Adherent endotoxin on dental implant surfaces: a reappraisal.

Osteoimmunology is the crosstalk between cells from the immune and skeletal systems, suggesting a role of pro-inflammatory cytokines in the stimulation of osteoclast activity. Endotoxin or bacterial challenges to inflammatory cells are directly relevant to dental implant pathologies involving bone resorption, such as osseointegration failure and peri-implantitis. While the endotoxin amount on implant devices is regulated by standards, it is unknown whether commercially available dental implants elicit different levels of adherent-endotoxin stimulated cytokines. The objective of this work is to develop a model system and evaluate endotoxin-induced expression of pro-inflammatory cytokine genes relevant to osteoclast activation on commercially available dental implants. Murine J774-A1 macrophages were cultured on Ti disks with different level of lipopolysaccharide (LPS) contamination to define the time-course of the inflammatory response to endotoxin, as evaluated by reverse transcription polymerase chain reaction analysis. The developed protocol was then used to measure adherent endotoxin on commercially available packaged and sterile dental implants in the "as-implanted" condition. Results show that tested dental implants induce variable expression of endotoxin-stimulated genes, sometimes above the level expected to promote bone resorption in vivo. Results are unaffected by the specific surface treatment; rather, they likely reflect care in cleaning and packaging protocols. In conclusion, expression of genes that enhance osteoclast activity through endotoxin stimulation of inflammatory cells is widely different on commercially available dental implants. A reappraisal of the clinical impact of adherent endotoxins on dental (and bone) implant devices is required in light of increasing knowledge on crosstalk between cells from the immune and skeletal systems.

Expressions of RANKL/RANK and M-CSF/c-fms in root resorption lacunae in rat molar by heavy orthodontic force.

The differentiation and functions of osteoclasts are regulated by receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL) system that stimulates osteoclasts formation. Macrophage colony-stimulating factor (M-CSF) is also essential for osteoclastogenesis. A recent immunocytochemical study reported that RANKL/RANK and M-CSF/c-fms were localized in the periodontal ligament of rat molars during experimental orthodontic tooth movement. The present study focused on the expressions of RANKL/RANK and M-CSF/c-fms in root resorption area during experimental tooth movement in rats. Forty 6-week-old male Wistar rats were subjected to an orthodontic force of 10 or 50 g with a closed coil spring (wire size: 0.005 inch, diameter: 1/12 inch) ligated to the maxillary first molar cleat by a 0.008 inch stainless steel ligature wire to induce a mesial tipping movement of the upper first molars. Experimental tooth movement was undertaken for 10 days. Each sample was sliced into 6 µm continuous sections in a horizontal direction and prepared for haematoxylin and eosin (H and E) and immunohistochemistry staining for tartrate-resistant acid phosphatase (TRAP), RANK, RANKL M-CSF, and c-fms in root resorption area. Statistical analysis was carried out using a Mann-Whitney U-test with a significance level of P<0.01. On days 7 and 10, immunoreactivity for RANKL/RANK and M-CSF/c-fms was detected in odontoclasts with an orthodontic force of 50 g, but not 10 g. Therefore, RANKL/RANK and M-CSF/c-fms systems may be involved in the process of root resorption by heavy orthodontic force.

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. MATERIAL AND METHODS: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real-time PCR, and protein expression was analyzed using ELISA. RESULTS: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and regulated upon activation, normal T-cell expressed and secreted (RANTES). Macrophage colony-stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL-6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. CONCLUSION: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.

Profiling of inflammatory cytokines produced by gingival fibroblasts after human cytomegalovirus infection.

INTRODUCTION: The purpose of this study was to determine the profile of inflammatory cytokines that are produced after in vitro infection of gingival fibroblasts with human cytomegalovirus (HCMV). MATERIALS AND METHODS: Gingival fibroblasts were infected with the Towne strain of HCMV and the cytokine profile in the supernatant was studied using a human inflammation antibody array. Expression of messenger RNA (mRNA) using reverse transcription-polymerase chain reaction for interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was also analyzed in infected gingival fibroblasts and gingival specimens from subjects with and without periodontitis according to HCMV detection. HCMV was determined in subgingival samples by nested polymerase chain reaction. RESULTS: Gingival fibroblasts produced mainly IL-1alpha, IL-12p40, IL-12p70, IL-6, TNF-alpha, and IL-1beta after HCMV infection. Expression of mRNA for IL-1beta and TNF-alpha was increased after HCMV infection. Production of IL-1beta and TNF-alpha was increased in HCMV-positive periodontitis specimens. In addition, infected gingival fibroblasts produced more IL-8, monocyte chemoattractant protein 1, macrophage inflammatory proteins 1alpha, and 1beta over time postinfection in comparison to baseline. The lowest production of all cytokines studied corresponded to IL-2, IL-4, IL-13, and interferon-gamma. A decreasing production pattern was observed for granulocyte-macrophage colony-stimulating factor, IL-7, and IL-17 while IL-11 and macrophage colony-stimulating factor were increased at 72 h postinfection. CONCLUSIONS: HCMV infection in gingival fibroblasts upregulated the production of proinflammatory-related cytokines and chemokines. The expression of IL-1beta and TNF-alpha was increased both in vitro and in specimens from HCMV-positive subjects with periodontitis. The overproduction of proinflammatory cytokines and chemokines as a result of viral infection should be considered an important pathogenic mechanism linking HCMV to periodontitis in vivo.

Salivary and Serum Markers Related to Innate Immunity in Generalized Aggressive Periodontitis.

BACKGROUND: Host inflammatory and immune responses play an important role in aggressive periodontitis (AgP). Thus, this study aims to evaluate levels of the innate immunity-related markers calprotectin, colony-stimulating factor (CSF)-1, macrophage migration inhibitory factor (MIF), monokine induced by interferon-γ (MIG), and matrix metalloproteinase (MMP)-8 in serum and saliva from patients with generalized AgP and those with gingivitis or a healthy periodontium. METHODS: This study enrolled 40 individuals (17 males and 23 females; mean age 33.30 ± 9.31 years), 15 with generalized AgP, 15 with gingivitis, and 10 who were periodontally healthy. Full-mouth periodontal examinations were performed, and serum and saliva were collected. Levels of calprotectin, CSF-1, MIF, MIG, and MMP-8 were measured using enzyme-linked immunosorbent assays. RESULTS: In serum, mean levels of calprotectin were 2.06-fold higher in patients with AgP than in healthy patients (P = 0.01). Serum levels of MMP-8 were significantly elevated in patients with AgP compared with both healthy patients and those with gingivitis, by 2.60-fold and 2.77-fold, respectively (P = 0.03 and P = 0.009, respectively). In saliva, levels of MMP-8 were 5.66-fold higher in patients with AgP than in healthy patients (P = 0.02). CSF-1, MIF, and MIG levels in both serum and saliva did not differ significantly among the groups. CONCLUSIONS: Serum levels of calprotectin and MMP-8 are elevated in patients with AgP. MMP-8 levels are also increased in saliva from patients with AgP. These results support involvement of innate immune response in the pathogenesis of AgP.

Topography Influences Adherent Cell Regulation of Osteoclastogenesis.

The importance of osteoclast-mediated bone resorption in the process of osseointegration has not been widely considered. In this study, cell culture was used to investigate the hypothesis that the function of implant-adherent bone marrow stromal cells (BMSCs) in osteoclastogenesis is influenced by surface topography. BMSCs isolated from femur and tibia of Sprague-Dawley rats were seeded onto 3 types of titanium surfaces (smooth, micro, and nano) and a control surface (tissue culture plastic) with or without osteogenic supplements. After 3 to 14 d, conditioned medium (CM) was collected. Subsequently, rat bone marrow-derived macrophages (BMMs) were cultured in media supplemented with soluble receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as well as BMSC CM from each of the 4 surfaces. Gene expression levels of soluble RANKL, osteoprotegerin, tumor necrosis factor α, and M-CSF in cultured BMSCs at different time points were measured by real-time polymerase chain reaction. The number of differentiated osteoclastic cells was determined after tartrate-resistant acid phosphatase staining. Analysis of variance and t test were used for statistical analysis. The expression of prominent osteoclast-promoting factors tumor necrosis factor α and M-CSF was increased by BMSCs cultured on both micro- and nanoscale titanium topographies (P < 0.01). BMSC CM contained a heat-labile factor that increased BMMs osteoclastogenesis. CM from both micro- and nanoscale surface-adherent BMSCs increased the osteoclast number (P < 0.01). Difference in surface topography altered BMSC phenotype and influenced BMM osteoclastogenesis. Local signaling by implant-adherent cells at the implant-bone interface may indirectly control osteoclastogenesis and bone accrual around endosseous implants.

CSF-1, RANKL and OPG regulate osteoclastogenesis during murine tooth eruption.

During tooth eruption, osteoclast-mediated bone resorption predominates in alveolar bone along the occlusal surface rather than in bone basal to the tooth. CSF-1, RANKL and OPG, regulatory molecules essential for osteoclastogenesis, are expressed during eruption. However, it is unclear if these cytokines exhibit an expression pattern that correlates with sites of osteoclastogenesis in vivo. To address this issue, mouse mandibles, isolated from 1 to 14 days postnatal, were analysed for osteoclast activity using tartrate-resistant acid phosphatase (TRAP) staining as well as colony-stimulating factor-1 (CSF-1), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) mRNA expression using in situ hybridisation. Results showed that CSF-1, RANKL and OPG are expressed in a distinct temporal and spatial manner. In the occlusal region, osteoclast activity was maximal at day 5 and correlated with a relative high expression of CSF-1 and RANKL compared to OPG. In basal bone at this time point, osteoclast activity decreased despite persistent CSF-1 expression and was associated with increased expression of OPG compared to RANKL. By day 8, osteoclastogenesis declined and correlated with upregulation of OPG at the occlusal and basal regions, with this effect continuing throughout eruption. These findings suggest that the spatiotemporal pattern and relative abundance of CSF-1, RANKL and OPG during eruption are key determinants of site-specific osteoclast activity in bone surrounding the tooth. Targeting these cytokines to specific regions in alveolar bone may provide a mechanism for regulating osteoclastogenesis in dental disorders associated with altered tooth eruption.

An in vitro comparison of implant materials cell attachment, cytokine and osteocalcin production.

Bone deposition, for any implant system, is the deciding factor for the success. The biochemical signals at the cellular level will help elucidate the direction of host response. In this report, intercellular messenger, cytokines, that are regulatory for osteoblast and osteoclast function, were measured. Production of osteocalcin, a marker for osteoblast maturation was also estimated. Human osteoblast-like cells from osteosarcoma cell line MG 63 were grown in wells in the presence of titanium (Ti), titanium alloy (Ti6A14V) and stainless steel implant materials incubated at 37 degrees C. Interleukin-1alpha (IL-1alpha), IL-6, IL-8, IL-11 and osteocalcin were quantitated using standard enzyme linked immunosorbant assay (ELISA) kits from the growth media extracted at specific intervals over the critical ten day period. In all dishes, cells were seen adhering to the base after 24 hours and to confluence at 96 hours. Both IL-1alpha and IL-11 were not produced in sufficient quantities to be measured in the assay (< pg/ml). Interleukin-6 production was significantly higher for stainless steel than for titanium and the alloy. There was a progressive rise in osteocalcin production for titanium contrasted to a basal rate for stainless steel and alloy. Interleukin-8 levels for all metals and controls increased markedly after two days implicating inherent cellular characteristics. A relatively high constant range for macrophage colony stimulating factor from the first day was seen for all metals, including the controls. In conclusion, it appears that titanium implants activate osteocalcin production while stainless steel activates IL-6.

Metabolic syndrome exacerbates inflammation and bone loss in periodontitis.

Clinical studies have shown that metabolic syndrome (MetS) is associated with increased risk of developing periodontitis. However, the underlying mechanisms remain largely unknown. Since it is known that lipopolysaccharide (LPS)-activated toll-like receptor 4 signaling pathways play a crucial role in periodontitis, we hypothesized that MetS enhances LPS-induced periodontal inflammation and alveolar bone loss. In this study, we induced MetS in C57BL/6 mice by feeding them high-fat diet (HFD), and we induced periodontitis by periodontal injection of Aggregatibacter actinomycetemcomitans LPS. We found that mice fed a HFD had significantly increased body weight, plasma lipids, insulin, and insulin resistance when compared with mice fed regular chow, indicating that the mice developed MetS. We also found that a HFD markedly increased LPS-induced alveolar bone loss, osteoclastogenesis, and inflammatory infiltration. Analysis of gene expression in periodontal tissue revealed that HFD and LPS injection cooperatively stimulated expression of cytokines that are known to be involved in periodontal tissue inflammation and osteoclastogenesis-such as interleukin 6, monocyte-chemotactic protein 1, receptor activator of nuclear factor kappa-B ligand, and macrophage colony-stimulating factor. To further understand the potential mechanisms involved in MetS-boosted tissue inflammation, our in vitro studies showed that palmitic acid-the most abundant saturated fatty acid (SFA) and the major SFA in the HFD used in our animal study-potently enhanced LPS-induced proinflammatory gene expression in macrophages. In sum, this study demonstrated that MetS was associated with increased periodontal inflammation and alveolar bone loss in an LPS-induced periodontitis animal model. This study also suggests that SFA palmitic acid may play an important role in MetS-associated periodontitis by enhancing LPS-induced expression of inflammatory cytokines in macrophages.

Mesenchymal dental pulp cells attenuate dentin resorption in homeostasis.

Dentin in permanent teeth rarely undergoes resorption in development, homeostasis, or aging, in contrast to bone that undergoes periodic resorption/remodeling. The authors hypothesized that cells in the mesenchymal compartment of dental pulp attenuate osteoclastogenesis. Mononucleated and adherent cells from donor-matched rat dental pulp (dental pulp cells [DPCs]) and alveolar bone (alveolar bone cells [ABCs]) were isolated and separately cocultured with primary rat splenocytes. Primary splenocytes readily aggregated and formed osteoclast-like cells in chemically defined osteoclastogenesis medium with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) and 50 ng/mL of receptor activator of nuclear factor κB ligand (RANKL). Strikingly, DPCs attenuated osteoclastogenesis when cocultured with primary splenocytes, whereas ABCs slightly but significantly promoted osteoclastogenesis. DPCs yielded ~20-fold lower RANKL expression but >2-fold higher osteoprotegerin (OPG) expression than donor-matched ABCs, yielding a RANKL/OPG ratio of 41:1 (ABCs:DPCs). Vitamin D3 significantly promoted RANKL expression in ABCs and OPG in DPCs. In vivo, rat maxillary incisors were atraumatically extracted (without any tooth fractures), followed by retrograde pulpectomy to remove DPCs and immediate replantation into the extraction sockets to allow repopulation of the surgically treated root canal with periodontal and alveolar bone-derived cells. After 8 wk, multiple dentin/root resorption lacunae were present in root dentin with robust RANKL and OPG expression. There were areas of dentin resoprtion alternating with areas of osteodentin formation in root dentin surface in the observed 8 wk. These findings suggest that DPCs of the mesenchymal compartment have an innate ability to attenuate osteoclastogenesis and that this innate ability may be responsible for the absence of dentin resorption in homeostasis. Mesenchymal attenuation of dentin resorption may have implications in internal resorption in the root canal, pulp/dentin regeneration, and root resorption in orthodontic tooth movement.

In vivo effect of interleukin-1 alpha on colony-stimulating factor-1 gene expression in the dental follicle of the rat molar.

Interleukin-1 alpha (IL-1 alpha) acts in vitro to enhance the gene expression of colony-stimulating factor-1 (CSF-1) of dental follicle cells. Because the dental follicle is required for tooth eruption and because CSF-1 appears to be a key molecule in initiating eruption, it was the aim of this study to determine if IL-1 alpha could enhance the expression of the CSF-1 gene in the dental follicle in vivo. To determine this, rats were injected with IL-1 alpha at different ages postnatally and the total RNA was isolated from the dental follicles of the first mandibular molars. Reverse transcription-polymerase chain reaction showed that IL-1 alpha enhanced the expression of CSF-1 in the follicle. Thus, IL-1 alpha may play a part in the cascade of molecular signals that initiate eruption by its effect upon the CSF-1 gene.

Effects of pulpectomy on the amount of root resorption during orthodontic tooth movement.

INTRODUCTION: Previous studies have revealed that orthodontic force affects dental pulp via the rupture of blood vessels and vacuolization of pulp tissues. We hypothesized that pulp tissues express inflammatory cytokines and regulators of odontoclast differentiation after excess orthodontic force. The purpose of this study was to investigate the effects of tensile force in human pulp cells and to measure inflammatory root resorption during tooth movement in pulpless rat teeth. METHODS: After cyclic tensile force application in human pulp cells, gene expression and protein concentration of macrophage colony-stimulating factor, receptor activator of nuclear factor kappa-B ligand, interleukin-1 beta, and tumor necrosis factor alpha were determined by real-time polymerase chain reaction and enzyme-linked immunoassay. Moreover, the role of the stretch-activated channel was evaluated by gadolinium (Gd(3+)) treatment. The upper right first molars of 7-week Wistar rats were subjected to pulpectomy and root canal filling followed by mesial movement for 6 months. RESULTS: The expression of cytokine messenger RNAs and proteins in the experimental group peaked with loading at 10-kPa tensile force after 48 hours (P < .01). Gd(3+) reduced the expression of these cytokine messenger RNAs and protein concentrations (P < .01). The amount of inflammatory root resorption was significantly larger in the control teeth than the pulpectomized teeth (P < .05). CONCLUSIONS: This study shows that tensile forces in the pulp cells enhance the expression of various cytokines via the S-A channel, which may lead to inflammatory root resorption during tooth movement. It also suggests that root canal treatment is effective for progressive severe inflammatory root resorption during tooth movement.

Changes of temporomandibular joint and semaphorin 4D/Plexin-B1 expression in a mouse model of incisor malocclusion.

AIMS: To investigate the changes in condylar cartilage and subchondral bone of the temporomandibular joint (TMJ) in a mouse model of incisor malocclusion. METHODS: By bonding a single (single group) or a pair (pair group) of metal tube(s) to the left incisor(s), a crossbite-like relationship was created between left-side incisors in mice. The morphological changes in the TMJ condyles were examined by hematoxylin and eosin and toluidine blue staining. Indices of osteoclastic activity, including tartrate-resistant acid phosphatase (TRAP) staining and macrophage colony stimulating factor (M-CSF) were investigated by histochemistry or real-time polymerase chain reaction (PCR). The osteoblastic activity was indexed by osteocalcin expression. Expressions of semaphorin 4D and its receptor, Plexin-B1, were detected by real-time PCR. Two-way analysis of variance was used to assess the differences between groups. RESULTS: One week and 3 weeks after bonding the metal tube(s), cartilage degradation and subchondral bone loss were evident histologically. Both indices of osteoclastic activity (TRAP and M-CSF) were significantly increased in cartilage and subchondral bone after bonding the metal tube(s). Osteocalcin expression in cartilage was significantly increased at week 3, while its expression in subchondral bone was significantly increased at week 1 but decreased at week 3. The semaphorin 4D expression in cartilage and subchondral bone was significantly decreased at week 1 but significantly increased at week 3. For Plexin-B1 expression, a significant increase was detected in subchondral bone at week 3. CONCLUSION: Bonding a single or a pair of metal tube(s) to left incisor(s) is capable of inducing remodeling in the TMJ, which involved cartilage degradation and alteration of osteoclastic and osteoblastic activity.

Compressive stress induced the up-regulation of M-CSF, RANKL, TNF-α expression and the down-regulation of OPG expression in PDL cells via the integrin-FAK pathway.

OBJECTIVES: This study was performed to elucidate the involvement of integrin-FAK (focal adhesion kinase) pathway in compressive stress-induced mRNA expression of macrophage colony stimulating factor (M-CSF), tumour necrosis factor (TNF)-α, receptor activator of nuclear factor κB (RANKL) and osteoprotegerin (OPG) and to further confirm the role of the integrin-FAK complex as a mechanoreceptor in PDL cells. DESIGN: Periodontal ligament (PDL) cells were obtained from patients having healthy first premolars extracted for orthodontic purposes. Cultured PDL cells were divided into three groups: the control group in which compressive stress was administered; the negative control group in which mechanical stress was administered after transfection of negative control siRNA; and FAK knockdown group in which mechanical stress was administered after FAK siRNA treatment. Compressive stress (2g/cm(2)) was for various time durations (0.5, 2, 6, 24, 48h). Total RNA was collected after the experiment and real-time PCR analysis was performed to determine the mRNA expression levels of M-CSF, TNF-α, RANKL and OPG. Also the supernatant was analysed with ELISA to detect the corresponding cytokine concentrations. RESULTS: The cells of the control group and the negative control group expressed higher mRNA levels of M-CSF, TNF-α, and RANKL but a lower mRNA level of OPG compared to those of baseline. FAK knockdown cells showed lower mRNA expression levels of M-CSF, TNF-α, and RANKL but a higher mRNA expression level of OPG than that in the control. The OPG mRNA expression level in FAK knockdown cells was even higher than that of baseline. ELISA results showed similar pattern of cytokine concentration changes. CONCLUSIONS: Results of this study indicate that the integrin-FAK pathway regulates compressive stress-induced expression of M-CSF, TNF-α, RANKL and OPG and suggests that the integrin-FAK complex acts as a mechanoreceptor in PDL cells.

Immunolocalization of CSF-1, RANKL and OPG in the enamel-related periodontium of the rat incisor and their implications for alveolar bone remodeling.

The enamel-related periodontium (ERP) in rat incisors is related to bone resorption. In these teeth the face of the socket related to the enamel is continuously removed at the inner side and newly formed at the outer side. CSF-1, RANKL and OPG are regulatory molecules essential for osteoclastogenesis. To verify the effects of impeded eruption on bone remodeling, the tooth eruption was prevented by immobilization of lower rat incisor and CSF-1, RANKL and OPG distribution in the ERP was analyzed after 18 days of immobilization and in normal eruption. The region of the alveolar crest of the rat incisor was used. Immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) were performed. The immunostaining of the dental follicle was quantified using Leica QWin software. Positive-TRAP osteoclasts were counted, and both groups were compared. In the normal incisor, the number of osteoclasts was significantly greater than in the immobilized tooth. In the dental follicle, there was no significant difference in the immunostaining intensity for CSF-1 and OPG between the groups (p > 0.05), but for RANKL the immobilized incisor group showed immunostaining intensity smaller than the normal incisor group (p < 0.01). These findings suggest that changes in the ERP, in the immobilized incisor, modify the RANKL/OPG ratio, in the presence of CSF-1, altering the metabolism of cells that participate in the bone remodeling.

The role of dental pulp cells in resorption of deciduous teeth.

OBJECTIVE: To address the question whether dental pulp cells of exfoliating human deciduous teeth have some roles for controlling or regulating the root resorption via secreting key molecules (OPG, RANKL, CSF-1, TGFbeta, MCP-1 and Cbfa-1) in osteoclastogenesis, we used a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) method for detection of mRNA expressions for the cytokines listed. STUDY DESIGN: The dental pulps were retrieved from incisor and molar teeth in the late stage of shedding (n = 30) and from sound premolar teeth extracted for orthodontic reasons (control group; n = 30). The RT-PCR assays were used to identify targeted gene expression. RESULTS: Of the cytokines examined, RANKL and CSF-1 expressions showed significantly higher occurrence in deciduous dental pulps than in permanent teeth pulpal tissues (P < .040). CONCLUSIONS: The findings may suggest an interactive role for pulp tissue cells in the physiologic root resorption process. The cells of dental pulp may have some cytokine-producing cells which mediate monocyte-macrophage lineage to form osteo/odontoclasts, and the RANKL/RANK system might be involved in human deciduous teeth resorption.

Human cytomegalovirus, Epstein-Barr virus and bone resorption-inducing cytokines in periapical lesions of deciduous teeth.

BACKGROUND: A connection of herpesvirus periapical infection with symptomatic and large-size periapical lesions has been recognized in adult patients, but no data exist about a possible involvement of herpesviruses in severe periapical pathosis in children. Herpesviruses have the potential to elicit potent bone resorption-inducing cytokines in mammalian cells. AIM: This study aimed to determine the occurrence of human cytomegalovirus and Epstein-Barr virus DNA, and mRNA transcripts of receptor activator of nuclear kappa B ligand (RANKL), osteoprotegerin, core binding factor alpha-1, colony stimulating factor-1, transforming growth factor-beta, and monocyte chemoattractant protein-1 in periapical symptomatic pathosis of deciduous teeth. MATERIAL AND METHODS: Twelve deciduous molar teeth from patients aged 2-8 years were extracted due to severe periapical infection, and granulomatous tissue adherent to the root tip of the extracted teeth was collected using a surgical knife. Non-diseased pulpal tissue, obtained from 12 teeth extracted for orthodontic reasons, served as negative control. Polymerase chain reaction assays were employed to identify herpesvirus DNA and cytokine gene expression, using established polymerase chain reaction primers and procedures. RESULTS: Seven (58%) of the periapical lesions yielded human cytomegalovirus and eight (67%) Epstein-Barr virus. Only one (8%) periapical lesion showed neither human cytomegalovirus nor Epstein-Barr virus. In healthy pulpal tissue, one (8%) specimen demonstrated human cytomegalovirus and another (8%) specimen revealed Epstein-Barr virus. Of the cytokines examined, RANKL expression showed significantly higher occurrence in periapical pathosis than in healthy pulpal tissue (P < 0.040). No relationship was identified between the type of herpesvirus and cytokine expression in the periapical lesions studied. CONCLUSIONS: The present findings provide evidence of a putative role of human cytomegalovirus and Epstein-Barr virus in the pathogenesis of symptomatic periapical pathosis in deciduous teeth. Increased RANKL expression in periapical lesions may be of pathogenetic significance.

Cytokine secretion of periodontal ligament fibroblasts derived from human deciduous teeth: effect of mechanical stress on the secretion of transforming growth factor-beta 1 and macrophage colony stimulating factor.

The periodontal ligament may play an important role in tooth eruption, root development and resorption. The tissue physiologically receives mechanical force during mastication. We focused on the effects of intermittent mechanical strain on the cytokine synthesis of periodontal ligament (PDL) fibroblasts in vitro. The cells were derived from human periodontal ligament of deciduous teeth (HPLF-Y) and permanent teeth (HPLF). The two kinds of PDL cells and human gingival fibroblasts (HGF) were cultured in flexible bottomed culture plates. The cells were mechanically stretched at 5% elongation, 3-cycles/min for 24 h on d 7 in culture using a Flexercell strain unit. After the stretching, we measured DNA content and alkaline phosphatase activity in the cell layer, transforming growth factor beta 1 (TGF-beta 1) and macrophage colony stimulating factor (M-CSF) contents in the conditioned medium. The TGF-beta 1 level in the conditioned medium of HPLF was significantly higher than that of HPLF-Y and HGF. It was stimulated by mechanical stretching only on HPLF, whereas no significant effect was observed on HPLF-Y and HGF. M-CSF secretion was inhibited by the stretching on all of HPLF, HPLF-Y and HGF. 1 alpha, 25 dihydroxy vitamin D3 (D3) stimulated M-CSF secretion into the culture medium of both HPLF and HPLF-Y, but the stretching inhibited M-CSF secretion and completely blocked the enhancement by D3. These data suggest that periodontal ligament cells synthesize and secrete the molecules as autocrine or paracrine factors that affect bone remodelling and root resorption and the level of those factors change in response to mechanical stress.

Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1 beta and tumour necrosis factor alpha.

OBJECTIVE: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1 beta and TNF alpha. METHODS: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. RESULTS: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1 beta, and greatly inhibited induction by TNF alpha. CONCLUSION: IL18, IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1 beta, but less potent than TNF alpha.

An in vitro comparison of implant materials cell attachment, cytokine and osteocalcin production

Bone deposition, for any implant system, is the deciding factor for the success. The biochemical signals at the cellular level will help elucidate the direction of host response. In this report, intercellular messenger, cytokines, that are regulatory for osteoblast and osteoclast function, were measured. Production of osteocalcin, a marker for osteoblast maturation was also estimated. Human osteoblast-like cells from osteosarcoma cell line MG 63 were grown in wells in the presence of titanium (Ti), titanium alloy (Ti6A14V) and stainless steel implant materials incubated at 37 degrees C. Interleukin-1alpha (IL-1alpha), IL-6, IL-8, IL-11 and osteocalcin were quantitated using standard enzyme linked immunosorbant assay (ELISA) kits from the growth media extracted at specific intervals over the critical ten day period. In all dishes, cells were seen adhering to the base after 24 hours and to confluence at 96 hours. Both IL-1alpha and IL-11 were not produced in sufficient quantities to be measured in the assay (< pg/ml). Interleukin-6 production was significantly higher for stainless steel than for titanium and the alloy. There was a progressive rise in osteocalcin production for titanium contrasted to a basal rate for stainless steel and alloy. Interleukin-8 levels for all metals and controls increased markedly after two days implicating inherent cellular characteristics. A relatively high constant range for macrophage colony stimulating factor from the first day was seen for all metals, including the controls. In conclusion, it appears that titanium implants activate osteocalcin production while stainless steel activates IL-6.