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INTRODUCTION: Basic research in orthodontics is commonly conducted in rodents. However, experimental studies on orthodontic tooth movement (OTM) lack a standard method to examine OTM and periodontal changes. This study describes a unifying protocol for the analysis of OTM and associated bone microarchitectural changes in mice using microcomputed tomography (µCT). METHODS: Mice (10 animals/group) were divided into control and OTM groups. OTM was generated by anchoring a nickel-titanium closed-coil spring to the upper incisors to pull the upper left first molar. A third group of TNFαâ-/- mice was added since these are known to have slower OTM. Using µCT, we implemented and tested a number of methods to measure OTM distance and examine 3D bone morphometric parameters associated with OTM in mice. RESULTS: In total, we tested five methods to measure the OTM distance in mice. The results indicated that measuring the intermolar diastema, and assessing tooth movement relative to the anterior root of the zygomatic arch, displayed the lowest standard deviation and enabled optimal detection of intergroup differences. We also developed two protocols for µCT analysis of the periradicular bone that yielded no false-positive results. Our results revealed that including the width of the periodontal ligament rather than excluding it from the region of interest in mice detected more statistically significant differences in the morphometric parameters between the OTM and control sides and between WT and TNFαâ-/- mice despite more subtle differences. CONCLUSIONS: We, therefore, propose new guidelines for a standardized µCT-based method to analyse OTM and the extent of the periradicular bone structural changes in mice.
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Osteoclastos , Técnicas de Movimentação Dentária , Animais , Remodelação Óssea , Humanos , Camundongos , Ligamento Periodontal/diagnóstico por imagem , Técnicas de Movimentação Dentária/métodos , Microtomografia por Raio-XRESUMO
INTRODUCTION: Force application (FA) during orthodontic tooth movement is mediated through periodontal ligament (PDL) fibroblasts. FA on deciduous teeth has an inherent risk of root resorption, which is less in permanent teeth. Currently, the root resorption mechanism is poorly understood. We hypothesized that FA alters the morphology and gene expression of PDL fibroblasts. This study was designed to achieve homogenous PDL fibroblast cultures, establish an in-vitro FA model, analyze fibroblast morphology after FA, and compare the gene expressions of PDL fibroblasts of deciduous and permanent teeth after FA. METHODS: Fibroblasts were sorted from primary cultures of deciduous and permanent tooth PDLs. Cell viability was evaluated in the Opticell (Thermo Scientific, Waltham, Mass) FA model. Cellular morphology was analyzed using immunofluorescence staining for actin and focal adhesion complexes. Gene expressions of untreated or pressure-treated PDL fibroblasts of deciduous and permanent teeth were compared by gene array and confirmed by real-time polymerase chain reaction. RESULTS: Cell sorting resulted in cultures containing 98% of PDL fibroblasts. The Opticell model showed 94% cell survival after FA. FA increased fibroblasts' adhesion. Gene arrays and real-time polymerase chain reactions indicated greater up-regulation of DKK2 mRNA in untreated PDL fibroblasts of deciduous teeth and greater up-regulation of ADAMTS1 mRNA in pressurized PDL fibroblasts of deciduous and permanent teeth. CONCLUSIONS: Cell sorting is an efficient method to establish homogenous PDL fibroblast cultures. Using the Opticell FA model allows the maintenance of excellent cell viability. FA increased the surface adherence of fibroblasts. Up-regulation of ADAMTS1 after FA may indicate its involvement in the remodeling of the periodontium during orthodontic tooth movement. Understanding root resorption mechanisms under FA will help to prevent it during orthodontic treatment.
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Fibroblastos/citologia , Fibroblastos/fisiologia , Expressão Gênica , Ligamento Periodontal/citologia , Pressão , Técnicas de Movimentação Dentária , Proteína ADAMTS1/genética , Adolescente , Adulto , Sobrevivência Celular , Células Cultivadas , Criança , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , RNA Mensageiro/genética , Dente/fisiologia , Dente Decíduo/fisiologia , Regulação para Cima , Adulto JovemRESUMO
Orthodontic tooth movement (OTM) is a "sterile" inflammatory process. The present study aimed to reveal the underlying biological mechanisms, by studying the force associated-gene expression changes, in a time-dependent manner. Ni-Ti springs were set to move the upper 1st-molar in C57BL/6 mice. OTM was measured by µCT. Total-RNA was extracted from tissue blocks at 1,3,7 and 14-days post force application, and from two control groups: naïve and inactivated spring. Gene-expression profiles were generated by next-generation-RNA-sequencing. Gene Set Enrichment Analysis, K-means algorithm and Ingenuity pathway analysis were used for data interpretation. Genes of interest were validated with qRT-PCR. A total of 3075 differentially expressed genes (DEGs) were identified, with the greatest number at day 3. Two distinct clusters patterns were recognized: those in which DEGs peaked in the first days and declined thereafter (tissue degradation, phagocytosis, leukocyte extravasation, innate and adaptive immune system responses), and those in which DEGs were initially down-regulated and increased at day 14 (cell proliferation and migration, cytoskeletal rearrangement, tissue homeostasis, angiogenesis). The uncovering of novel innate and adaptive immune processes in OTM led us to propose a new term "Immunorthodontics". This genomic data can serve as a platform for OTM modulation future approaches.
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Expressão Gênica , Dente Molar/imunologia , Animais , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ortodontia , RNA/genética , RNA/imunologia , Técnicas de Movimentação DentáriaRESUMO
BACKGROUND: The aim of the study is to examine bone healing following augmentation with allograft or ß-tricalcium phosphate (ß-TCP) and evaluate orthodontic tooth movement (OTM) into the augmented sites. METHODS: The study included two parts. Part I included the alveolar bone regeneration model. Osseous defects were created by extraction of the maxillary first molars in C57BL/6 mice, and the sockets were filled with allograft, ß-TCP, or left unfilled (n = 6/group). Mouse allograft was prepared by a novel method using long bones. Maxillae were collected at 2, 4, and 6 weeks for microcomputed tomography (µCT) and histological analysis. In Part II, OTM was performed after full bone healing, through grafted and unfilled sockets (n = 10/group), and the second molar shift was assessed using µCT. RESULTS: Bone volume and trabeculation were reduced in ß-TCP compared with allograft and non-grafted groups at 2 and 4 weeks post-grafting, but similar at 6 weeks. Graft particles could be detected at 2 weeks post-grafting for ß-TCP, and at 2 and 4 weeks for allograft. Increased osteoclasts' presence was observed in the ß-TCP group at 2 and 4 weeks compared with allograft and control. OTM was similar in the two graft groups, but impaired versus the non-grafted controls. CONCLUSION: ß-TCP and allograft induce full normal healing but alter OTM into the regenerated sites.
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BACKGROUND: The aim of this study was to investigate the biological mechanisms underlying alveolar bone regeneration (ABR) and orthodontic tooth movement into bovine bone (BB) regenerated sites. METHODS: Two mouse models were established in C57BL/6 mice. The ABR model was based on osseous defects filled with BB. The orthodontic tooth movement-ABR model was used to move a molar into the regenerated site. Osseous morphometric analysis and tooth movement distance were evaluated with micro-CT. Histologic characteristics and osteoclast (OCS) accumulation were evaluated by hematoxylin and eosin and tartrate-resistant acid phosphatase staining (TRAP). Expression and location of the receptor activator of nuclear factor-kappa B (RANKL) and of osteoprotegerin (OPG) were evaluated by immunofluorescent staining. RESULTS: Bone healing peaked at 4 weeks. The distance of the orthodontic tooth movement into the bovine bone was significantly reduced versus that of the nonbovine bone controls. BB particles accumulated along the root's pressure side during orthodontic treatment. Despite the osteoclasts' presence adjacent to the BB particles, no BB resorption was observed. Increased RANKL expression was seen at the orthodontic tooth movement pressure zone, without any change in OPG expression. CONCLUSION: The two novel mouse models show that the lack of resorption of BB xenografts renders them inadequate for proper orthodontic tooth movement at a later stage.
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Osteoprotegerina , Técnicas de Movimentação Dentária , Animais , Regeneração Óssea , Bovinos , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos , Ligante RANK , Fosfatase Ácida Resistente a Tartarato , CicatrizaçãoRESUMO
Tissue damage in chronic periodontal disease is driven by the host response to a dysbiotic microbiota, and not by bacteria directly. Among chronic inflammatory diseases of the oral cavity, inflammation and tissue damage around dental implants (peri-implantitis) is emerging as a major clinical challenge, since it is more severe and less responsive to treatment compared to inflammation around natural teeth. We tested whether oral fibroblasts from the periodontal ligament (PDLF), which are present around natural teeth but not around dental implants, actively regulate inflammatory responses to bacterial stimulation. We show that human PDLF down-regulate TNF-α post-transcriptionally in macrophages stimulated with the oral pathogen Porphyromonas gingivalis. Cell contact and secretion of IL-6 and IL-10 contribute to the modulation of inflammatory cytokine production. Although fibroblasts decreased TNF-α secretion, they enhanced the ability of macrophages to phagocytose bacteria. Surprisingly, donor matched oral fibroblasts from gingival tissues, or fibroblasts from peri-implant inflamed tissues were at least as active as PDLF in regulating macrophage responses to bacteria. In addition, priming fibroblasts with inflammatory mediators enhanced PDLF regulatory activity. A further understanding of the spectrum of fibroblast activities in inflammatory lesions is important in order to design ways to control inflammatory tissue damage.