Hypertension modifies OPG, RANK, and RANKL expression during the dental socket bone healing process in spontaneously hypertensive rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relevance of the Myeloid Differentiation Factor 88 (MyD88) on RANKL, OPG, and Nod Expressions Induced by TLR and IL-1R Signaling in Bone Marrow Stromal Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The RANK/RANKL/OPG interaction in the repair of autogenous bone grafts in female rats with estrogen deficiency

The aim of this study was to evaluate the resorption process during the repair of autogenous bone grafts with or without coverage by an expanded polytetrafluoroethylene (e-PTFE) membrane in female rats with estrogen deficiency using the immunohistochemical technique. Eighty female rats were randomly divided into two groups (OVX and SHAM). The 40 female rats in the OVX group were subjected to ovariectomy, and the 40 female rats in the SHAM group were subjected to simulated ovariectomy. The two groups were further divided in subgroup E, which was subjected to surgery for placement of autogenous bone graft (ABG), and subgroup ME, in which the ABG was covered with an e-PTFE membrane. The animals were killed at 0, 7, 21,45 and 60 days. The specimens were analyzed using immunohistochemistry for the bone resorption markers RANK, RANK-L and Osteoprotegerin (OPG). A higher remodeling rate was observed at 7 and 21 days after the autogenous bone grafts, when the markers were more intensely expressed. At the final time point, the specimens presented similar characteristics to those observed at the initial time point. The expression of immunohistochemical markers was not altered by the estrogen deficiency. The presence of the e-PTFE membrane delayed the bone resorption process, influencing the immunohistochemical expression of markers.

Are skeletally mature female rats a suitable model to study osteoporosis?

OBJECTIVE: To analyze if female Wistar rats at 56 weeks of age are a suitable model to study osteoporosis. MATERIALS AND METHODS: Female rats with 6 and 36 weeks of age (n = 8 per group) were kept over a 20-week period and fed a diet for mature rodents complete in terms of Ca, phosphorous, and vitamin D. Excised femurs were measured for bone mass using dual-energy x-ray absorptiometry, morphometry, and biomechanical properties. The following serum mar-kers of bone metabolism were analyzed: parathyroid hormone (PTH), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor Κappa B ligand (RANKL), C-terminal peptides of type I collagen (CTX-I), total calcium, and alkaline phosphatase (ALP) activity. RESULTS: Rats at 56 weeks of age showed important bone metabolism differences when compared with the younger group, such as, highest diaphysis energy to failure, lowest levels of OC, CTX-I, and ALP, and elevated PTH, even with adequate dietary Ca. CONCLUSION: Rats at 26-week-old rats may be too young to study age-related bone loss, whereas the 56-week-old rats may be good models to represent the early stages of age-related changes in bone metabolism.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

RANKL is a mediator of bone resorption in idiopathic hypercalciuria

Background and objectives: This study aimed to determine the expression of osteoprotegerin, receptor activator of nuclear factor kappa B ligand, interleukin-la, transforming growth factor-beta, and basic fibroblast growth factor in stone-forming patients with idiopathic hypercalciuria. Design, setting, participants, & measurements: Immunohistochemical analysis was performed in undecalcified bone samples previously obtained from 36 transiliac bone biopsies of patients who had idiopathic hypercalciuria and whose histomorphometry had shown lower bone volume, increased bone resorption, and prolonged mineralization lag time. Results: Bone expression of receptor activator of nuclear factor kappa B ligand and osteoprotegerin was significantly higher in patients with idiopathic hypercalciuria versus control subjects. Transforming growth factor-beta immunostaining was lower in patients with idiopathic hypercalciuria than in control subjects and correlated directly with mineralization surface. Interleukin-la and basic fibroblast growth factor staining did not differ between groups. Receptor activator of nuclear factor kappa B ligand bone expression was significantly higher in patients who had idiopathic hypercalciuria and exhibited higher versus normal bone resorption. Conclusion: A higher expression of receptor activator of nuclear factor kappa B ligand in bone tissue suggests that increased bone resorption in patients with idiopathic hypercalciuria is mediated by receptor activator of nuclear factor kappa B ligand. Osteoprotegerin bone expression might have been secondarily increased in an attempt to counteract the actions of receptor activator of nuclear factor kappa B ligand. The low bone expression of transforming growth factor-beta could contribute to the delayed mineralization found in such patients.

Bone mineral apparent density in juvenile dermatomyositis: the role of lean body mass and glucocorticoid use

Objective: To analyse bone mineral density (BMD) in juvenile dermatomyositis (JDM) and its possible association with body composition, disease activity, duration of disease, glucocorticoid (GC) use, and biochemical bone parameters, including osteoprotegerin (OPG) and receptor activator of nuclear factor B (RANKL). Methods: Twenty girls with JDM and 20 controls matched for gender and age were selected. Body composition and BMD were analysed by dual-energy X-ray absorptiometry (DXA) and bone mineral apparent density (BMAD) was calculated. Duration of disease, cumulative GC, and GC pulse therapy use were determined from medical records. Disease activity and muscle strength were measured by the Disease Activity Score (DAS), the Childhood Myositis Assessment Scale (CMAS), and the Manual Muscle Test (MMT). Inflammatory and bone metabolism parameters were also analysed. OPG and RANKL were measured in patients and controls using an enzyme-linked immunosorbent assay (ELISA). Results: A lower BMAD in the femoral neck (p< 0.001), total femur (p< 0.001), and whole body (p=0.005) was observed in JDM patients compared to controls. Body composition analysis showed a lower lean mass in JDM compared to controls (p=0.015), but no difference was observed with regard to fat mass. A trend of lower serum calcium was observed in JDM (p=0.05), whereas all other parameters analysed, including OPG and RANKL, were similar. Multiple linear regression analysis revealed that, in JDM, lean mass (p< 0.01) and GC pulse therapy use (p< 0.05) were independent factors for BMAD in the hip region. Conclusions: This study has identified low lean mass and GC pulse therapy use as the major factors for low hip BMAD in JDM patients.

Usefulness of a quick decalcification of bone sections embedded in methyl metacrylate: an improved method for immunohistochemistry

Immunohistochemistry of undecalcified bone sections embedded in methyl methacrylate (MMA) is not commonly employed because of potential destruction of tissue antigenicity by highly exothermic polymerization. The aim of the present study was to describe a new technique in which a quick decalcification of bone sections embedded in MMA improves the results for immunohistochemistry. The quality of interleukin 1 alpha (IL-1 alpha) immunostaining according to the present method was better than the conventional one. Immunostaining for osteoprotegerin (OPG) and the receptor activator of NF-kappa B ligand (RANKL) in bone sections of chronic kidney disease patients with mineral bone disorders (CKD-MBD) was stronger than in controls (postmortem healthy subjects). The present study suggested that this method is easy, fast, and effective to perform both histomorphometry and immunohistochemistry in the same bone fragment, yielding new insights into pathophysiological aspects and therapeutic approaches in bone disease.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface

The aim of this study was to evaluate the development of the osteoblastic phenotype in human alveolar bone-derived cells grown on collagen type I-coated titanium (Ti) surface (Col-Ti) obtained by plasma deposition acrylic acid grafting compared with machined Ti (M-Ti). Osteoblastic cells were cultured until subconfluence and subcultured on Col-Ti and M-Ti for periods of up to 21 days. Cultures grown on Col-Ti and M-Ti exhibited similar cell morphology. Cell adhesion, total protein content, and alkaline phosphatase (ALP) activity were not affected by Ti surface modification in all evaluated periods. Growth analyses indicated that there were significantly more cells in cultures grown on Col-Ti at day 3. Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteoprotegerin (OPG) mRNA expression of cells subcultured on Col-Ti was higher, whereas collagen type I (COL) was lower compared with M-Ti. Ti surface modification neither affected the osteocalcin (OC), ALP and receptor activator of NF-kappa B ligand (RANKL) mRNA expression nor the calcium content extracted from mineralized matrix. These results demonstrated that Col-Ti favours cell growth during the proliferative phase (day 3) and osteoblastic differentiation, as demonstrated by changes in mRNA expression profile during the matrix mineralization phase (day 14), suggesting that this Ti surface modification may affect the processes of bone healing and remodelling. To cite this article:Assis AF, Beloti MM, Crippa GE, de Oliveira PT, Morra M, Rosa AL. Development of the osteoblastic phenotype in human alveolar bone-derived cells grown on a collagen type I-coated titanium surface.Clin. Oral Impl. Res. 20, 2009; 240-246.doi: 10.1111/j.1600-0501.2008.01641.x.

Administration of Il-18bp by Gene Therapy Reduces Inflammation and Prevents Joint Destruction by Downregulation of Mmp9 In Rat Aia: Role ff Mmp9 In Bone and Joint Destruction in Arthritis

Background and objectives Interleukin 18 (IL-18) is a pleiotropic cytokine involved in rheumatoid arthritis (RA) pathogenesis. This study was carried out to evaluate the effi cacy of IL-18 binding protein (IL-18BP) gene therapy in the rat adjuvant- induced arthritis (AIA) model and to decipher the mechanisms by which IL-18BP delivery lessens bone destruction.Materials and methods Arthritis was induced in female Lewis rat by Mycobacterium butyricum and the mRNA expression of IL-18 and IL-18BP was determined in the joints. In a preventive study, rats were divided into an adenovirus producing IL-18BP-Fc (AdmIL-18BP-Fc) group (n=8) and an adenovirus producing green fl uorescent protein (AdGFP) group (n=7). On day 8 after AIA induction, adenoviruses were injected. Clinical parameters were assessed. At day 18, during maximal arthritis, the rats were euthanized, ankles were collected and x-rays were performed. mRNA and protein were extracted from joints for analysis by quantitative reverse transcriptase-PCR, multiplex, Western blot and zymography.Results The authors observed a decrease in the (IL-18BP/ IL-18) ratio from day 7 to 45. Administration of AdmIL-18BPd-Fc decreased clinical parameters and prevented bone and joint destruction compared to AdGFP administration. IL-18BP delivery reduced the (receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG)) ratio by 70%, the matrix metalloproteinase 9 (MMP9) level by 33% and the tartrate-resistant acid phosphatase (TRAP) level by 44% in the joint homogenates from AdmIL-18BPd-Fc compared to AdGFP treated rats.Conclusions In rat AIA, a decrease in the (IL-18BP/IL-18) ratio was observed. IL-18BP delivery prevented joint and bone destruction by downregulating MMP9, (RANKL/OPG) and TRAP, suggesting a potential benefi t of a similar therapy in RA.Abstract topics Towards novel therapeutic strategies.

PPARβ/δ governs Wnt signaling and bone turnover.

Peroxisome proliferator-activated receptors (PPARs) act as metabolic sensors and central regulators of fat and glucose homeostasis. Furthermore, PPARγ has been implicated as major catabolic regulator of bone mass in mice and humans. However, a potential involvement of other PPAR subtypes in the regulation of bone homeostasis has remained elusive. Here we report a previously unrecognized role of PPARβ/δ as a key regulator of bone turnover and the crosstalk between osteoblasts and osteoclasts. In contrast to activation of PPARγ, activation of PPARβ/δ amplified Wnt-dependent and β-catenin-dependent signaling and gene expression in osteoblasts, resulting in increased expression of osteoprotegerin (OPG) and attenuation of osteoblast-mediated osteoclastogenesis. Accordingly, PPARβ/δ-deficient mice had lower Wnt signaling activity, lower serum concentrations of OPG, higher numbers of osteoclasts and osteopenia. Pharmacological activation of PPARβ/δ in a mouse model of postmenopausal osteoporosis led to normalization of the altered ratio of tumor necrosis factor superfamily, member 11 (RANKL, also called TNFSF11) to OPG, a rebalancing of bone turnover and the restoration of normal bone density. Our findings identify PPARβ/δ as a promising target for an alternative approach in the treatment of osteoporosis and related diseases.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-alpha), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

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-kappa beta 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. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.