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Introduction Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. Methods An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. Results Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. Conclusions Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement.

OBJECTIVE: To investigate the effect of Lithothamnium sp (LTT) supplement, a calcium-rich alga widely used for mineral reposition, on strain-induced (orthodontic tooth movement [OTM]) and infection-induced bone resorption (periodontal disease [PD]) in mice. MATERIALS AND METHODS: Mice were divided into two bone resorption models: one with an orthodontic appliance and the other with PD induced by the oral inoculation of Aggregatibacter actinomycetencomitans (Aa). Both groups were fed a regular diet (vehicle), LTT-rich diet (LTT), or calcium-rich diet (CaCO(3)). Alveolar bone resorption (ABR), the number of osteoclasts, and the levels of tumor necrosis factor α (TNF-α), calcium, and vitamin D3 were evaluated. RESULTS: The number of osteoclasts was reduced in LTT and CaCO(3) mice, which led to diminished OTM and infection-induced alveolar bone loss. In addition, LTT- and calcium-treated groups also presented decreased levels of TNF-α in periodontal tissues and increased levels of calcium in serum. CONCLUSIONS: These results indicate that the LTT supplement influences ABR, probably due to its calcium content, by affecting osteoclast function and local inflammatory response, thus modulating OTM and PD.

Background and Objective The angiotensin type 1 (AT1) receptor has been implicated in the pathogenesis of inflammatory bone disorders. This study aimed to investigate the effect of an AT1 receptor antagonist in infection-induced and arthritis-associated alveolar bone loss in mice. Material and Methods Mice were subjected to Aggregatibacter actinomycetemcomitans oral infection or antigen-induced arthritis and treated daily with 10 mg/kg of the prototype AT1 antagonist, losartan. Treatment was conducted for 30 d in the infectious condition and for 17 d and 11 d in the preventive or therapeutic regimens in the arthritic model, respectively. The mice were then killed, and the maxillae, serum and knee joints were collected for histomorphometric and immunoenzymatic assays. In vitro osteoclast assays were performed using RAW 264.7 cells stimulated with A. actinomycetemcomitans lipopolysacharide (LPS). Results Arthritis and A. actinomycetemcomitans infection triggered significant alveolar bone loss in mice and increased the levels of myeloperoxidase and of TRAP+ osteoclasts in periodontal tissues. Losartan abolished such a phenotype, as well as the arthritis joint inflammation. Both arthritis and A. actinomycetemcomitans conditions were associated with the release of tumor necrosis factor alpha (TNF-α), interferon-gamma, interleukin-17 and chemokine (C-X-C motif) ligand 1 and an increased RANKL/osteoprotegerin ratio in periodontal tissues, but such expression decreased after losartan treatment, except for TNF-α. The therapeutic approach was as beneficial as the preventive one. In vitro, losartan prevented LPS-induced osteoclast differentiation and activity. Conclusion The blockade of AT1 receptor exerts anti-inflammatory and anti-osteoclastic effects, thus protecting periodontal tissues in distinct pathophysiological conditions of alveolar bone loss.

5-Lipoxygenase (5-LO) metabolites are important pro-inflammatory lipid mediators. However, much still remains to be understood about the role of such mediators in bone remodeling. This study aimed to investigate the effect of 5-LO metabolites, LTB4 and CysLTs, in a model of mechanical loading-induced bone remodeling. Strain-induced tooth movement and consequently alveolar bone resorption/apposition was achieved by using a coil spring placed on molar and attached to incisors of C57BL6 (wild-type-WT), 5-LO deficient mice (5-LO(-/-)) and mice treated with 5-LO inhibitor (zileuton-ZN) or with antagonist of CysLTs receptor (montelukast-MT). The amount of bone resorption and the number of osteoclasts were determined morphometrically. The expression of inflammatory and bone remodeling markers in periodontium was analyzed by qPCR. Osteoclast differentiation and TNF-α production were evaluated in vitro using RAW 264.7 cells treated with LTB4 or LTD4. Bone resorption, TRAP(+) cells and expression of Tnfa, Il10 and Runx2 were significantly diminished in 5-LO(-/-), ZN- and MT-treated mice. The expression of Rank was also reduced in 5-LO(-/-) and MT-treated mice. Accordingly, LTB4 and LTD4 in association with RANKL promoted osteoclast differentiation and increased TNF-α release in vitro. These data demonstrate that the absence of 5-LO metabolites, LTB4 and CysLTs reduces osteoclast recruitment and differentiation, consequently diminishing bone resorption induced by mechanical loading. Thus, 5-LO might be a potential target for controlling bone resorption in physiological and pathological conditions.

A aplicação de força mecânica sobre o tecido ósseo resulta em remodelação, caracterizada pela reabsorção e deposição óssea em sítios específicos. Esses processos são regulados pela liberação de mediadores inflamatórios, dentre esses os metabólitos derivados do ácido araquidônico (AA). O AA, quando metabolizado pela via 5 lipoxigenase (5-LO) resulta na formação de leucotrieno B4 (LTB4) e cisteinil leucotrienos (CysLTs). Esses metabólitos têm sido amplamente estudados na resposta inflamatória e, mais recentemente, na remodelação óssea. Sendo assim, os objetivos deste trabalho foram: (i) desenvolver um modelo de remodelação óssea associada à movimentação dentária ortodôntica induzida por força mecânica; (ii) investigar o papel dos metabólitos da via 5-LO no processo de remodelação óssea, bem como no recrutamento e diferenciação de osteoclastos. Inicialmente, para definição da força mecânica ideal, camundongos C57BL/6 foram submetidos a uma força de intensidade entre 0,1 N e 0,5 N. A força ideal obtida, 0,35 N, foi aplicada nos demais grupos experimentais: C57BL/6 ou SV129 (Wild Type-WT); animais deficientes da enzima 5-LO (5-LO-/-), ou tratados com veículo (VH), zileuton (ZN) (inibidor da 5-LO) e montelucaste (MT) (antagonista do receptor de CysLTs). A quantidade movimentação dentária e o número de osteoclastos foram avaliados histomorfometricamente. A expressão de marcadores de remodelação óssea e citocinas foi avaliada em sítios de compressão e tensão do periodonto. O efeito dos metabólitos da via 5-LO na diferenciação de osteoclastos, bem como na expressão de TNF- foi avaliada in vitro após estímulo de células RAW 264.7 com LTB4 e LTD4. A força de 0,35 N promoveu movimentação dentária e recrutamento de osteoclastos sem induzir reabsorção radicular. A movimentação dentária e o número de osteoclastos estavam diminuídos na ausência ou inibição da enzima 5-LO e do receptor de CysLTs em comparação com os grupos WT e VH, respectivamente. Foi observada uma expressão diferenciada de citocinas e marcadores de remodelação óssea nos sítios de tensão e compressão. Catepsina K e o TNF- tiveram maior expressão nos sítios de compressão, enquanto OCN e RUNX2 no sítio de tensão. A expressão das moléculas TNF-, IL-10, RUNX2 e RANK estava diminuída nos grupos 5-LO-/- e MT em relação ao WT e ao VH, respectivamente. No grupo ZN a expressão reduzida de TNF-, IL-10 e RUNX2 foi observada em relação ao grupo VH. In vitro, LTB4 promoveu aumento da diferenciação de osteoclastos, enquanto LTB4 e LTD4 induziram a liberação de TNF-. Nossos resultados demonstram a contribuição dos metabólitos da via 5-LO, LTB4 e CysLTs, no processo de remodelação óssea por afetarem diretamente o recrutamento e diferenciação de osteoclastos. Bone remodeling, induced by mechanical force, results in bone resorption and bone deposition in specific sites and is regulated by the release of inflammatory mediators, among those, arachidonic acid (AA). AA, can be metabolized by 5-lipoxygenase (5-LO) pathway resulting in leukotriene B4 (LTB4) and cysteinyl leukotriene (CysLTs) formation. This metabolites of this pathway have long been studied in inflammatory response and, more recently, in bone remodeling. Thus, the aims of this study were i) develop a bone remodeling model during tooth movement induced by mechanical loading. ii) Investigate the role of 5-LO metabolites in bone remodeling and in osteoclast differentiation and recruitment. First, for ideal mechanical force definition for this model, C57BL/6 mice were submitted to orthodontic forces ranging from 0.1 N to 0.5 N. The ideal orthodontic force, 0.35 N, was used in the following groups: C57BL/6 or SV 129 (Wild Type-WT); 5-LO knockout (5-LO-/-) or treated with vehicle (VH), zileuton (ZN) (5-LO inhibitor) and montelukast (MT) (antagonist of the receptor of CysLTs). The amount of tooth movement and osteoclast numbers were evaluated histomorphometrically. Cytokine and bone remodeling expression was evaluated in compression and tension strains of the periodontium. The effect of 5-LO metabolites in osteoclast differentiation and also in TNF- release were evaluated in vitro in RAW 264.7 cells stimulated with LTB4 and LTD4. The 0.35 N force promoted tooth movement and osteoclast recruitment without root resorption. Tooth movement and osteoclast numbers were diminished in the absence or inhibition of 5-LO and treatment with CysLTs (MT) comparing to the respective WT and VH groups. It was observed the differential expression of cytokine and bone remodeling markers in tension and compression sites. Cathepsin K and TNF- were more expressed in compression site and OCN and RUNX2 in tension site. Moreover, TNF-, IL-10, RUNX2 and RANK expression diminished in 5-LO-/- and MT groups comparing to WT and VH, respectively. Also, TNF-, IL-10 and RUNX2, were diminished in ZN group compared to its VH. In vitro experiment demonstrated that LTB4 promoted more osteoclast differentiation than control and LTB4 and LTD4 increased TNF- release. Our results demonstrated the 5-LO metabolites, LTB4 and CysLTs, contribution to osteoclast recruitment and differentiation and consequently bone remodeling.

Bone remodeling is affected by mechanical loading and inflammatory mediators, including chemokines. The chemokine (C-C motif) ligand 3 (CCL3) is involved in bone remodeling by binding to C-C chemokine receptors 1 and 5 (CCR1 and CCR5) expressed on osteoclasts and osteoblasts. Our group has previously demonstrated that CCR5 down-regulates mechanical loading-induced bone resorption. Thus, the present study aimed to investigate the role of CCR1 and CCL3 in bone remodeling induced by mechanical loading during orthodontic tooth movement in mice. Our results showed that bone remodeling was significantly decreased in CCL3(-/-) and CCR1(-/-) mice and in animals treated with Met-RANTES (an antagonist of CCR5 and CCR1). mRNA levels of receptor activator of nuclear factor kappa-B (RANK), its ligand RANKL, tumor necrosis factor alpha (TNF-α) and RANKL/osteoprotegerin (OPG) ratio were diminished in the periodontium of CCL3(-/-) mice and in the group treated with Met-RANTES. Met-RANTES treatment also reduced the levels of cathepsin K and metalloproteinase 13 (MMP13). The expression of the osteoblast markers runt-related transcription factor 2 (RUNX2) and periostin was decreased, while osteocalcin (OCN) was augmented in CCL3(-/-) and Met-RANTES-treated mice. Altogether, these findings show that CCR1 is pivotal for bone remodeling induced by mechanical loading during orthodontic tooth movement and these actions depend, at least in part, on CCL3.

During orthodontic tooth movement (OTM), alveolar bone is resorbed by osteoclasts in compression sites (CS) and is deposited by osteoblasts in tension sites (TS). The aim of this study was to develop a standardized OTM protocol in mice and to investigate the expression of bone resorption and deposition markers in CS and TS. An orthodontic appliance was placed in C57BL6/J mice. To define the ideal orthodontic force, the molars of the mice were subjected to forces of 0.1N, 0.25N, 0.35N and 0.5N. The expression of mediators that are involved in bone remodeling at CS and TS was analyzed using a Real-Time PCR. The data revealed that a force of 0.35N promoted optimal OTM and osteoclast recruitment without root resorption. The levels of TNF-α, RANKL, MMP13 and OPG were all altered in CS and TS. Whereas TNF-α and Cathepsin K exhibited elevated levels in CS, RUNX2 and OCN levels were higher in TS. Our results suggest that 0.35N is the ideal force for OTM in mice and has no side effects. Moreover, the expression of bone remodeling markers differed between the compression and the tension areas, potentially explaining the distinct cellular migration and differentiation patterns in each of these sites.