Hidetoshi Shimauchi

Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21 : a randomized, double-blind, placebo-controlled study

AIMnThis randomized clinical study was designed to evaluate the effect of probiotic intervention using lactobacilli on the periodontal condition of volunteers without severe periodontitis.nnnMATERIAL AND METHODSnFreeze-dried Lactobacillus salivarius WB21 (WB21)-containing tablets or a placebo were given to volunteers in a double-blind randomized study. A total of 66 volunteers were finally enrolled and randomly assigned to receive tablets containing WB21 (6.7 x 10(8) CFU) with xylitol or xylitol alone (placebo) three times a day for 8 weeks. Periodontal clinical parameters and whole saliva samples were obtained at baseline (BL), 4 weeks, and the end of the interventional period (8 weeks). Salivary lactoferrin (Lf) levels were measured by enzyme-linked immunosorbent assay. Lactobacilli in saliva and plaque samples was detected by semi-quantitative RT-PCR using 16S rRNA primers.nnnRESULTSnPeriodontal clinical parameters were improved in both groups after an 8-week intervention. Current smokers in the test group showed a significantly greater improvement of plaque index and probing pocket depth from BL when compared with those in the placebo group. Salivary Lf level was also significantly decreased in the test group smokers.nnnCONCLUSIONnOur results indicate that probiotics could be useful in the improvement/maintenance of oral health in subjects at a high risk of periodontal disease.

Wnt signaling inhibits cementoblast differentiation and promotes proliferation

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Elevated extracellular calcium increases expression of bone morphogenetic protein-2 gene via a calcium channel and ERK pathway in human dental pulp cells.

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca(2+)Ca(2+)(o) has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca(2+)(o) signaling in odontogenesis remains unclear. We found that elevated Ca(2+)(o) increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca(2+) increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca(2+) channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca(2+), suggesting that the Ca(2+) influx from Ca(2+) channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca(2+)-sensing receptors (CaSR) and only respond slightly to other cations such as Sr(2+) and spermine, suggesting that dental pulp cells respond to Ca(2+)(o) to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca(2+)(o) among cations.

Comparative study on bone regeneration by synthetic octacalcium phosphate with various granule sizes

The present study was designed to investigate whether the granule size of synthetic octacalcium phosphate (OCP) and the resultant intergranular spaces between the granules formed by the filling affect its osteoconductive and biodegradable characteristics in a mouse calvaria critical-sized defect up to 10 weeks after implantation. Mercury intrusion porosimetry showed that OCP granules having distinct diameter sizes ranging from 53 to 300 (S-OCP), 300 to 500 (I-OCP) and 500 to 1000 microm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6 microm. The dissolution rate of OCP, estimated by the phosphate concentration in the culture medium, was the highest in S-OCP, followed by I-OCP and L-OCP, while the specific surface area of OCP decreased. Histological and histomorphometric analyses showed that bone formation around the implanted granules increased significantly with increasing granule size coupled with activating the appearance of TRAP- and cathepsin K-positive osteoclastic cells. The rate of new bone formation formed with L-OCP was two times higher than that formed with S-OCP at 10 weeks after implantation. The results indicated that the osteoconductive and biodegradable properties of OCP can be augmented by increasing the granule size, most probably by thus providing enough spaces between the granules, suggesting that the intergranular spaces formed by the granules may work similarly to pores, as reported in porous ceramic materials. It seems likely that the enhancement of bone formation by OCP is accompanied by simultaneous activation of osteoclastic resorption of OCP.

Phosphate increases bone morphogenetic protein-2 expression through cAMP-dependent protein kinase and ERK1/2 pathways in human dental pulp cells

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Elevated extracellular calcium increases fibroblast growth factor-2 gene and protein expression levels via a cAMP/PKA dependent pathway in cementoblasts

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Wnt3a signaling induces murine dental follicle cells to differentiate into cementoblastic/osteoblastic cells via an osterix-dependent pathway

BACKGROUND AND OBJECTIVEnDental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwigs epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial-mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells.nnnMATERIAL AND METHODSnThe expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression.nnnRESULTSnWe identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix-small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels.nnnCONCLUSIONnThese findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/β-catenin signaling pathway.

Nanohydroxyapatite increases BMP-2 expression via a p38 MAP kinase dependent pathway in periodontal ligament cells

OBJECTIVEnBone morphogenetic protein (BMP)-2 promotes the osteoblastic differentiation of human periodontal ligament (PDL) cells, which play a pivotal role in periodontal regeneration. Recently, nano-sized hydroxyapatite (nano-HA) has been highlighted due to its advantageous features over micro-sized materials.nnnDESIGN AND RESULTSnWe investigated the effect of nano-HA on BMP-2 expression in human PDL cells. Real time PCR analysis revealed that the expression of BMP-2 increased upon stimulation with nano-HA in dose- and time-dependent manners. An immunofluorescence assay demonstrated the synthesis of BMP-2 proteins. Concentrations of Ca(2+) as well as phosphate (Pi) in culture supernatants were unchanged, suggesting that nano-HA functioned as a nanoparticle rather than as a possible source for releasing Ca(2+) and/or Pi extracellularly, which were shown to also enhance the expression of BMP-2. Nano-HA-induced BMP-2 expression was dependent on the p38 MAP kinase pathway because increases in BMP-2 expression were inhibited by treatment with SB203580, a p38 inhibitor, and phosphorylation of p38 was detected by Western blotting.nnnCONCLUSIONSnThis novel mechanism of nano-HA will be important for the rational design of future periodontal regeneration.

Micro‐CT analysis of alveolar bone healing using a rat experimental model of critical‐size defects

OBJECTIVEnThis study was designed to establish a rat model of a critical size alveolar bone defect.nnnMATERIALS AND METHODSnStandardized buccal or mesiobuccal alveolar bone defects were made around the right first mandibular molar of 12-week-old rats, and the left was used as a control. Alveolar bone healing was examined quantitatively by three-dimensional micro-computed tomographic imaging. Bone matrix production of osteoblasts and osteocytes during repair of alveolar bone defects was examined with in situ hybridization for type I collagen.nnnRESULTSnBuccal defects were repaired significantly and the volume decreased by 88.3% in week 24, whereas mesiobuccal defects were repaired little. Osteoblasts and osteocytes expressed type I collagen in both defects in week 3 but showed little expression by week 6 and thereafter, leaving the mesiobuccal defects largely unrepaired.nnnCONCLUSIONnThe mesiobuccal defect is a critical-size defect that is not ultimately repaired with bone. It may be an appropriate experimental model for investigating the effectiveness of bone regenerative agents in human alveolar bone loss.