Fractalkine's dual role in inflammation and hard tissue formation in cultured human dental pulp cells.

This study aimed to explore the potential roles of fractalkine/CX3CR1, primarily expressed in vascular endothelial cells and has recently been identified in dental pulp cells at sites of pulp tissue inflammation, not only in inflammation but also in pulp hard tissue formation. To this end, cultured human dental pulp cells were grown in 10% FBS-supplemented α-MEM. Fractalkine was introduced to the culture, and COX-2 and dentin sialophosphoprotein (DSPP) expression levels were evaluated via western blotting. Real-time PCR was used to examine BMP-2 and Osterix mRNA expression. Calcified nodule formation was evaluated with Alizarin red staining. Results revealed that fractalkine increased COX-2 protein expression, calcified nodule formation, and BMP-2 and Osterix mRNA expression in a concentration- and time-dependent manner. DSPP protein expression also increased upon fractalkine addition. This effect of fractalkine on expression of DSPP protein was inhibited in the presence of the CX3CR1 inhibiter ADZ8797. In conclusion, our findings suggest a dual role for fractalkine in promoting pulp inflammation via COX-2 production and contributing to pulp hard tissue formation by stimulating the expression of hard tissue formation markers.

Successful Pulp-Preserving Treatment for Peri-invagination Periodontitis of Double Dens Invaginatus With Oehlers Type IIIA and IIIB: A Case Report.

Dens invaginatus (DI), which often occurs in the maxillary lateral incisor, is an important issue in endodontics because the treatment complexity increases depending on the degree of invagination and the vitality or nonvitality of the pulp. An 11-year-old female patient with a sinus tract in the gingiva of the maxillary lateral incisors showed peri-invagination periodontitis and double DI on radiography. Cone-beam computed tomographic imaging was used to examine the structure in the root canal in detail, and Oehlers type IIIA and IIIB DI was found. It was also shown that the patient's bone defect was caused by type IIIA. Because a healthy reaction was observed in the pulp test, the final diagnosis was peri-invagination periodontitis associated with type IIIA of the double DI with vital pulp. We expected the lesion to heal by treating only the type IIIA invaginated pseudo-root canal while preserving the healthy pulp. The invaginated root canal was cleaned under a microscope using ultrasonic instruments and nickel-titanium files to minimize irritation to the pulp. Because the lesion shrinkage was confirmed by cone-beam computed tomographic imaging taken 3 months after the start of treatment, vertical compaction of the warm gutta-percha technique was performed. At the 6-month postoperative recall, the pulp was normal, and the lesions were further improved. Treatment of the main root canal of double DI is complicated. However, proper diagnosis and careful cleaning of the invaginated root canal are essential for healing while preserving the pulp.

Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc.

Tumor necrosis factor α stimulates expression and secretion of urokinase plasminogen activator in human dental pulp cells.

Plasminogen activator (PA) is the enzyme responsible for converting plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. PA exists in two forms: urokinase-type PA (uPA) and tissue-type PA (tPA). Here we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNF-α) on PA production and secretion in human dental pulp cells. When the cells were stimulated with TNF-α (10 ng/mL), PA activity in the medium clearly increased in a time-dependent manner, and this activity was reduced after immunoprecipitation with anti-uPA antibody, but not with anti-tPA antibody. In TNF-α-stimulated cells, the expression of uPA mRNA was enhanced, but was lower than that of tPA mRNA. The expression of uPA mRNA and PA secretion stimulated by TNF-α were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFκB inhibitor pyrrolidine dithiocarbamate, but were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. In the presence of another inflammatory cytokine, interleukin 1ß (IL-1ß, 100 pg/mL), TNF-α-stimulated expression of uPA mRNA and secretion of uPA were enhanced. These observations suggest that TNF-α stimulates uPA production and secretion, and that this effect is regulated via activation of NFκB and tyrosine phosphorylation, apparently in conjunction with IL-1ß, during inflammation in human dental pulp.

Role of CTGF/CCN2 in reparative dentinogenesis in human dental pulp.

Connective tissue growth factor/CCN family 2 (CTGF/CCN2) has been considered to participate in tooth development. To date, the expression and role of CTGF/CCN2 in reparative dentinogenesis have been unclear. Our previous study revealed that matrix metalloproteinase-3 (MMP-3) stimulates cell migration via CTGF/CCN2 expression and secretion in human dental pulp cells, and that this is dependent on dynamin-related endocytosis and independent of protease activity. The objective of the present study was to determine the expression of CTGF/CCN2 in reparative dentin in human carious teeth and to examine the effect of CTGF/CCN2 on mineralization in cultured human dental pulp cells. Minimal expression of CTGF/CCN2 was evident in odontoblasts subjacent to the dentin-pulp junction in healthy teeth, whereas strong expression was detected in odontoblast-like cells lining the reparative dentin subjacent to dental caries. In human dental pulp cells, CTGF/CCN2 promoted mineralization but failed to induce proliferation, suggesting that this molecule has the ability to induce the differentiation of human dental pulp cells. Taken together, the data suggest that CTGF/CCN2 is likely involved in reparative dentinogenesis through formation of hard tissue in human carious teeth.

MMP-3 provokes CTGF/CCN2 production independently of protease activity and dependently on dynamin-related endocytosis, which contributes to human dental pulp cell migration.

Matrix metalloproteinase-3 (MMP-3) expression is promoted after pulpotomy, and application of MMP-3 to dental pulp after pulpotomy accelerates angiogenesis and hard tissue formation. However, the mechanism by which MMP-3 promotes dental pulp wound healing is still unclear. Connective tissue growth factor/CCN family 2 (CTGF/CCN2), a protein belonging to the CCN family, is considered to participate in wound healing, angiogenesis, and cell migration. In this study, we examined the involvement of CTGF/CCN2 in MMP-3-induced cell migration in human dental pulp (fibroblast-like) cells. In human dental pulp cells, MMP-3 promoted cell migration, but this effect was clearly blocked in the presence of anti-CTGF/CCN2 antibody. MMP-3 provoked mRNA and protein expression and secretion of CTGF/CCN2 in a concentration- and time-dependent manner. The MMP-3 inhibitor NNGH failed to suppress MMP-3-induced CTGF/CCN2 protein expression. The potent dynamin inhibitor dynasore clearly inhibited MMP-3-induced CTGF/CCN2 expression. These results strongly suggest that MMP-3 induces CTGF/CCN2 production independently of the protease activity of MMP-3 and dependently on dynamin-related endocytosis, which is involved in cell migration in human dental pulp cells.

Antioxidant effects of antioxidant biofactor on reactive oxygen species in human gingival fibroblasts.

The purpose of this study was to investigate the effects of antioxidant biofactor (AOB) on reactive oxygen species (ROS). Generation of superoxide radical (O(2) (•-)) and hydroxyl radical ((•)OH) was determined using an electron spin resonance (ESR) spin-trapping method. AOB was added at different concentrations to these free radical generating systems. The generation of both O(2) (•-) and (•)OH was scavenged by the addition of AOB in a dose-dependent manner. These results indicate that AOB has strong antioxidant properties against these radicals. We further investigated the anti-oxidative effect of AOB on human gingival fibroblasts (HGFs). HGFs were treated for 3 h with α-MEM containing a combination of AOB and H(2)O(2) (AOB + H(2)O(2) group), containing H(2)O(2) (H(2)O(2) group), or containing AOB alone (AOB group). Non-stimulated HGFs were used as a control group. The number of surviving cells was in the order of the AOB group > control group > AOB + H(2)O(2) group > H(2)O(2) group. The level of expression of type I collagen mRNA and production of collagen were also in the order of the AOB group > control group > AOB + H(2)O(2) group > H(2)O(2) group. In conclusion, our results suggest that AOB may protect HGFs against oxidative stress by reducing stress-induced ROS.

Stimulatory effects of low-concentration reactive oxygen species on calcification ability of human dental pulp cells.

The present study was conducted to investigate the effects of reactive oxygen species (ROS) on the calcification ability of human dental pulp (HDP) cells. HDP cells were treated with 100 mumol/L hydrogen peroxide (H(2)O(2)) for 5 or 10 minutes (5-min ROS group and 10-min ROS group) to investigate the mechanism of transmission to cells. Untreated cells were used as controls. Generation of free radicals was quantified by the electron spin resonance spin-trapping method and found to be increased by treatment with ROS. Formation of calcified nodules was also investigated by von Kossa staining and alizarin red S staining. Twenty-eight days after exposure, calcified nodules were present in cell cultures that had been treated with ROS for 5 or 10 minutes. Expression of mRNAs for osteopontin (OPN) and osteocalcin (OCN) was significantly greater in 10-min ROS group 6 and 9 days, respectively, after exposure than in controls. Production of OPN and OCN by 10-min ROS group was also greater 12 and 18 days, respectively, after exposure than in controls. These results suggested that calcification of HDP cells was stimulated by H(2)O(2) and by the ROS it generated.

Effects of Smads and BMPs induced by Ga-Al-As laser irradiation on calcification ability of human dental pulp cells.

We investigated the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells and on Smads and bone morphogenetic protein (BMP) production as one mechanism for the transmission of laser photochemical energy to cells. HDP cells in vitro were irradiated once with a Ga-Al-As laser at 1.0 W for 500 s, and calcified nodule formation was assessed by Alizarin red S staining. The laser irradiation was greater in the laser-irradiated group than in the non-irradiated group. Both calcium production and alkaline phosphatase (ALP) activity were higher after laser irradiation. Expression of mRNAs for Smad1, Smad7, BMPs, ALP, and osteocalcin was greater after laser irradiation, whereas expression of Smad6 mRNA was inhibited. Production of BMP-2 and BMP-4 in conditioned medium was also higher after laser irradiation. These results suggest that Smads and BMPs play important roles in ALP activity and calcification upon laser irradiation of HDP cells.

Plasmin is involved in inflammation via protease-activated receptor-1 activation in human dental pulp.

Plasmin is a proteolytic enzyme produced from plasminogen by plasminogen activators. We investigated the function of plasmin in human dental pulp fibroblast-like cells. Plasmin induced an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a concentration-dependent manner. Expression of mRNA for protease-activated receptor-1 (PAR-1) was detected, and the PAR-1 activating peptide SFLLRN induced an increase in [Ca(2+)](i) in the cells. The plasmin-induced increase in [Ca(2+)](i) was inhibited in the presence of the PAR-1 antagonist SCH79797. Plasmin stimulated the expression of interleukin-8 (IL-8) mRNA and prostaglandin E(2) release, which are involved in inflammation. These effects of plasmin on expression of IL-8 mRNA and prostaglandin E(2) release were inhibited in the presence of the PAR-1 antagonist SCH79797. These results suggest that plasmin activates PAR-1 and is involved in inflammation in human dental pulp.

Stimulatory Effects of CO(2) Laser, Er:YAG Laser and Ga-Al-As Laser on Exposed Dentinal Tubule Orifices.

We investigated the effects of lasers irradiation on the exposed dentinal tubule. Human tooth specimens with exposed dentinal tubule orifices were used. Three types of lasers (CO(2) laser, Er:YAG laser and Ga-Al-As laser) were employed. The parameters were 1.0 W in continuous-wave mode with an irradiation time of 30 s for the CO(2) laser, 30 mJ in continuous-wave mode with an irradiation time of 60 s for the Er:YAG laser, and 1.0 W in continuous-wave mode with an irradiation time of 60 s for the Ga-Al-As laser. A non-irradiated group was used as a control. After laser irradiation, the dentinal surface of each sample was observed using SEM. Afterwards, all samples were immersed in methylene blue dye solution in order to evaluate the penetration of the dye solution and observe the change in dentinal permeability after laser irradiation. SEM observation showed that the control group had numerous exposed dentinal tubule orifices, whereas these orifices were closed in the laser-irradiated groups. There was consistent dye penetration into the pulp chamber in the control group, whereas no dye penetration was evident in the laser-irradiated groups. Therefore, laser appears to be a promising treatment for reducing permeation through exposed dentinal tubules.

Protein kinase C synergistically stimulates tumor necrosis factor-alpha-induced secretion of urokinase-type plasminogen activator in human dental pulp cells.

Plasminogen activator (PA) is the enzyme converting plasminogen to its active form, plasmin, involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). When human dental pulp cells were stimulated by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), PA activity in the conditioned medium was increased, indicating that TNF-alpha provoked PA secretion. The TNF-alpha-induced PA release was significantly enhanced in the presence of phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator. The PKC inhibitor Ro31-8220 abolished the effect of PMA on the PA release. The activity of PA secreted from the cells stimulated by TNF-alpha and PMA was reduced by immunoprecipitation using anti-uPA antibody. PMA failed to enhance the TNF-alpha-induced expression of uPA mRNA. These results suggest that protein kinase C synergistically enhances the secretion of uPA in TNF-alpha-stimulated human dental pulp cells.

Stimulatory effects of hydroxyl radical generation by Ga-Al-As laser irradiation on mineralization ability of human dental pulp cells.

The present study was conducted to investigate the effects of Ga-Al-As laser irradiation on the mineralization ability of human dental pulp (HDP) cells. HDP cells in vitro were irradiated once with a Ga-AL-As laser at 0.5 W for 500 s and at 1.0 W for 500 s in order to investigate free radicals as one mechanism for transmission of laser photochemical energy to cells. Production of the hydroxyl radical (*OH) was measured using the ESR spin-trapping method and was found to be increased by laser irradiation. The DMPO-OH was not detected in the presence of dimethyl sulfoxide (DMSO), a *OH scavenger. The formation of calcification nodule was also investigated by von Kossa staining. The number of calcified nodules was increased by 1.0 W-laser irradiation. Alkaline phosphatase (ALP) activity was higher in the 1.0 W-laser irradiation group. Expression of mRNAs for heat shock protein 27, bone morphogenetic proteins (BMPs) and ALP were greater in the 1.0 W-laser irradiation group. Expression of BMPs in the conditioned medium was also higher in the 1.0 W-laser irradiation group. In particular, DMSO decreased the number of calcified nodule produced by 1.0 W-laser irradiation. These results supposed that the mineralization of HDP cells is stimulated by laser irradiation, and that *OH generated by laser irradiation is a trigger for promotion of HDP cell mineralization.

IL-1beta stimulates urokinase-type plasminogen activator expression and secretion in human dental pulp cells.

Plasminogen activator (PA) is the enzyme that converts plasminogen to its active form, plasmin, which is involved in various physiological and pathological phenomena. The conversion is catalyzed by two types of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). We investigated the effect of the inflammatory cytokine interleukin-1beta (IL-1beta) on PA secretion in human dental pulp cells. When the cells were stimulated by IL-1beta, PA activity in the medium was clearly increased in a time- and dose-dependent manner. This PA activity in the medium was reduced after immunoprecipitation with anti-uPA antibody, and uPA protein was detected in the immunoprecipitated fraction by Western blotting. However, no such effect was observed with anti-tPA antibody. In the IL-1beta-stimulated cells, expression of uPA mRNA was enhanced whereas expression of tPA mRNA was less. The IL-1beta-stimulated uPA mRNA expression and PA activities in the cell lysate and medium were reduced by the tyrosine kinase inhibitors herbimycin A and genistein, and by the NFkappaB inhibitor pyrolidinedithiocarbamate, and were augmented by the tyrosine phosphatase inhibitor sodium orthovanadate. These observations suggest that IL-1beta stimulates uPA production via activation of NFkappaB and tyrosine phosphorylation, and also secretion of the enzyme, and that the uPA/plasmin system appears to be involved in inflammation in human dental pulp.

Regulation of ALP activity by TNF-alpha on human dental pulp.

In this study, we examined the effects of TNF-alpha on Bone morphogenetic protein (BMP-2), Smads (which play intracellular signaling of BMPs) expression and alkaline phosphatase (ALP) activity of human dental pulp (HDP) cells to clarify the mechanism of tertiary dentin formation. The quantity of RT-PCR product for BMP-2 from the HDP cells stimulated by TNF-alpha is increased. However, ALP activity was not increased on the cells incubated with TNF-alpha. On the other hand, ALP activity was significantly increased on HDP cells treated with Ammonium Pyrrolidinedithiocarbamate (PDTC, NF- kappaB inhibitor) groups and combined supplementation of TNF-alpha and PDTC groups. Furthermore, we examined the effect of TNF-alpha and PDTC on Smad7 expression using RT-PCR and western blot analysis. Smad7 expression in HDP cells was increased by TNF-alpha, but decreased by PDTC treatment. These results suggest that NF- kappaB and Smad7 play an important role in the down regulation of ALP activity by TNF-alpha on HDP cells.

Effects of pH on mineralization ability of human dental pulp cells.

The purpose of this study was to investigate the effect of alkaline pH on calcification in human dental pulp (HDP) cells. HDP cells were cultured in pH 7.8 conditioned medium, and alkaline phosphatase (ALP) activity was measured. The ALP activity was higher in the pH 7.8 conditioned medium group than in the pH 7.2 conditioned medium group. Expression of mRNAs for bone morphogenetic protein (BMP)-2 was measured by the RT-PCR technique. The expression of BMP-2 in the pH 7.8 groups was greater than that in the pH 7.2 group. Furthermore, we determined Calcified nodule formation by von Kossa staining. The number of calcified nodules was increased in the pH 7.8 conditioned medium. These results suggest that HDP cell mineralization was enhanced in alkaline pH (pH 7.8) conditioned medium.