in both excitatory cells and non‐excitatory cells, is involved in the differentiation and proliferation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs), MSCs derived from dental pulp, exhibit multipotent characteristics similar to those of MSCs. The aim of the present study was to examine the contribution of Cav1.2 and its distal C‐terminus (DCT) to the commitment of rat DPSCs (rDPSCs) toward chondrocytes and adipocytes in vitro. The expression of Cav1.2 was obviously elevated in chondrogenic differentiation but did not differ significantly in adipogenic differentiation. The chondrogenic differentiation but not adipogenic of rDPSCs was inhibited by either blocking LTCC using nimodipine or knockdown of Cav1.2 via short hairpin RNA (shRNA). Overexpression of DCT rescued the inhibition by Cav1.2‐shRNA during chondrogenic differentiation, indicating that DCT is essential for the chondrogenic differentiation of rDPSCs. However, the protein level of DCT decreased after chondrogenic differentiation in wild‐type cells, and overexpression of DCT in rDPSCs inhibited the phenotype. These data suggest that DCT is indispensable for chondrogenic differentiation of rDPSCs but that superfluous DCT inhibits this process. Through the analysis of differentially expressed genes using RNA‐seq data, we speculated that the regulation of DCT might be mediated by the mitogen‐activated protein kinase/extracellular‐regulated kinase and c‐Jun N‐terminal kinase signaling pathways, or Chondromodulin‐1.