Evaluation of the cytocompatibility of methacrylate resin-based root canal sealers with osteoblast-like cells.

This study compared the cytocompatibilities of three methacrylate resin-based root canal sealers [MetaSEAL Soft (MSS), Hybrid Root SEAL (HRS), and Superbond Sealer (SBS)] in either freshly mixed or set conditions using the Kusa A1 osteoblastic cell line. The three sealers and an epoxy resin-based sealer (AH Plus; AHP) were extracted in culture medium; cell growth and osteogenic properties were analyzed. Cell adhesion on set sealers was analyzed with scanning electron microscopy. The respective extents of cell growth were as follows in freshly mixed and set sealer extracts: SBS>MSS>AHP>HRS and SBS=AHP>MSS>HRS. Light irradiation of MSS and HRS increased the cell growth of set sealer extracts. Set SBS, MSS, and AHP did not alter expression of osteogenic genes or formation of mineralized nodules. Attached cells were observed only on SBS. In conclusion, the four sealers exhibited varying degrees of compatibility to osteoblasts; SBS and HRS were the most and least compatible, respectively.

A Novel Bioactive Endodontic Sealer Containing Surface-Reaction-Type Prereacted Glass-Ionomer Filler Induces Osteoblast Differentiation.

Surface­reaction­type prereacted glass-ionomer (S­PRG) fillers exhibit bioactive properties by the release of multiple ions. This study examined whether a novel endodontic sealer containing S­PRG fillers (PRG+) has the capacity to induce osteoblast differentiation. Kusa­A1 osteoblastic cells were cultured with extracts of PRG+, PRG- (an experimental sealer containing S­PRG­free silica fillers), AH Plus (an epoxy-resin­based sealer), and Canals N (a zinc-oxide noneugenol sealer). Cell viability and mineralized nodule formation were determined using WST­8 assay and Alizarin red staining, respectively. Osteoblastic-marker expression was analyzed with RT­qPCR and immunofluorescence. Phosphorylation of extracellular signal­regulated kinase (ERK) and p38 mitogen­activated protein kinase (MAPK) was determined with Western blotting. Extracts of freshly mixed PRG+, PRG-, and AH Plus significantly decreased cell growth, but extracts of the set samples were not significantly cytotoxic. Set PRG+ significantly upregulated mRNAs for alkaline phosphatase and bone sialoprotein (IBSP) compared to set PRG-, and upregulation was blocked by NPS2143, a calcium­sensing receptor antagonist. Set PRG+ significantly accelerated IBSP expression, mineralized nodule formation, and enhanced the phosphorylation of ERK and p38 compared with set PRG-. In conclusion, PRG+ induced the differentiation and mineralization of Kusa­A1 cells via the calcium-sensing receptor-induced activation of ERK and p38 MAPK.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Author Correction: Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HIF1α inhibits LPS-mediated induction of IL-6 synthesis via SOCS3-dependent CEBPß suppression in human dental pulp cells.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPß), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPß.

Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

This study examined the effects and mechanisms of strontium ranelate (SrRn)-a drug used to treat osteoporosis-on the proliferation and differentiation/mineralization of cloned dental pulp-like cells (mouse dental papillae cells; MDPs). It also determined whether topical application of SrRn to exposed dental pulp tissue promotes the formation of mineralized tissue in vivo. The MDPs were cultured with or without SrRn, and cell proliferation, odonto-/osteoblastic gene expression, mineralized nodule formation, and Akt phosphorylation were evaluated. The formation of mineralized tissue in SrRn-treated pulp tissue in rat upper first molars was evaluated histologically. The SrRn up-regulated cell proliferation and expression of Alp (alkaline phosphatase), Bsp (bone sialoprotein), Dmp (dentin matrix acidic phosphoprotein)-1, Dspp (dentin sialophosphoprotein), and Oc (osteocalcin) in a dose-dependent manner. Mineralized nodule formation was also enhanced by SrRn. NPS-2143, a calcium-sensing receptor (CaSR) antagonist, and siRNA against the CaSR gene blocked SrRn-induced proliferation, odonto-/osteoblastic gene expression, and mineralized nodule formation. SrRn induced Akt phosphorylation, and this was blocked by NPS-2143. Topical application of SrRn to exposed rat molar pulps induced the formation of osteodentin-like mineralized tissue. Our study revealed for the first time that SrRn promotes proliferation and odonto-/osteogenic differentiation/mineralization of MDPs via PI3K/Akt signaling activated by CaSR in vitro; mineralized tissue forms from the dental pulp in vivo.

Semisynthesis of human ghrelin: condensation of a Boc-protected recombinant peptide with a synthetic O-acylated fragment.

The creation of peptide using a combination of recombinant expression and chemical synthesis can be a powerful tool for the production of a wide variety of polypeptides modified by phosphorylation, glycosylation, etc. We have developed a new method for the preparation of a recombinant peptide with a free N(alpha)-amino group and protected N(epsilon)-amino groups, and have used this method in the semisynthesis of human ghrelin. Ghrelin, a natural ligand for growth hormone secretagogue receptor, is a 28-residue peptide with an essential n-octanoyl modification on Ser3. A 7-residue N-terminal fragment of ghrelin containing the octanoyl modification was prepared by Fmoc chemistry. In the preparation of it, all reactions were performed on the 2-chlorotrityl resin. Additionally, TBDMS and tBu turned out to be the most effective protection groups for the Ser3 and the Ser2, Ser6, respectively. For preparation of a 21-residue C-terminal fragment, we established a two-step protease processing method for the partially protected segment. A recombinant precursor peptide was Boc protected and subsequently cleaved using two distinct proteases, OmpT and Kex2. The peptides were then coupled to each other and, after deprotection, resulted in fully active human ghrelin.

Senescence marker protein-30 as a novel antiaging molecule.

Senescence marker protein-30 (SMP30), composed of 299 amino acids, has an approximate molecular mass of 32-34 kDa and has a pI 4.9 in charge. The amino acid alignment from various animal species revealed a highly conserved structure. SMP30 has an enzyme activity hydrolyzing sarin, soman, and tabun, known as lethal toxic nerve chemicals. We analyzed the organophosphatase activity of SMP30 using DFP as a substrate. This DFPase activity is revealed in a dose-dependent manner in the presence of magnesium ions. We investigated the intracellular localization of SMP30. It is localized in both the cytoplasm and nucleus. To confirm the presence of SMP30 in the nucleus, we prepared nuclear and cytoplasmic extracts from isolated cultured hepatocytes. Western blotting showed that SMP30 was detected in both extracts. Because the expression is reduced by carbon tetrachloride, one can speculate that the expression is modulated by oxidative stress increased with aging.

Senescence marker protein-30 knockout mouse as an aging model.

A mouse strain lacking SMP30 can be regarded as a strain showing ultimate decrease of the SMP30 molecule. After three months of age, SMP30-KO mice had an increased mortality rate, compared with the SMP30-WT mice, all of which remained alive. Electron microscopic observation of the hepatocytes from 12-month-old SMP30-KO mice revealed many empty vacuoles, presumably lipid droplets, abnormally enlarged mitochondria with indistinct cristae, and exceptionally large lysosomes filled with electron-dense bodies. The total hepatic triglyceride concentration of SMP30-KO mice was approximately 3.6-fold higher than that of the age-matched wild type. Similarly, the total hepatic cholesterol of SMP30-KO mice reached an approximate 3.3-fold greater value than that of the comparative group. Total hepatic phospholipids of SMP30-KO mice achieved an approximately 3.7-fold higher level compared with that of the wild-type mice. The cells from SMP30-KO mice were sensitive to apoptotic reagents. Those results supported the idea that SMP30 has an antiapoptotic function with wide spectrum. These findings indicate that SMP30-KO mice are highly susceptible to various harmful reagents. This strain might be a useful tool for aging and biological monitoring.

Human peptidylarginine deiminase type II: molecular cloning, gene organization, and expression in human skin.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Rodents have four isoforms of PAD (types I, II, III, and IV), each of which is distinct in substrate and tissue specificity. In fact, the only tissue in which all four PAD mRNAs have been detected is the epidermis. In this study, we found PAD activity in HSC-1 human cutaneous squamous carcinoma cells in vitro, and this activity increased during cultivation. Using a homology-based strategy, we cloned a full-length cDNA encoding human PAD type II. The cDNA was 2348 bp long and encoded a 665-amino-acid sequence with a predicted molecular mass of 75 kDa. The predicted protein shared 93% identity with the rat and mouse PAD type II sequence. Alignment of the amino acid sequences from both species revealed notable conservation in the C-terminal region, suggesting the presence of a functional region such as an enzyme catalytic site and/or a calcium-binding domain. Gene organization analysis established that human PAD type II on chromosome 1p35.2-p35.21 spanned more than 50 kb and contained 16 exons and 15 introns. A recombinant PAD protein subsequently produced in Escherichia coli proved to be enzymatically active, with substrate specificities similar to those of the rat PAD type II. In an immunohistochemical study of human skin, the type II enzyme was expressed by all the living epidermal layers, suggesting that PAD type II is functionally important during terminal differentiation of epidermal keratinocytes.