Abstract Objectives This study aimed to investigate the osseointegration of titanium (Ti) implants with micro-nano textured surfaces functionalized with strontium additions (Sr) in a pre-clinical rat tibia model. Methodology Ti commercially pure (cp-Ti) implants were installed bilaterally in the tibia of 64 Holtzman rats, divided into four experimental groups (n=16/group): (1) Machined surface - control (C); (2) Micro-nano textured surface treatment (MN); (3) Micro-nano textured surface with Sr2+ addition (MNSr); and (4) Micro-nano textured surface with a higher complementary addition of Sr2+ (MNSr+). In total, two experimental euthanasia periods were assessed at 15 and 45 days (n=8/period). The tibia was subjected to micro-computed tomography (μ-CT), histomorphometry with the EXAKT system, removal torque (TR) testing, and gene expression analysis by PCR-Array of 84 osteogenic markers. Gene expression and protein production of bone markers were performed in an in vitro model with MC3T3-E1 cells. The surface characteristics of the implants were evaluated by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and laser scanning confocal microscopy. Results SEM, confocal, and EDS analyses demonstrated the formation of uniform micro-nano textured surfaces in the MN group and Sr addition in the MNSr and MNSr+ groups. TR test indicated greater osseointegration in the 45-day period for treated surfaces. Histological analysis highlighted the benefits of the treatments, especially in cortical bone, in which an increase in bone-implant contact was found in groups MN (15 days) and MNSr (45 days) compared to the control group. Gene expression analysis of osteogenic activity markers showed modulation of various osteogenesis-related genes. According to the in vitro model, RT-qPCR and ELISA demonstrated that the treatments favored gene expression and production of osteoblastic differentiation markers. Conclusions Micro-nano textured surface and Sr addition can effectively improve and accelerate implant osseointegration and is, therefore, an attractive approach to modifying titanium implant surfaces with significant potential in clinical practice. (Ti micronano micro nano (Sr preclinical pre cpTi cp (cp-Ti 6 rats n=16/group n16group ngroup n 16 (n=16/group) 1 (1 C (C) 2 (2 Micronano Micro (MN) 3 (3 Sr2 (MNSr) 4 (4 MNSr+. . (MNSr+) total n=8/period. n8period nperiod n=8/period 8 (n=8/period) microcomputed computed μCT, μCT μ CT , (μ-CT) system (TR testing PCRArray PCR Array MC3T3E1 MCTE MC3T3 E1 MC T E MC3T3-E cells SEM (SEM) energydispersive energy dispersive EDS, (EDS) 45day day boneimplant osteogenesisrelated osteogenesis related genes RTqPCR RT qPCR is therefore practice (n=16/group ( (C (MN (MNSr (MNSr+ (n=8/period (μ-CT MC3T3E MCT MC3T (SEM (EDS

Resumo A autofagia é um mecanismo de autodigestão das células, que está relacionado ao estresse celular. Ela permite a sobrevivência das células, mantendo a homeostase celular ou iniciando a morte celular. O objetivo deste estudo foi investigar a sinalização intracelular da regulação da autofagia induzida por pressão nas células do ligamento periodontal humano (PDL) e analisar o envolvimento de Rho kinases (ROCK) e espécies reativas de oxigênio (ROS) em particular. As células do ligamento periodontal humano foram tratadas com o inibidor de ROCK Y-27632 e o eliminador de ROS N-acetilcisteína (NAC) em combinação com magnitudes de pressão de 2, 6 e 8 g/cm2 durante 16 horas. As células tratadas com rapamicina serviram como controle positivo e as células não tratadas como grupo de controle. O kit de detecção de autofagia Cyto-ID® foi usado para análise de citometria de fluxo. A análise estatística foi realizada usando ANOVA e testes post-hoc. Os resultados mostram que a autofagia induzida por pressão foi afetada de forma diferente pelos dois inibidores (p<0,05). A aplicação do Y-27632 levou a uma redução significativa da autofagia em todos os grupos de pressão. A aplicação de NAC levou à redução da autofagia nas pressões de 2 g/cm2 e 6 g/cm2. A 8 g/cm2, esse efeito não estava mais presente. No grupo de controle, a autofagia foi significativamente reduzida pelo Y-27632 e significativamente aumentada pela NAC. Nossos dados sugerem que tanto a Rho-kinase quanto as espécies reativas de oxigênio podem influenciar a regulação da autofagia induzida pela pressão nas células PDL. PDL (PDL (ROCK (ROS particular Y27632 Y 27632 Y-2763 Nacetilcisteína N acetilcisteína (NAC gcm2 gcm g cm2 cm g/cm 1 horas CytoID® CytoID Cyto ID® ID Cyto-ID fluxo posthoc. posthoc post hoc. hoc post-hoc p<0,05. p005 p p<0,05 . 0 05 (p<0,05) presente Rhokinase kinase Y2763 2763 Y-276 p00 p<0,0 (p<0,05 Y276 276 Y-27 p0 p<0, (p<0,0 Y27 27 Y-2 p<0 (p<0, Y2 Y- p< (p<0 (p< (p

Abstract Autophagy is a self-digestion mechanism of cells, which is related to cell stress. It enables cell survival by maintaining cellular homeostasis or initiates cell death. This study aimed to investigate the intracellular signaling of pressure-induced autophagy regulation in human periodontal ligament (PDL) cells and to analyze the involvement of Rho kinases (ROCK) and reactive oxygen species (ROS) in particular. Human PDL cells were treated with the ROCK inhibitor Y-27632 and the ROS scavenger N-acetylcysteine (NAC) in combination with pressure magnitudes of 2, 6, and 8 g/cm2 over 16 hours. Cells treated with rapamycin served as a positive control and untreated cells as a control group. The Cyto-ID® Autophagy Detection Kit was used for flow cytometric analysis. Statistical analysis was performed using ANOVA and post-hoc tests. The results show that the pressure-induced autophagy was affected differently by the two inhibitors (p<0.05). The application of Y-27632 led to a significant reduction in autophagy in all pressure groups. The application of NAC led to reduced autophagy at pressures of 2 g/cm2 and 6 g/cm2. At 8 g/cm2, this effect was no longer present. In the control group, autophagy was significantly reduced by Y-27632 and significantly increased by NAC. Our data suggest that both Rho-kinase and reactive oxygen species could influence pressure-induced autophagy regulation in PDL cells. selfdigestion self digestion stress death pressureinduced induced (PDL (ROCK (ROS particular Y27632 Y 27632 Y-2763 Nacetylcysteine N acetylcysteine (NAC gcm2 gcm g cm2 cm g/cm 1 hours group CytoID® CytoID Cyto ID® ID Cyto-ID posthoc post hoc tests p<0.05. p005 p p<0.05 . 0 05 (p<0.05) groups present Rhokinase kinase Y2763 2763 Y-276 p00 p<0.0 (p<0.05 Y276 276 Y-27 p0 p<0. (p<0.0 Y27 27 Y-2 p<0 (p<0. Y2 Y- p< (p<0 (p< (p

Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.