Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Resumo Introdução Durante o reparo de enxertos ósseos, diferentes taxas de reabsorção são mediadas pelos osteoclastos e podem ser afetadas pelos bisfosfonatos, que são drogas que agem como inibidores da reabsorção óssea. Objetivo Avaliar a taxa de reabsorção dos enxertos ósseos de calota craniana de coelhos, com e sem o uso alendronato de sódio. Material e método Trinta e dois coelhos Nova Zelândia foram divididos igualmente em 2 grupos (grupo controle e grupo alendronato de sódio) e subdivididos em 4 períodos (7, 14, 30 e 60 dias). O grupo controle não recebeu alendronato, enquanto os animais do grupo experimental receberam 4 mg de alendronato de sódio por semana, em dose única, após a cirurgia. Um bloco de osso de diâmetro de 8 mm foi retirado o osso parietal e fixado com parafuso no osso parietal contralateral. Após cirurgia, nos períodos de 7, 14, 30 e 60 dias, os animais foram eutanasiados e as peças removidas para análise. Análises morfológica e histomorfométrica foram utilizados para comparar a espessura do enxerto e para avaliar a interface de osso recém formado entre o enxerto ósseo e o sítio receptor. Os testes de Wilcoxon e Mann-Whitney foram utilizados para as análises estatísticas. Resultado Todos os enxertos repararam e integraram sem intercorrências; não foram detectadas diferenças estatisticamente significativas nas taxas de reabsorção ou deposição óssea, após a incorporação final do enxerto em ambos os grupos. Conclusão Alendronato de sódio parece não diminuir a taxa de reabsorção, porém houve uma tendência de resultados melhores no grupo controle tanto na reabsorção quanto na neoformação óssea em enxertos ósseos autógenos de calota craniana de coelhos.

Abstract Introduction Different rates of resorption are mediated by osteoclasts that may be affected by bisphosphonates during bone graft repair. Bisphosphonates are drugs that act as inhibitors of bone resorption. Objective The aim of the present study was to evaluate the rate of resorption of skullcap grafts in rabbits with and without the use of alendronate sodium. Material and method Thirty two New Zealand rabbits were divided into two groups (control group and alendronate group) and divided again into four periods (7, 14, 30 and 60 days). The control group did not receive alendronate, while animals of the experimental group received 4 mg of alendronate sodium weekly after the surgery. An 8 mm diameter bone block was removed from the parietal bone and fixed by screws to the contralateral parietal bone. During the periods of 7, 14, 30 and 60 days, the animals had undergone euthanasia and samples were removed for further analysis. Morphological and histomorphometric tests were used to compare graft thicknesses and to evaluate the newly formed bone at the interface between the graft and receptor site. The Wilcoxon and Mann-Whitney tests were used for statistical analyses. Result All grafts healed and integrated uneventfully and no statistically significant differences in resorption rates or bone deposition were detected after the final incorporation of the graft in both groups. Conclusion Alendronate Sodium did not decrease the bone graft resorption rates, but there was a tendency for better results in the control group regarding the resorption and neoformation in autogenous calvarial bone grafts in rabbits.

Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas. Methods: The expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas. Results: The expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05). Conclusion: There is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread cytokine expression seems to be a feature of chronic lesions, hierarchical cluster analysis demonstrates the association of TNF-α, IL-21, IL-17 and IFN-γ with lesions activity, and the association of FOXP3, IL-10, IL-9, IL-4 and IL-22 with lesions inactivity.