CCN2/CTGF expression via cellular uptake of BMP-1 is associated with reparative dentinogenesis.

OBJECTIVE: CCN family member 2/connective tissue growth factor (CCN2/CTGF) is known as an osteogenesis-related molecule and is thought to be implicated in tooth growth. Bone morphogenetic protein-1 (BMP-1) contributes to tooth development by the degradation of dentin-specific substrates as a metalloprotease. In this study, we demonstrated the correlations between CCN2/CTGF and BMP-1 in human carious teeth and the subcellular dynamics of BMP-1 in human dental pulp cells. MATERIALS AND METHODS: Expression of CCN2/CTGF and BMP-1 in human carious teeth was analyzed by immunohistochemistry. BMP-1-induced CCN2/CTGF protein expression in primary cultures of human dental pulp cells was observed by immunoblotting. Intracellular dynamics of exogenously administered fluorescence-labeled BMP-1 were observed using confocal microscope. RESULTS: Immunoreactivities for CCN2/CTGF and BMP-1 were increased in odontoblast-like cells and reparative dentin-subjacent dental caries. BMP-1 induced the expression of CCN2/CTGF independently of protease activity in the cells but not that of dentin sialophosphoprotein (DSPP) or dentin matrix protein-1 (DMP-1). Exogenously added BMP-1 was internalized into the cytoplasm, and the potent dynamin inhibitor dynasore clearly suppressed the BMP-1-induced CCN2/CTGF expression in the cells. CONCLUSION: CCN2/CTGF and BMP-1 coexist beneath caries lesion and CCN2/CTGF expression is regulated by dynamin-related cellular uptake of BMP-1, which suggests a novel property of metalloprotease in reparative dentinogenesis.

Effects of JNK1/2 on the inflammation cytokine TNF-α-enhanced production of MMP-3 in human dental pulp fibroblast-like cells.

AIM: To investigate the effects of the c-Jun N-terminal kinase (JNK1/2) on the inflammation cytokine tumour necrosis factor-alpha (TNF-α)-enhanced production of matrix metalloproteinase-3 (MMP-3) in human dental pulp fibroblast-like cells (HPFs). METHODOLOGY: HPFs were grown from pulp explants from healthy donors. Primary cultures were established by culturing the cells for 20 to 30 days. The experiments with HPFs were performed between passages 3 and 10. The HPFs were incubated in serum-free medium containing TNF-α for 24 h. The medium in each well was prepared in SDS sample buffer and was analysed for MMP-3 by Western blotting. RESULTS: JNK inhibitor SP601245 markedly inhibited the production of MMP-3 in TNF-α-stimulated human dental pulp fibroblasts. MMP-3 production was enhanced by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. cAMP response element-binding protein (CREB) was activated by TNF-α in HPFs; silencing JNK1 and JNK2 expression inhibited this activation. CONCLUSION: The activation of CREB via JNK pathways in the presence of TNF-α occurred with enhancement of MMP-3 production in dental pulp fibroblasts.

Mechanotransduction activates α5ß1 integrin and PI3K/Akt signaling pathways in mandibular osteoblasts.

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and ß-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)ß(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)ß(1) integrin to upregulate expression of the proteins ß-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)ß(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)ß(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.

NF-kappaB activation in human dental pulp stem cells by TNF and LPS.

Post-natal human dental pulp stem cells (DPSCs) represent a unique precursor population in the dental pulp, which has multipotential and can regenerate a dentin/pulp-like structure. Because the dental pulp is frequently infected by oral bacteria due to dental decay, in this study, we examined whether lipopolysaccharide (LPS) and tumor necrosis factor (TNF) activated the immunologic transcription factor nuclear factor kappa B (NF-kappaB) in DPSCs. We found that both TNF and LPS activated the I-kappa B kinase complex (IKK) in DPSCs to induce the phosphorylation and degradation of IkappaBalpha, resulting in the nuclear translocation of NF-kappaB. Consistently, both TNF and LPS rapidly induced the expression of the NF-kappaB-dependent gene interleukin-8 (IL-8). However, unlike in monocytes, we found that LPS could not induce the phosphorylation of the NF-kappaB active subunit p65 in DPSCs. In summary, our studies suggest that DPSCs may be involved in immune responses during pulpal infection through activating NF-kappaB.

Clinical and radiographic evaluation of root-canal obturation with obtura II.

This study evaluated clinical and radiographic healing of 236 root-canal treatments in 131 cases obturated with the Obtura II system. One operator performed all canal preparation and obturation with sealer. A standardized apical-coronal preparation technique instrumented all canals. Clinical symptoms, periodontal condition, and radiographic findings were evaluated at 3, 6, and 12 months. Radiographs taken immediately postobturation were compared to recall radiographs. The level of the final root filling was classified as short (more than 2 mm short of the apex), flush (within 2 mm), or over (beyond) in 12.7%, 81.4%, and 5.9% of cases, respectively. More than 96% of cases were treated successfully by the Obtura II system. Where roots were filled flush, over, or short, lesions healed in 97%, 93%, and 93% of cases, respectively, with no significant differences (p < 0.05). Root filling excess had no impact on the healing process.

The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells.

Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development.

Inhibition of osteoclast differentiation and bone resorption by cathepsin K antisense oligonucleotides.

We confirmed the expression of cathepsin K, the most abundant and specific cysteine protease found in osteoclasts, at the mRNA level in most of our cases of breast cancer, and even at the protein level in bone metastatic lesions. Therefore, we investigated the functions of cathepsin K in osteoclasts with special attention to bone metastasis from breast cancer. Mouse osteoclast-like cells (OCLs) were established by coculture of mouse bone marrow cells and osteoblastic cells. Rodent cathepsin K antisense (AS) or random control (CL) oligonucleotides were added on day 0, 3, or 6 of culture. Tartrate-resistant acid phosphatase staining confirmed the formation of OCLs after 9 d of incubation. AS treatment significantly reduced both the number of TRAP-positive cells and the percentage of multinuclear cells. For the pit-forming assay, after 9 d of incubation, mature OCLs were collected and incubated on ivory slices with AS or CL for 48 h. The antisense oligonucleotides also inhibited the bone-resorbing activity of OCLs. CL treatment did not affect either the number of TRAP-positive cells or pit formation. Cathepsin K may play important roles in bone resorption as well as in differentiation of osteoclasts. These findings indicate that the inhibition of this enzyme may prevent the development of bone metastasis from breast cancer.

Autocrine regulation of osteoclast formation and bone resorption by IL-1 alpha and TNF alpha.

Bone resorption is regulated by the cytokines within marrow cells that mediate osteoclast formation and activation. IL-1 and TNF induce bone resorption by stimulating the production of osteoclast-like multinucleated cells and by increasing the bone-resorbing activity of formed osteoclasts. This study was designed to detect IL-1 and TNF in osteoclasts in vitro and to determine whether these cytokines up-regulate osteoclast differentiation and bone resorption. The production of IL-1 alpha, -beta, and TNF alpha, beta in osteoclasts was examined immunohistochemically and by in situ hybridization. In the co-culture of C57BL/6N mouse bone marrow and MC3T3-G2/PA6 cells, a colony of osteoclasts formed, and IL-1 alpha and TNF alpha were detected. However, IL-1 beta and TNF beta were not detected. To investigate the role of IL-1 alpha and TNF alpha from osteoclasts, we enumerated TRAP-positive cells and measured the resorption pit areas in the presence of antibodies against IL-1 alpha and TNF alpha. The addition of antibodies against IL-1 alpha and TNF alpha to the co-culture system decreased the number of TRAP-positive colonies at seven days after incubation (anti-IL-1 alpha, 25.0 +/- 2.3%; anti-TNF alpha, 41.7 +/- 3.7%; anti-IL-1 alpha + anti-TNF alpha, 40.5 +/- 1.3%; and control, 100%), and the ratio of mononuclear to multinuclear cells had changed (anti-IL-1 alpha, 90:10; anti-TNF alpha, 75:25; anti-IL-1 alpha+ anti-TNF alpha, 88:12; and control, 60:40). The total pit areas per dentin slice also decreased with the addition of antibodies (anti-IL-1 alpha, 28,828; anti-TNF alpha, 49,249; anti-IL-1 alpha + anti-TNF alpha, 30,685; and control, 303,139 mm2). These results suggest that local production of IL-1 alpha and TNF alpha by osteoclasts is an important mechanism for regulating the osteoclast differentiation and bone resorptive process.

Osteopontin antisense deoxyoligonucleotides inhibit bone resorption by mouse osteoclasts in vitro.

Osteopontin (OPN) is an acidic phosphoprotein synthesized by osteoblasts and osteoclastic cells that are localized in the mineralized phase of bone matrix. OPN is thought to bind to the vitronection receptor on the osteoclast membrane and regulates bone resorption by the osteoclast. In this study, we investigated whether or not OPN can relate to osteoclast differentiation and bone resorption in a co-culture system. When C57Black/6N mouse bone marrow cells suspended on ivory slices coated with collagen were inoculated onto a MC3T3-G2/PA6 cell layer, colonies containing TRAP(+) mononuclear and multinuclear cells were formed in the presence of 1 alpha, 25-dihydroxyvitamin D3 and dexamethasone. At the end of culture period the number of TRAP(+) osteoclast-like cells were counted and the resorption pits were evaluated by reflected light microscopy. The mRNA of OPN was detected by in situ hybridization. Osteoclast-like cells expressed OPN mRNA. The addition of an OPN antisense oligomer (5' AAT CAC TGC CAA TCT CAT 3') at the start of the co-culture period decreased the number of TRAP(+) cells present after 7 d (30.3 +/- 3.4 vs 56.9 +/- 12.4), and the ratio of mononuclear and multinucleated cells was changed (77.6:23.2 vs 60.8:39.3). The total area of pits per ivory slice was also decreased by adding the OPN antisense oligomer (246813 vs 303139 microns2). These results showed that OPN can be an important mechanism for regulating differentiation and bone resorption.

Bone-resorptive cytokine gene expression in periapical lesions in the rat.

Periapical bone destruction is an important pathogenic sequela of pulpal infection. Recent findings from this laboratory have demonstrated that most bone-resorbing activity in extracts of rat periapical lesions can be neutralized by an anti-interleukin (IL)-1 alpha antiserum. To further clarify pathogenic mechanisms, bone-resorptive cytokine messenger RNA (mRNA) expression was analyzed in developing rat periapical lesions. The molar teeth of 20 Sprague-Dawley rats were surgically exposed and left open to permit infection from the oral environment. Total cell RNA was isolated from periapical granuloma tissue obtained on days 3, 7, 15 and 30 after exposure. mRNA for IL-1 alpha, IL-1 beta and tumor necrosis factor alpha (TNF-alpha) was amplified by reverse transcription polymerase chain reaction, and levels were approximated by comparison to the parallel amplification of the housekeeping gene glyceraldehyde phosphate dehydrogenase. IL-1 alpha and TNF-alpha mRNA were both highly expressed beginning on day 7, increased on day 15, and declined somewhat on day 30. In contrast, IL-1 beta mRNA was expressed at much lower levels, but with similar kinetics. The kinetics of steady state IL-1 alpha and TNF-alpha mRNA levels were confirmed using the quantitative RNase protection assay, whereas IL-1 beta mRNA could not be detected by this technique. IL-1 alpha mRNA-expressing cells were identified using in situ hybridization and included infiltrating macrophages, as well as resident fibroblasts, endothelial cells and osteoclasts. These results demonstrate that the IL-1 alpha and TNF-alpha genes are highly expressed in developing periapical lesions in the rat and confirm previous studies at the protein level in this model.

Histological findings of human leprosy periapical granulomas.

Histological analysis of human leprosy periapical granulomas was conducted to study the histological responses to Mycobacterium leprae. Many Langhans-type giant cells and epithelioid cell tubercles were observed, although M. leprae were not detected in leprosy periapical granulomas. Although numerous T- and B-cells were infiltrated in leprosy periapical granuloma, the T-/B-cell ratio of leprosy is not changed to that of periapical granulomas isolated from normal patients. These findings suggested that human leprosy periapical granulomas develop as a result of immunological responses to M. leprae.

Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure.

The bone-resorptive cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of many chronic inflammatory diseases, including pulpitis and apical periodontitis.To further elucidate their role in these disorders, we have identified cells that express IL-1 alpha and TNF alpha in infected pulps and in developing rat periapical lesions after surgical pulp exposure. As detected by immunohistochemistry, IL-1 alpha- and TNF alpha-positive cells were present as early as 2 days after pulp exposure in both the pulp and periapical region. The numbers of cytokine-expressing cells increased up to day 4 in the pulp and up to day 30 in the periapex. In contrast, cells expressing IL-1 beta and TNF beta, the homologous forms of these mediators, were not found in pulp or periapical lesions during this period. Cells expressing IL-1 alpha and TNF alpha were identified primarily as macrophages and fibroblasts, with occasional staining of polymorphonuclear leukocytes. Osteoblasts and osteoclasts were also positive, whereas lymphocytes were negative. In general, cytokine-expressing cells were located proximal to abscesses and the root apex. These findings demonstrate that cells that express bone-resorptive cytokines IL-1 alpha and TNF alpha are present immediately after pulp exposure in this model, which supports the hypothesis that these mediators play a key role in pulpal and periapical pathogenesis, including the concomitant bone destruction. They also indicate that both resident connective tissue cells as well as infiltrating cells express bone-resorptive cytokines in response to infection in these lesions.

Pathogenesis of induced rat periapical lesions.

Studies of the mechanisms of pathogenesis of periapical lesions were undertaken using a rat model of surgical pulp exposure. In this model, periapical lesions develop rapidly between days 0 and 15 (active phase) and more slowly thereafter (chronic phase). A Gram-negative anaerobic flora, similar to that seen in human beings, are quickly established. Lesions contain a mixed inflammatory cell infiltrate consisting of T cells, neutrophils, B cells, macrophages, and plasma cells. Helper T cells predominate during the active phase, whereas suppressor T cells are more frequent in the chronic phase. Extracts of periapical lesions contain bone-resorbing activity, the highest levels of which are present when lesions are actively expanding. Most bone-resorbing activity is mediated by the cytokine interleukin-1 alpha, as determined by biochemical criteria and antibody neutralization studies. Prostaglandin2 accounts for 10% to 15% of resorptive activity. Cells that express interleukin-1 alpha were identified in pulp beginning on day 2 after exposure and in periapical tissue beginning on day 7, as determined by in situ hybridization and immunostaining. Macrophages, fibroblasts, neutrophils, and osteoclasts were positive for interleukin-1 alpha mRNA and protein. Cells that express tumor necrosis factor alpha were also detected, whereas cells expressing interleukin-1 beta or tumor necrosis factor beta were absent. Finally, periapical bone destruction was inhibited by 60% by treatment with interleukin-1 receptor antagonist. These studies establish a key role for interleukin-1 alpha in the pathogenesis of periapical lesions in the rat model.

Changes in root canal microbiota during the development of rat periapical lesions.

Periapical lesions are reproducibly induced in rats by pulp exposure and infection from the oral cavity. Lesions expand rapidly between day 7 and day 15-20 (active phase), with slowed expansion thereafter. In the present study we characterized the root canal microbiota present during the active phase of lesion development in this system. The mandibular first molars of Sprague-Dawley rats were exposed on day 0. The teeth were extracted after 7 days (n = 10 animals) and 15 days (n = 10), and the microbiota present in roots was isolated and characterized. The number of colonies isolated per tooth was similar on day 7 (1.53 +/- 0.64 colony-forming units x 10(3)) and day 15 (1.49 +/- 0.37 colony-forming units x 10(3)). No colonies were isolated from unexposed control teeth. Anaerobic bacteria increased significantly between day 7 (24.3 +/- 5.7%) and day 15 (47.3 +/- 7.5%), and the proportion of gram-negative organisms increased from day 7 (24.3 +/- 6.1) to day 15 (46.9 +/- 6.8). The predominant bacteria included, on day 7: Streptococcus and Bacteroides species; on day 15: species of Streptococcus, Bacteroides, Prevotella, Neisseria and Peptostreptococcus. Streptococcus oralis, Streptococcus mitis, Streptococcus rattus, Bacteroides pneumosintes and Bacteroides ureolyticus were frequently isolated at both points. Although approximately the same mean number of different species (approximately 3.5) was isolated per tooth on both day 7 and 15, the overall diversity of the isolates increased on day 15.(ABSTRACT TRUNCATED AT 250 WORDS)