The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity.

Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (valproic acid, trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 µM); while low concentrations (1 µM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14 days) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14 days. 59% of genes were down-regulated at 24 h and 36% at 14 days, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7 d, 14 days) and enzyme activity (48 h, 14 days). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. J. Cell. Physiol. 231: 798-816, 2016. © 2015 Wiley Periodicals, Inc.

The possibility of genistein as a new direct pulp capping agent.

Genistein, kind of soy isoflavones, is well-known as natural ingredients and consumed as health foods and supplements. They are expected to improve renal function. They have high-affinity to estrogen receptor ß expressed predominantly in bone tissue, they prevent osteoporosis specifically and safely. We examined whether genistein can be a new direct capping agent. In this study, we examined the effect of genistein for the proliferation and differentiation of rat dental pulp cells in vitro and the ability of tertiary dentin formation in vivo. As a result, rat dental pulp cells with genistein were increased activity of ALPase and showed alizarin red positive-staining. Calcification-related genes expression has been confirmed by the addition of genistein. From in vivo study, high quality of tertiary dentin formation and minor pulp reaction were observed. From these findings, it was suggested that genistein may be useful agent for direct pulp capping.

Combination of astaxanthin and fish oil supplementation alters antioxidant enzyme profile of dental pulp tissue.

AIM: To evaluate the effect of administration of astaxanthin (ASTA) and fish oil (FO) on enzymatic antioxidant parameters of dental pulp tissue from healthy rats. METHODOLOGY: Thirty-two healthy Wistar rats were divided into four groups: untreated control, ASTA-treated (1 mg kg(-1) body weight), FO-treated (10 mg eicosapentaenoic acid per kg BW and 7 mg docosahexaenoic acid per kg BW) and FO plus ASTA-treated. A prophylactic dose was administered in each group daily by gavage, 5 days a week, for 45 days. After treatment, the rats were killed and all incisor dental pulps were removed. Superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase and reductase activities were determined. Data were compared by anova and the Tukey's post-test ( P  < 0.05). RESULTS: Treatment with FO, ASTA and FO plus ASTA caused a reduction in SOD and GSH reductase activities in dental pulp tissue compared to untreated control rats ( P  < 0.05). ASTA partially stimulated catalase activity. CONCLUSIONS: The preventive administration of ASTA and FO changed the enzymatic antioxidant system of dental pulp tissue, possibly by controlling oxidative stress.

In vivo astaxanthin treatment partially prevents antioxidant alterations in dental pulp from alloxan-induced diabetic rats.

AIM: To evaluate the effect of astaxanthin on antioxidant parameters of dental pulp from diabetic rats. The hypothesis tested was that supplementation of diabetic rats with astaxanthin might eliminate, or at least attenuate, the defect in their antioxidative status. METHODOLOGY: Wistar rats (n=32) were divided into four groups: untreated control, treated control, untreated diabetic and treated diabetic rats. A prophylactic dose of astaxanthin (20 mg kg(-1) body weight) was administered daily by gavage for 30 days. On day 23, diabetes was induced by injection of alloxan (60 mg kg(-1) body weight). After 7 days of diabetes induction, the rats were killed, and pulp tissue from incisor teeth removed. Superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and reductase activities were determined. Data were compared by anova and the Newman-Keuls test (P<0.05). RESULTS: Diabetes caused a reduction in SOD, GPx and reductase activity in dental pulp tissue. Astaxanthin had no effect on SOD and catalase activities; however, it stimulated GPx in control and diabetic rats. CONCLUSIONS: Diabetes altered the antioxidant system in dental pulp tissue; astaxanthin partially improved the diabetic complications.

Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons.

The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.

Neodymium-doped yttrium-aluminium-garnet laser irradiation abolishes the increase in interleukin-6 levels caused by peptidoglycan through the p38 mitogen-activated protein kinase pathway in human pulp cells.

The anti-inflammatory effects of low-power laser irradiation have previously been reported. However, how the laser irradiation regulates the expression of inflammatory cytokines remains unknown. In the present study, to elucidate the mechanism behind the anti-inflammatory effect, we examined the effects of low-power neodymium-doped yttrium-aluminium-garnet (Nd:YAG) laser irradiation on interleukin (IL)-6 expression in human pulp (HP) cells stimulated by peptidoglycan (PGN) and focused on intracellular signaling pathways. Low-power Nd:YAG laser irradiation obviated the PGN-induced increase in IL-6 levels in HP cells. A p38 mitogen-activated protein kinase inhibitor, SB203580, also inhibited the increase in IL-6 messenger RNA levels. PGN stimulated the activity of phosphorylated p38 in HP cells. Low-power laser irradiation inhibited the activity. Thus, suppression of the phosphorylated p38 activity by low-power laser irradiation in HP cells culminates in inhibition of the increase in IL-6 induced by PGN, suggesting that low-power laser irradiation regulates intracellular signaling molecule activities to exert its anti-inflammatory effect.

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues.

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.

Matrix vesicles isolated from apical pulp of rat incisors: crystal formation in low Ca x Pi ion-product medium containing beta-glycerophosphate.

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.