Calcium-sensing receptor regulates the angiogenic differentiation of LPS-treated human dental pulp cells via the phosphoinositide 3-kinase/Akt pathway in vitro.

AIM: The purpose of this study was to investigate the role of calcium-sensing receptor (CaSR) in the angiogenic differentiation of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). METHODOLOGY: The LPS-induced hDPCs were cultured in the medium with different combinations of CaSR agonist R568 and antagonist Calhex231. The cell proliferation, migration, and angiogenic capacity were measured by Cell Counting Kit-8 (CCK-8), scratch wound healing, and tube formation assays, respectively. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot were conducted to determine the gene/protein expression of CaSR, inflammatory mediators, and angiogenic-associated markers. The activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) was assessed by western blot analysis. RESULTS: The cell proliferation was elevated in response to R568 or Calhex231 exposure, but an enhanced cell migration was only found in cultures supplemented with Calhex231. Furthermore, R568 was found to potentiate the formation of vessel-like structure, up-regulated the protein expression of tumour necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and stromal cell-derived factor (SDF)-1; comparable influences were also observed in R568-stimulated cells in the presence of PI3K inhibitor LY294002. In contrast, Calhex231 obviously inhibited the tube formation and VEGF protein level, whereas promoted the production of IL-6, TNF-α, and eNOS; however, in the presence of LY294002, Calhex231 showed a significant promotion on the protein expression of CaSR, VEGF, and SDF-1. In addition, R568 exhibited a promotive action on the Akt phosphorylation, which can be reversed by LY294002. CONCLUSIONS: Our results demonstrated that CaSR can regulate the angiogenic differentiation of LPS-treated hDPCs with an involvement of the PI3K/Akt signalling pathway.

A role for the calcium-sensing receptor in the expression of inflammatory mediators in LPS-treated human dental pulp cells.

The aim of this study is to investigate the role of calcium-sensing receptor (CaSR) in the expression of inflammatory mediators of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The expression profile of CaSR in LPS-simulated hDPCs was detected using immunofluorescence, real time quantitative PCR (RT-qPCR), and Western blot analyses. Then, its regulatory effects on the expression of specific inflammatory mediators such as interleukin (IL)-1ß, IL-6, cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and IL-10 were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). LPS significantly downregulated the gene expression of CaSR, but upregulated its protein expression level in hDPCs. Treatments by CaSR agonist R568 or its antagonist Calhex231, and their combinations with protein kinase B (AKT) inhibitor LY294002 showed obvious effects on the expression of selected inflammatory mediators in a time-dependent manner. Meanwhile, an opposite direction was found between the action of R568 and Calhex231, as well as the expression of the pro- (IL-1ß, IL-6, COX2-derived PGE2, and TNF-α) and anti-inflammatory (IL-10) mediators. The results provide the first evidence that CaSR-phosphatidylinositol-3 kinase (PI3K)-AKT-signaling pathway is involved in the release of inflammatory mediators in LPS-treated hDPCs, suggesting that the activation or blockade of CaSR may provide a novel therapeutic strategy for the treatment of pulp inflammatory diseases.

Nitric Oxide in Dental Pulp Tissue: From Molecular Understanding to Clinical Application in Regenerative Endodontic Procedures.

Nitric oxide (NO), which is synthesized by the enzyme NO synthase (NOS), is a versatile endogenous molecule with multiple biological effects on many tissues and organs. In dental pulp tissue, NO has been found to play multifaceted roles in regulating physiological activities, inflammation processes, and tissue repair events, such as cell proliferation, neuronal degeneration, angiogenesis, and odontoblastic differentiation. However, there is a deficiency of detailed discussion on the NO-mediated interactions between inflammation and reparative/regenerative responses in wounded dental pulp tissue, which is a central determinant of ultimate clinical outcomes. Thus, the purpose of this review is to outline the current molecular understanding on the roles of Janus-faced molecule NO in dental pulp physiology, inflammation, and reparative activities. Based on this knowledge, advanced physicochemical techniques designed to manipulate the therapeutic potential of NOS and NO production in endodontic regeneration procedures are further discussed. Impact statement The interaction between inflammation and reparative/regenerative responses is very important for regenerative endodontic procedures, which are biologically based approaches intended to replace damaged tissues. Inside dental pulp tissue, endogenous nitric oxide (NO) is generated mainly by immunocompetent cells and dental pulp cells and mediates not only inflammatory/immune activities but also signaling cascades that regulate tissue repair and reconstruction, indicating its involvement in both tissue destruction and regeneration. Thus, it is feasible that NO acts as one of the indicators and modulators in dental pulp repair or regeneration under physiological and pathological conditions.

CaSR as a Therapeutic Target and Tool in Human Dental Pulp: A Concise Review and Novel Hypothesis.

PURPOSE: To review the essential characteristics of calcium sensing receptor (CaSR) and explore the hypothesis that elevated extracellular calcium ions (Ca2+) may affect the odontogenic/osteogenic differentiation and mineralisation of human dental pulp cells (hDPCs) through the CaSR signal. MATERIALS AND METHODS: Based on a literature search of databases using different combinations of the key words and our previous researches, we gleaned the following important viewpoints. RESULTS: The Ca2+ released from pulp capping materials plays an essential role in maintaining the viability and function of human dental pulp, and elevated extracellular Ca2+ concentrations can promote the odontogenic/osteogenic differentiation and mineralisation of hDPCs. Ca2+ is the primary physiological ligand of the CaSR, which has been reported to be widely expressed in a broad range of cells, including various osteoblast-like cell lines, osteoprogenitor cells, and mature osteoblasts. hDPCs consist of different subpopulations and have been shown to share phenotypical features with osteoblasts. Thus, we speculated that hDPCs also express CaSR and respond to extracellular Ca2+ via this receptor. Calcimimetics are indirect allosteric regulators of CaSR function and can increase the receptor's sensitivity to ambient Ca2+. CONCLUSION: The local use of calcimimetics and calcium-based pulp capping materials could create an option for promoting the Ca2+ influx of hDPCs from the extracellular space via the CaSR. Such elevated Ca2+ concentrations could enhance the odontogenic/osteogenic differentiation and mineralisation of hDPCs and eventually improve the success rate of direct pulp capping treatments in patients suffering from accidental dental pulp exposure.

Identification of a Calcium-sensing Receptor in Human Dental Pulp Cells That Regulates Mineral Trioxide Aggregate-induced Mineralization.

INTRODUCTION: The purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). METHODS: The expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). RESULTS: CaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 µmol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 µmol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 µmol/L R-568-treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568- and Calhex 231-treated hDPCs. CONCLUSIONS: Our data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.

The emerging role of extracellular Ca2+ in osteo/odontogenic differentiation and the involvement of intracellular Ca 2+ 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.

Enterococcus faecalis lipoteichoic acid regulates macrophages autophagy via PI3K/Akt/mTOR pathway.

Enterococcus faecalis (E. faecalis) infection is considered an important etiological factor for the development of persistent apical periodontitis (PAP), but the exact mechanisms of autophagy between E. faecalis and immune cells remain unknown. In this study, we elucidated how E. faecalis lipoteichoic acid (LTA) is associated with macrophages autophagy. We found that E. faecalis LTA apparently activated macrophage autophagy with significant increase of autophagosomes and autophagy relative protein. Meanwhile, we noticed significantly decreasing expression of p-Akt and p-mTOR. However, these effect were absent in macrophages knockdown of Beclin1. In summary, these findings suggested E. faecalis LTA may increased macrophages autophagy via inhibiting PI3K/Akt/mTOR pathway and this process was Beclin1 dependent.

Promotive Effect of Zinc Ions on the Vitality, Migration, and Osteogenic Differentiation of Human Dental Pulp Cells.

Zinc is an essential trace element for proper cellular function and bone formation. However, its exact role in the osteogenic differentiation of human dental pulp cells (hDPCs) has not been fully clarified before. Here, we speculated that zinc may be effective to regulate their growth and osteogenic differentiation properties. To test this hypothesis, different concentrations (1 × 10-5, 4 × 10-5, and 8 × 10-5 M) of zinc ions (Zn2+) were added to the basic growth culture medium and osteogenic inductive medium. Cell viability and migration were measured by cell counting kit-8 (CCK-8) and transwell migration assay in the basic growth culture medium, respectively. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expression levels of selective osteogenic differentiation markers and zinc transporters. Alkaline phosphatase (ALP) activity analysis and alizarin red S staining were used to investigate the mineralization of hDPCs. Exposure of hDPCs to Zn2+ stimulated their viability and migration capacity in a dose- and time-dependent manner. RT-qPCR assay revealed elevated expression levels of osteogenic differentiation-related genes and zinc transporters genes in various degrees. ALP activity was also increased with elevated Zn2+ concentrations and extended culture periods, but enhanced matrix nodules formation were observed only in 4 × 10-5 and 8 × 10-5 M Zn2+ groups. These findings suggest that specific concentrations of Zn2+ could potentiate the vitality, migration, and osteogenic differentiation of hDPCs. We may combine optimum zinc element into pulp capping materials to improve their biological performance.

Characterization of human periodontal ligament cells cultured on three-dimensional biphasic calcium phosphate scaffolds in the presence and absence of L-ascorbic acid, dexamethasone and ß-glycerophosphate in vitro.

The aim of this study was to evaluate the effect of porous biphasic calcium phosphate (BCP) scaffolds on the proliferation and osteoblastic differentiation of human periodontal ligament cells (hPDLCs) in the presence and absence of osteogenic inducer (L-ascorbic acid, dexamethasone and ß-glycerophosphate). The cell growth within the scaffolds in the absence of osteogenic inducers was studied by cell counting kit-8 (CCK-8) assay and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and osteoblastic differentiation markers of hPDLCs in BCP scaffolds were examined in the presence and absence of osteogenic inducers. The cell number of hPDLCs in the BCP scaffolds was less than that of hPDLCs cultured in microplates (control). SEM images showed that cells successfully adhered to the BCP scaffolds and spread amongst the pores; they also produced abundant extracellular cell matrix. In the presence and absence of osteogenic inducers, the ALP activity of hPDLCs within BCP scaffolds was suppressed in varying degrees at all time-points. In the absence of osteogenic inducers, hPDLCs in BCP scaffolds express significant higher levels of osteopontin (OPN) mRNA than the control, and there were no significant differences for Runx2 and osteocalcin (OCN) mRNA levels compared with those cultured in microplates. In the presence of osteogenic inducers, Runx2 expression levels were significantly higher than those in control. OPN and OCN mRNA levels were downregulated slightly. Three-dimensional porous BCP scaffolds are able to stimulate the osteoblastic differentiation of hPDLCs in the presence and absence of osteogenic inducer and may be capable of supporting hPDLC-mediated bone formation.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers.

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF­2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF­2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and ß­glycerophosphate). FGF­2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type Ⅰ, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF­2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT­qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF­2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF­2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

Short-term effects of calcium ions on the apoptosis and onset of mineralization of human dental pulp cells in vitro and in vivo.

Calcium ions (Ca2+) are a major constituent of most pulp-capping materials and have an important role in the mineralization of human dental pulp cells (hDPCs). A previous study by our group has shown that increased levels of Ca2+ can promote hDPC-mediated mineralization in long-term cultures (21 days). However, the initiation of mineralization occurs in the early stage of osteogenic inductive culture, and the effects of Ca2+ on the mineralization of hDPCs in short-term cultures (five days) have not been studied in detail. Furthermore, the underlying mechanism by which Ca2+ stimulates the mineralization of hDPCs has remained controversial. A strong correlation between mineralization and cell apoptosis and/or death has been identified. Thus, the present study hypothesized that Ca2+ may promote the onset of hDPC-mediated mineralization through inducing their apoptosis and/or death. To verify this hypothesis, Ca2+ was added to the growth culture medium and osteogenic culture medium at various concentrations. Alizarin Red S staining and reverse transcription-polymerase chain reaction analysis were used to evaluate the onset of mineralization. Furthermore, the cell counting kit-8 and fluorescein isothiocyanate-Annexin V/propidium iodide double-staining method were adopted to detect the proliferation and apoptosis of hDPCs in the growth culture medium. An animal experiment and scanning electron microscopic observation of ceramic graft implants were applied to measure the mineralization in vivo. The results showed that 5.4 and 9.0 mM Ca2+ accelerated the onset of mineralized matrix nodule formation, promoted osteopontin mRNA expression and induced marked cell apoptosis and necrosis, but had no obvious effect on cell proliferation. These findings indicated a positive association between cell apoptosis and/or death and the timing of formation as well as the quantity of extracellular mineralization induced by Ca2+ in short-term cultured hDPCs.

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on ß-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro.

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. ß-tricalcium phosphate (ß-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous ß-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell-Counting kit-8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the ß-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the ß-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the ß-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on ß-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements.

Calcium ions promote osteogenic differentiation and mineralization of human dental pulp cells: implications for pulp capping materials.

Calcium (Ca) is the main element of most pulp capping materials and plays an essential role in mineralization. Different pulp capping materials can release various concentrations of Ca ions leading to different clinical outcomes. The purpose of this study was to investigate the effects of various concentrations of Ca ions on the growth and osteogenic differentiation of human dental pulp cells (hDPCs). Different concentrations of Ca ions were added to growth culture medium and osteogenic inductive culture medium. A Cell Counting Kit-8 was used to determine the proliferation of hDPCs in growth culture medium. Osteogenic differentiation and mineralization were measured by alkaline phosphatase (ALP) assay, Alizarin red S/von kossa staining, Ca content quantitative assay. The selected osteogenic differentiation markers were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Within the range of 1.8-16.2 mM, increased concentrations of Ca ions had no effect on cell proliferation, but led to changes in osteogenic differentiation. It was noted that enhanced mineralized matrix nodule formation was found in higher Ca ions concentrations; however, ALP activity and gene expression were reduced. qRT-PCR results showed a trend towards down-regulated mRNA expression of type I collagen and Runx2 at elevated concentrations of Ca ions, whereas osteopontin and osteocalcin mRNA expression were significantly up-regulated. Ca ions content in the culture media can significantly influence the osteogenic properties of hDPCs, indicating the importance of optimizing Ca ions release from dental pulp capping materials in order to achieve desirable clinical outcomes.

[Comparative study of sealing ability and fracture resistance of roots endodontically treated with bonded and unbonded filling materials].

OBJECTIVE: To compare the sealing ability and fracture resistance of roots endodontically treated with bonded and unbonded filling materials. METHODS: One hundred and fifteen straight mandibular premolar teeth with single canal were divided randomly into 6 experimental groups, with 15 samples each, and 3 control groups. The sealing ability was evaluated using a glucose quantitative microleakage mode and fracture resistance was tested by universal testing machine. RESULTS: The microleakage results showed that the bonded filling material had the lowest value while the unbonded filling material had the highest value in all groups. There were significant differences in microleakage value among the groups (P < 0.01), but no significant difference was noted in the fracture resistance among the testing groups (P = 0.7016). CONCLUSIONS: Bonded filling material enhanced the sealing ability but could not reinforce the fracture resistance of endodontically treated teeth.

[An in vitro study on the incidence of the second mesiobuccal canal in the mesiobuccal root of the first and second maxillary molars].

OBJECTIVE: To investigate the incidence of the second mesiobuccal canal (MB2) in the mesiobuccal root of the maxillary first and second molars in Chinese population using three techniques, including the clearing technique, spiral CT scanning and serial root sections. METHODS: A total of 216 extracted human first and 334 second maxillary molars were randomly divided into two groups respectively: group A and B. The teeth in group A were cleared. The specimens in group B were subjected to spiral CT scanning, and then the roots were cross-sectioned every 1 mm from the root apex. Under the Dental operating microscope (DOM), the incidence of MB2 were recorded. RESULTS: (1) The incidence of MB2 in the first and the second maxillary molars were 81.48% and 49.70% respectively by clearing, and 77.78% and 47.31% from S-CT scanning, 88.89% and 53.89% respectively from serial root section. The occurrence of MB2 in maxillary first molars was statistically higher than in maxillary second molars (P < 0.05, chi square test). (2) There was no significant difference among the three approaches for detecting the MB2 canal (P > 0.05, chi square test). CONCLUSIONS: Both the maxillary first molars and the second molars have high incidence of MB2.