M-CSF Priming of Osteoclast Precursors Can Cause Osteoclastogenesis-Insensitivity, Which Can be Prevented and Overcome on Bone

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. J. Cell. Physiol. 229: 210-225, 2014. (c) 2014 Wiley Periodicals, Inc.

Triiodothyronine (T3) does not induce RANKL expression in rat ROS 17/2.8 cells

Osteoclastogenesis may be regulated via activation of the RANK/RANKL (receptor activator of nuclear factor-kappa B/ receptor activator of nuclear factor-kappa B ligand) system, which is mediated by osteoblasts. However, the bone loss mechanism induced by T3 (triiodothyronine) is still controversial. In this study, osteoblastic lineage rat cells (ROS 17/2.8) were treated with T3 (10(-8) M 10(-9) 10 M, and 10(-10) M), and RANKL mRNA (messenger RNA) expression was measured by semiquantitative RT-PCR. Our results show that T3 concentrations used did not significantly enhance RANKL expression compared to controls without hormone treatment. This data suggests that other mechanisms, unrelated to the RANK/RANKL system, might be to activate osteoclast differentiation in these cells.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappa B ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappa B ligand (RANKL) expression by murine periodontal ligament cells.Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor κB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright © 2007 John Wiley & Sons, Ltd.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: Calvarial bone

Objectives: Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods: Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-κβ ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results: The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P≤0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions: These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. © 2009 John Wiley & Sons A/S.

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine-treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H1- and H2-receptors. Cimetidine is a H2-receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100mg kg-1 of cimetidine for 50days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate-resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) method and immunohistochemical reactions for detecting caspase-3 and RANKL were performed. The number of TRAP-positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL-positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP-positive osteoclasts with TUNEL-positive nuclei and caspase-3-immunolabeled osteoclasts were found. A significant reduction in the number of TRAP-positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL-positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine-induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis. © 2012 The Authors Journal of Anatomy © 2012 Anatomical Society.

Guided implant surgery: What is the influence of this new technique on bone cell viability?

Purpose: To evaluate the effect of implant osteotomy on immediate bone cell viability, comparing guided surgery for implant placement with the classic drilling procedure. Materials and Methods: For this study, 20 rabbits were used. The animals were divided into a guided surgery group (GG) and a control group (CG) and were then divided into 4 subgroups - subgroups 1, 2, 3, and 4 - corresponding to drills used 10, 20, 30, and 40 times, respectively. All animals received 5 osteotomies in each tibia, by use of the classic drilling procedure in one tibia and guided surgery in the other tibia. The osteotomized areas were removed and processed immunohistochemically for detection of osteocalcin, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and caspase 3. Results: Immunohistochemical analysis showed that osteocalcin expression was initially higher in the CG and remained constant after drill reutilization. Although the expressions of RANKL and OPG were not statistically different for the GG and CG, the RANKL/OPG ratio tended to be higher for the GG. Moreover, caspase 3 expression was elevated in the GG, proportionally to the number of osteotomies, indicating an increase in the apoptosis index in the GG. Conclusions: The classic drilling procedure is more favorable to cell viability than guided surgery.© 2013 American Association of Oral and Maxillofacial Surgeons.

Adjunctive antimicrobial photodynamic treatment of experimentally induced periodontitis in rats with ovariectomy

Background: The aim of this study is to compare antimicrobial photodynamic therapy (aPDT) as an adjunctive therapy to scaling and root planing (SRP) for the treatment of experimentally induced periodontitis in rats with ovariectomy (OVX) that are or are not treated with estrogen replacement. Methods: A total of 270 female rats were divided into three groups: 1) normal rats; 2) rats with OVX; and 3) rats with OVX with estrogen replacement. Periodontal disease was induced through the introduction of a cotton thread around the mandibular left first molar. After 7 days, the ligature was removed, and the rats were randomly divided into the following treatment groups: 1) SRP plus saline solution; 2) SRP plus low-level laser therapy (LLLT); and 3) SRP plus toluidine blue O irrigation followed by LLLT. Ten rats from each group were euthanized at days 7, 15, and 30 after dental treatment. Bone loss (BL) in the furcation region was evaluated using histometric and immunohistochemical analyses. Results: aPDT treatment resulted in reduced BL compared with SRP treatment at all time points. Additionally, rats treated with aPDT exhibited reduced numbers of tartrate-resistant acid-phosphatase-positive cells and more proliferating cell nuclear antigen-positive cells in all treatment groups regardless of estrogen status. Whereas rats treated with aPDT showed weak immunoreactivity to the receptor activator of nuclear factor-k B ligand at day 7 post-treatment, strong osteoprotegerin immunoreactivity was observed at day 15 post-treatment. Conclusion: aPDT is an effective adjunctive therapy for the treatment of periodontitis in rats with OVX that are or are not given estrogen replacement therapy.

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC 50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14+ human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK. © FASEB.

The role of cytokines in inflammatory bone loss

Chronic inflammatory processes close to bone often lead to loss of bone in diseases such as rheumatoid arthritis, periodontitis, loosened joint prosthesis and tooth implants. This is mainly due to local formation of bone resorbing osteoclasts which degrade bone without any subsequent coupling to new bone formation. Crucial for osteoclastogenesis is stimulation of mononuclear osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) which induces their differentiation along the osteoclastic lineage and the fusion to mature, multinucleated osteoclasts. M-CSF and RANKL are produced by osteoblasts/ osteocytes and by synovial and periodontal fibroblasts and the expression is regulated by pro- and anti-inflammatory cytokines. These cytokines also regulate osteoclastic differentiation by direct effects on the progenitor cells. In the present overview, we introduce the basic concepts of osteoclast progenitor cell differentiation and summarize the current knowledge on cytokines stimulating and inhibiting osteoclastogenesis by direct and indirect mechanisms. © Informa Healthcare USA, Inc.

Influência do FK-506 sobre a expressão de RANKL e OPG na doença periodontal induzida: estudo in vivo e in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Papel das proteínas intracelulares Nod e da proteína adaptadora MyD88 na regulação de espressão de RANKL e modulação da resposta inflamatória induzidos por antígenos bacterianos in vitro: estudo em células relevantes de periodonto

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evidências da associação entre testosteronas e doença periodontal no sexo masculino

Pós-graduação em Odontologia - FOAR

Efeito de um curcumin modificado quimicamente sobre a reabsorção óssea inflamatória e apoptose: estudos in vivo e in vitro

Pós-graduação em Odontologia - FOAR

RANKL expression is differentially modulated by TLR2 and TLR4 signaling in fibroblasts and osteoblasts

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.