miR-20a: a key regulator of orthodontic tooth movement via BMP2 signaling pathway modulation.

AIM: In this study, we aimed to establish a rat tooth movement model to assess miR-20's ability in enhancing the BMP2 signaling pathway and facilitate alveolar bone remodeling. METHOD: 60 male SD rats had nickel titanium spring devices placed between their left upper first molars and incisors, with the right side serving as the control. Forces were applied at varying durations (18h, 24h, 30h, 36h, 42h, 1d, 3d, 5d, 7d, 14d), and their bilateral maxillary molars and surrounding alveolar bones were retrieved for analysis. Fluorescent quantitative PCR was conducted to assess miR-20a, BMP2, Runx2, Bambi and Smad6 gene expression in alveolar bone, and western blot was performed to determine the protein levels of BMP2, Runx2, Bambi, and Smad6 after mechanical loading. RESULT: We successfully established an orthodontic tooth movement model in SD rats and revealed upregulated miR-20a expression and significantly increased BMP2 and Runx2 gene expression and protein synthesis in alveolar bone during molar tooth movement. Although Bambi and Smad6 gene expression did not significantly increase, their protein synthesis was found to decrease significantly. CONCLUSION: MiR-20a was found to be involved in rat tooth movement model alveolar bone remodeling, wherein it promoted remodeling by reducing Bambi and Smad6 protein synthesis through the BMP2 signaling pathway.

Oxytocin alleviates periodontitis in adult rats.

OBJECTIVE: The objective of the study was to evaluate the expression of oxytocin receptors in normal and inflamed gingiva, as well as the effects of systemic administration of oxytocin in bone loss and gum inflammatory mediators in a rat model of experimental periodontitis. BACKGROUND DATA: Current evidence supports the hypothesis of a disbalance between the oral microbiota and the host's immune response in the pathogenesis of periodontitis. Increased complexity of the microbial biofilm present in the periodontal pocket leads to local production of nitrogen and oxygen-reactive species, cytokines, chemokines, and other proinflammatory mediators which contribute to periodontal tissue destruction and bone loss. Oxytocin has been suggested to participate in the modulation of immune and inflammatory processes. We have previously shown that oxytocin, nitric oxide, and endocannabinoid system interact providing a mechanism of regulation for systemic inflammation. Here, we aimed at investigating not only the presence and levels of expression of oxytocin receptors on healthy and inflamed gingiva, but also the effects of oxytocin treatment on alveolar bone loss, and systemic and gum expression of inflammatory mediators involved in periodontal tissue damage using ligature-induced periodontitis. Therefore, anti-inflammatory strategies oriented at modulating the host's immune response could be valuable adjuvants to the main treatment of periodontal disease. METHODS: We used an animal model of ligature-induced periodontitis involving the placement of a linen thread (Barbour flax 100% linen suture, No. 50; size 2/0) ligature around the neck of first lower molars of adult male rats. The ligature was left in place during the entire experiment (7 days) until euthanasia. Animals with periodontitis received daily treatment with oxytocin (OXT, 1000 µg/kg, sc.) or vehicle and/or atosiban (3 mg/kg, sc.), an antagonist of oxytocin receptors. The distance between the cement-enamel junction and the alveolar bone crest was measured in stained hemimandibles in the long axis of both buccal and lingual surfaces of both inferior first molars using a caliper. TNF-α levels in plasma were determined using specific rat enzyme-linked immunosorbent assays (ELISA). OXT receptors, IL-6, IL-1ß, and TNF-α expression were determined in gingival tissues by semiquantitative or real-time PCR. RESULTS: We show that oxytocin receptors are expressed in normal and inflamed gingival tissues in male rats. We also show that the systemic administration of oxytocin prevents the experimental periodontitis-induced increased gum expression of oxytocin receptors, TNF-α, IL-6, and IL-1ß (p < .05). Furthermore, we observed a reduction in bone loss in rats treated with oxytocin in our model. CONCLUSIONS: Our results demonstrate that oxytocin is a novel and potent modulator of the gingival inflammatory process together with bone loss preventing effects in an experimental model of ligature-induced periodontitis.

IFT80 promotes early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone-derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1Cre;IFT80f/f). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP-positive osteoblastic activity, TRAP-positive osteoclastic activity, and OSX-/COL I-/OCN-positive areas in tooth extraction sockets of Prx1Cre; IFT80f/f mice compared with IFT80f/f littermates. Furthermore, aBMSCs from Prx1Cre; IFT80f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1Cre; IFT80f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway.

The relationship between the uptake of alveolar bone inflammation and of cervical lymph nodes on fluoro-2-deoxy-D-glucose positron emission tomography.

OBJECTIVES: To elucidate the relationships between the maximum standardized uptake value (SUVmax) of alveolar bone and those of lymph nodes (LNs) around the neck on 18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET). METHODS: The SUVmax values of alveolar bone and of level IA, level IB, and level IIA LNs of 174 patients, including those with and without active odontogenic inflammation, on PET/CT performed for a health check were retrospectively evaluated. The upper and lower jaws were divided into four blocks (right maxilla, left maxilla, right mandible, and left mandible). The SUVmax values of each block and of the LNs were calculated. The differences in the SUVmax of each LN level between patients with and without odontogenic inflammation, and the relationship between the SUVmax values of alveolar bone and of the LNs were analysed statistically. RESULTS: Significant differences in SUVmax values of bilateral level IB and IIA LNs were found between patients with and without odontogenic inflammation (Mann-Whitney U test: right level IB, P = .008; left level IB, P = .006; right level IIA, P < .001; left level IIA, P = .002), but not in bilateral level IA LNs (Mann-Whitney U test: right level IA, P = .432; left level IA, P = .549). The inflammatory site with the highest SUVmax in level IB LNs was the ipsilateral mandible (multivariate analysis: right, beta = 0.398, P < .001; left, beta = 0.472, P < .001), and the highest SUVmax in level IIA LNs was the ipsilateral maxilla (multivariate analysis: right, beta = 0.223, P = .002; left, beta = 0.391, P < .001). CONCLUSIONS: The SUVmax values of level IB and IIA LNs were associated with a tendency towards a higher SUVmax value of alveolar bone on 18F-FDG-PET.

Spatio-temporal immunolocalization of VEGF-A, Runx2, and osterix during the early steps of intramembranous ossification of the alveolar process in rat embryos.

Vascular endothelial growth factor A (VEGF-A) is expressed by several cell types and is a crucial factor for angiogenic-osteogenic coupling. However, the immunolocalization of VEGF-A during the early stages of the alveolar process formation remains underexplored. Thus, we analyzed the spatio-temporal immunolocalization of VEGF-A and its relationship with Runt-related transcription factor 2 (Runx2) and osterix (Osx) during the early steps of intramembranous ossification of the alveolar process in rat embryos. Embryo heads (E) of 16, 18 and 20-day-old rats were processed for paraffin embedding. Histomorphometry and immunohistochemistry to detect VEGF-A, Runx2, and Osx (osteoblast differentiation markers) were performed. The volume density of bone tissue including bone cells and blood vessels increased significantly in E18 and E20. Cells showing high VEGF-A immunoreactivity were initially observed within a perivascular niche in the ectomesenchyme; afterwards, these cells were diffusely located near bone formation sites. Runx2-and Osx-immunopositive cells were observed in corresponded regions of cells showing strong VEGF-A immunoreactivity. Although these immunostained cells were observed in all specimens, this immunolocalization pattern was more evident in E16 specimens and gradually decreased in E18 and E20 specimens. Double immunofluorescence labelling showed intracellular co-localization of Osx and VEGF-A in cells surrounding the developing alveolar process, indicating a crucial role of VEGF-A in osteoblast differentiation. Our results showed VEGF-A immunoexpression in osteoblasts and its precursors during the maxillary alveolar process formation of rat embryos. Moreover, the VEGF-A-positive cells located within a perivascular niche at the early stages of the alveolar process development suggest a crosstalk between endothelium and ectomesenchymal cells, reinforcing the angiogenic-osteogenic coupling in this process.

Roles and mechanisms of YAP/TAZ in orthodontic tooth movement.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators encoded by paratactic homologous genes, shuttle-crossing between cytoplasm and nucleus to regulate the gene expression and cell behavior and standing at the center place of the sophisticated regulatory networking of mechanotransduction. Orthodontic tooth movement (OTM) is a process in which extracellular mechanical stimuli are transformed into intracellular biochemical signals to regulate cellular responses and tissue remodeling. Literature studies have confirmed that YAP/TAZ plays an important role not only in embryonic development, homeostasis and tumorigenesis, but also in mechanical-biochemical signal transduction of periodontal tissues under the mediation of various signal molecules in its upstream and downstream. Herein, we review the advances in the roles and mechanisms of YAP/TAZ in OTM to provide insights for better understanding and further study of the OTM and possible targeted clinical intervention in orthodontic treatment.

Effect of VEGFC on lymph flow and inflammation-induced alveolar bone loss.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1)+ /podoplanin (PDPN)+ lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)-associated periodontitis and ligature-induced periodontitis. Further, PDPN+ /alpha-smooth muscle actin (αSMA)- lymphatic capillaries are increased, whereas PDPN+ /αSMA+ collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP-positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno-associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN+ /αSMA+ collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1+ /αSMA- lymphatic capillaries are increased, whereas LYVE1+ /αSMA+ collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation-mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.

Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 µm in diameter, lacunae around 10-20 µm as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.

Study of bone remodeling in corticotomy-assisted orthodontic tooth movement in rats.

The goal of this study was to determine the structure change of the alveolar bone and the expression of a group of bone remodeling-related factors. Sixty healthy male Wistar rats were randomly divided into three groups. Selective alveolar decortication (SAD), tooth movement (TM), and "combined therapy" (SAD+TM) was performed in group I, II, and III, respectively. On days 0, 7, 14, 21, and 42, a Micro-CT scan was performed on the maxillary alveolar bone and tooth. In addition, on days 0, 7, 14, 21, 28, and 42, some of the rats were killed by cervical dislocation and tissues were harvested. Analysis of scan data revealed a significant decrease in bone density of the alveolar bone at 14 days post-surgery, and increased at 42 days post-surgery to a level higher than that before the surgery. Microarray and bioinformatics analysis were performed to explore gene expression profile in three groups (SAD, TM, and SAD+TM), and a large number of differentially expressed genes were identified. In addition, real-time polymerase chain reaction was performed to determine the expression of bone remodeling-related factors. The expression of osteoblast-related cytokines, including osteopontin, bone sialoprotein, and osteocalcin, and osteoclast regulators macrophage-colony stimulating factor (M-CSF) and RANKL (activator of nuclear factor KB receptor ligand) were increased in group III, suggesting that there was increased bone synthesis and activation of bone absorption. Moreover, group III had a unique alveolar bone remodeling pattern: RANKL and osteoprotegerin-promoted alveolar remodeling. In conclusion, during the early stage of orthodontic tooth movement, corticotomy can accelerate the movement of teeth, modulate the state of bone metabolism, and activate osteogenesis and osteoclast, which support the theory of regional acceleratory phenomenon.

Differential osteopontin expression in human osteoblasts derived from iliac crest and alveolar bone and its role in early stages of angiogenesis.

In our previous study, we revealed significant differences of osteopontin (OPN) gene expression in primary human osteoblasts (HOBs) derived from iliac crest bone (iHOBs) and alveolar bone (aHOBs). The present study aims at assigning this discriminative expression to a possible biologic function. OPN is known to be involved in several pathologic and physiologic processes, among others angiogenesis. Therefore, we studied the reaction of human umbilical vein endothelial cells (HUVECs) to HOB-derived OPN regarding angiogenesis. To this end, human primary explant cultures of both bone entities from ten donors were established. Subsequent transcription analysis detected higher gene expression of OPN in iHOBs compared to aHOBs, thereby confirming the results of our previous study. This difference was particularly apparent when cultures were derived from female donors. Hence, OPN protein expression as well as the angiogenic potential of OPN was analyzed, originating from HOBs of one female donor. In accordance to the gene expression level, secreted OPN was more abundant in the supernatant of iHOBs than in aHOBs. Moreover, secreted OPN was found to stimulate migration of HUVECs, but not proliferation or tube formation. These results indicate an involvement in very early stages of angiogenesis and a functional distinction of OPN from HOBs derived from different bone entities.

Influence of omega-3 fatty acid on orthodontic tooth movement in rats: A biochemical, histological, immunohistochemical and gene expression study.

OBJECTIVE: The aim of this study was to investigate the effects of omega-3 fatty acids on orthodontic tooth movement. SETTING AND SAMPLE POPULATION: For this study, 56 12-week-old adult male Wistar albino rats from the Animal Laboratory at Adnan Menderes University, Faculty of Medicine, were used. MATERIAL AND METHODS: Rats were randomly divided into seven groups (n = 8 each): control group (without any treatment), tooth movement groups (three groups of animals with only tooth movement) and omega groups (three groups of animals with tooth movement and omega-3 administration). Omega-3 fatty acids were administered to the rats systemically during the tooth movement period. On the 3rd, 7th and 14th days after the orthodontic tooth movement, the rats were sacrificed and biochemical, histological, immunohistochemical andgene expression examinations were performed. RESULTS: On the 14th experimental day, the amount of tooth movement in the omega groups was significantly lower than the tooth movement groups (P = 0.012). Biochemical experimentsshowed that the omega groups had significantly lower total oxidant levels and higher total antioxidant levels compared to the tooth movement group on the 14th experimental day (P = 0.001). The levels of RANKL, IL-6 and IL-1ß in the omega groups were significantly lower than the tooth movement groups on all experimental days (P < 0.05). CONCLUSION: Systemic administration of omega-3 fatty acids showed antioxidant and antiinflammatory effects and decelerate the orthodontic tooth movement.

Interaction between the Wnt/ß-catenin signaling pathway and the EMMPRIN/MMP-2, 9 route in periodontitis.

BACKGROUND AND OBJECTIVES: In periodontitis, the Wnt/ß-catenin signaling pathway is related to the metabolism of the alveolar bone; further, extracellular matrix metalloproteinase inducer (EMMPRIN) expression is correlated with matrix metalloproteinases (MMPs) expression and inflammation severity. The aim of this study was to perform a preliminary investigation of the interaction between the Wnt/ß-catenin signaling pathway and the EMMPRIN/MMPs route in periodontitis. MATERIAL AND METHODS: Chronic periodontitis and healthy gingival tissues were obtained to detect the expression of Wnt3a, ß-catenin, EMMPRIN and MMP-2, 9 by using immunohistochemical analysis. The human immortalized oral epithelial cell/human gingival fibroblast direct co-culture model was treated with 10 µg/mL Porphyromonas gingivalis lipopolysaccharide (Pg. LPS). Anti-EMMPRIN antibody was used to block the effect of EMMPRIN. Dickkopf-1 (DKK-1) and Wnt3a were used as the inhibitor and activator of the Wnt/ß-catenin signaling pathway, respectively. Immunofluorescence was performed to visualize the localization of ß-catenin and EMMPRIN. Expression of the EMMPRIN, MMP-2, 9 and Wnt pathway's components was confirmed by western blotting and quantitative real-time polymerase chain reaction. RESULTS: Higher levels of Wnt3a, ß-catenin, EMMPRIN and MMP-2, 9 were observed in chronic periodontitis gingival tissues compared with controls. Pg. LPS significantly enhanced ß-catenin, p-GSK-3ß, EMMPRIN and MMP-2, 9 inductions in the human immortalized oral epithelial cell/human gingival fibroblast co-culture model. Anti-EMMPRIN antibody markedly reduced the expression of MMP-2, 9 only in the presence of Pg. LPS. Co-expression of ß-catenin and EMMPRIN was detected in the co-culture model. DKK-1 inhibited Wnt pathway, but upregulated the EMMPRIN/MMP-2, 9 routes. In contrast, activating Wnt pathway by Wnt3a repressed the EMMPRIN/MMP-2, 9 routes. The promotion effect of DKK-1 on MMP-2, 9 expressions was partially inhibited by the anti-EMMPRIN antibody. In addition, anti-EMMPRIN antibody led to a drastic decrease in ß-catenin and p-GSK-3ß. CONCLUSION: In periodontitis, EMMPRIN regulates MMP-2, 9 expressions, the activation of Wnt/ß-catenin signaling pathway downregulates the EMMPRIN/MMP-2, 9 routes and the blockade of EMMPRIN attenuates Wnt/ß-catenin signaling pathway.

Functions of Periostin in dental tissues and its role in periodontal tissues' regeneration.

The goal of periodontal regenerative therapy is to predictably restore the tooth's supporting periodontal tissues and form a new connective tissue attachment of periodontal ligament (PDL) fibers and new alveolar bone. Periostin is a matricellular protein so named for its expression primarily in the periosteum and PDL of adult mice. Its biological functions have been widely studied in areas such as cardiovascular physiology and oncology. Despite being initially identified in the dental tissues and bone, investigations of Periostin functions in PDL and alveolar-bone-related physiopathology are less abundant. Recently, several studies have suggested that Periostin may be an important regulator of periodontal tissue formation. By promoting collagen fibrillogenesis and the migration of fibroblasts and osteoblasts, Periostin might play a pivotal part in regeneration of the PDL and alveolar bone following periodontal surgery. The aim of this article is to provide an extensive review of the implications of Periostin in periodontal tissue biology and its potential use in periodontal tissue regeneration.

Clastic cells are absent around the root surface in pulp-exposed periapical periodontitis lesions in mice.

INTRODUCTION: Clastic cells, originating from the monocyte-macrophage lineage, resorb mineralized tissues. In periapical periodontitis, alveolar bone around the tooth apex becomes resorbed; however, the roots of the teeth are often left intact by yet unknown mechanisms. Here, we examined the status of clastic cells in a periapical periodontitis model in mice. METHODS: Periapical periodontitis was induced by performing pulp exposure on the maxillary first molar. The contralateral maxillary first molar was used as a control. The maxillae were harvested, fixed, and subjected to µCT scanning and three-dimensional volumetric analysis. TRAP staining was performed, and osteoclasts were quantified. Immunohistochemical staining was performed for RANKL, OPG, and F4/80, a marker for macrophages. RESULTS: At the apex of the tooth, pulp exposure resulted in periapical radiolucency with mineralized tissues at the surrounding bone surfaces but not on the root surfaces. Histologically, clastic cells were present on the bone surfaces but absent around the root surfaces. Expression of F4/80 and RANKL was not found at close proximity to the root surfaces, but OPG was globally expressed. CONCLUSION: The absence of clastic cells around the root surface of pulp-exposed teeth, in part, is associated with the lack of macrophages and RANKL expression.

Biological Effects of Orthodontic Tooth Movement Into the Grafted Alveolar Cleft.

PURPOSE: Functional stimulus during orthodontic tooth movement into the grafted bone can lead to better alveolar bone grafting outcomes. The aim of this study was to analyze the biological effects of orthodontic tooth movement into the grafted alveolar cleft area with histologic staining, fluorescence staining, and real-time polymerase chain reaction (PCR). MATERIALS AND METHODS: An animal model of orthodontic tooth movement into the grafted alveolar cleft area was established in 8-week-old Sprague-Dawley rats. The animals were divided into the experimental group and the control group. Four checkpoints were observed: before orthodontic stimuli, day 1 after orthodontic stimuli, day 3 after orthodontic stimuli, and day 5 after orthodontic stimuli. The cleft bone formation conditions, including the collagen fibers and the activities of the osteoclasts and osteoblasts, were evaluated by histologic staining. The expression of tartrate-resistant acid phosphatase (TRAP), receptor activator nuclear factor κB ligand, and Runt-related transcription factor 2 was detected by real-time PCR in both groups. RESULTS: Hematoxylin-eosin staining showed that the remodeling process of iliac autografts was completed when the orthodontic stress was applied, whereas the bone tissues first showed osteoclastogenesis and then osteogenesis. On the basis of TRAP staining, the osteoclasts increased to the maximal amount on day 3 and decreased thereafter. Evidence from tetracycline fluorescence staining indicated that no obvious changes in osteoblast activity were detected at the early stage; however, it gradually increased, especially in the region close to the root surface. According to real-time PCR, the expression of TRAP increased in both the early and middle stages, that of receptor activator nuclear factor κB ligand increased in the early stage, and that of Runt-related transcription factor 2 increased in the late stage. Moreover, the results showed significant differences between the experimental and control groups. CONCLUSIONS: Orthodontic tooth movement into the alveolar cleft bone graft area promoted bone remodeling of embedded bone, thus inducing bone resorption and subsequent deposition.

Comparison of Clinical, Radiographic, and Immunologic Inflammatory Parameters around Crestally and Subcrestally Placed Dental Implants: 5-Year Retrospective Results.

PURPOSE: To compare changes in clinical (bleeding on probing [BOP] and probing pocket depth [PPD]), radiographic (crestal bone loss [CBL]), and immunologic inflammatory (interleukin-1beta [IL-1ß] and matrix metalloproteinase-9 [MMP-9]) parameters around crestally and subcrestally placed dental implants 5 years after implant placement. MATERIALS AND METHODS: Fifty-two patients were divided into 2 groups: group 1 (n = 27): patients with single implants placed approximately 2 mm below the alveolar crest; group 2 (n = 25): patients with single implants placed at bone level. In both groups, peri-implant BOP, PPD, and CBL were measured, and levels of IL-1ß and MMP-9 were determined in duplicates using enzyme-linked immunosorbent assay. Full-mouth debridement was performed biannually in both groups. Statistical analysis was performed using the Mann-Whitney U test (significance set at p < 0.05). RESULTS: All measurements in groups 1 and 2 were performed 5.3 ± 0.2 and 5.2 ± 0.1 years after implant placement, respectively. The mean CBL was 1.2 ± 0.2 mm and 1.4 ± 0.2 mm in groups 1 and 2, respectively. There was no significant difference in mean BOP, PPD, CBL and in levels of IL-1ß, and MMP-9 among implants in both groups. CONCLUSION: Clinical, radiographic, and immunologic inflammatory parameters are comparable around crestally and subcrestally placed single dental implants up to 5 years after placement. The depth of implant placement appears to have no effect on clinical status and performance of single dental implants.

Effects of vascular formation during alveolar bone process morphogenesis in mice.

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-ß. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

Bone metabolic microarray analysis of ligature-induced periodontitis in streptozotocin-induced diabetic mice.

BACKGROUND AND OBJECTIVE: Periodontal disease is a chronic infectious disease that results in bone loss. Many epidemiological studies have reported the progression of periodontal tissue destruction in patients with diabetes; however, the associated mechanism remains unclear. In this study, we comprehensively investigated how diabetes affects the periodontal tissue and alveolar bone loss using a ligature-induced periodontitis model in streptozotocin-induced diabetic (STZ) mice. MATERIAL AND METHODS: Diabetes was induced by intraperitoneal injection with streptozotocin in 6-wk-old C57/BL6J male mice. A silk ligature was tied around the maxillary left second molar in 9-wk-old wild-type (WT) and STZ mice. Bone loss was evaluated at 3 and 7 d after ligation. mRNA expression levels in the gingiva between the two groups were examined by DNA microarray and quantitative polymerase chain reaction at 1, 3 and 7 d post-ligation. Tartrate-resistant acid phosphatase and alkaline phosphatase staining of the periodontal tissue was performed for evaluation of osteoclasts and osteoblasts in histological analysis. RESULTS: In the gingiva, hyperglycemia upregulated the osteoprotegerin (Opg) mRNA expression and downregulated Osteocalcin mRNA expression. In the ligated gingiva, tumor necrosis factor-α (Tnf-α) mRNA expression was upregulated at 1 d post-ligation in STZ mice but not in WT mice. At 3 d post-ligation, alveolar bone loss was observed in STZ mice, but not in WT mice. Significantly severe alveolar bone loss was observed in STZ mice compared to WT mice at 7 d post-ligation. Bone metabolic analysis using DNA microarray showed significant downregulation in the mRNA expression of glioma-associated oncogene homologue 1 (Gli1) and collagen type VI alpha 1 (Col6a1) at the gingiva of the ligated site in STZ mice compared to that in WT mice. Quantitative polymerase chain reaction showed that Gli1 and Col6a1 mRNA expression levels were significantly downregulated in the gingiva of the ligated site in STZ mice compared to WT mice. Histological analysis showed lower alkaline phosphatase activity in STZ mice. In addition, an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the ligated sites in STZ mice. CONCLUSIONS: These results suggest that an imbalance of bone metabolism causes osteoclastosis in insulin-deficient diabetes, and that alveolar bone loss could occur at an early phase under this condition.

A novel chemically modified curcumin reduces inflammation-mediated connective tissue breakdown in a rat model of diabetes: periodontal and systemic effects.

BACKGROUND AND OBJECTIVE: Periodontal disease is the most common chronic inflammatory disease known to mankind (and the major cause of tooth loss in the adult population) and has also been linked to various systemic diseases, particularly diabetes mellitus. Based on the literature linking periodontal disease with diabetes in a "bidirectional manner", the objectives of the current study were to determine: (i) the effect of a model of periodontitis, complicated by diabetes, on mechanisms of tissue breakdown including bone loss; and (ii) the response of the combination of this local and systemic phenotype to a novel pleiotropic matrix metalloproteinase inhibitor, chemically modified curcumin (CMC) 2.24. MATERIAL AND METHODS: Diabetes was induced in adult male rats by intravenous injection of streptozotocin (nondiabetic rats served as controls), and Escherichia coli endotoxin (lipopolysaccharide) was repeatedly injected into the gingiva to induce periodontitis. CMC 2.24 was administered by oral gavage (30 mg/kg) daily; untreated diabetic rats received vehicle alone. After 3 wk of treatment, the rats were killed, and gingiva, jaws, tibia and skin were collected. The maxillary jaws and tibia were dissected and radiographed. The gingival tissues of each experimental group (n = 6 rats/group) were pooled, extracted, partially purified and, together with individual skin samples, analyzed for matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography; MMP-8 was analyzed in gingival and skin tissue extracts, and in serum, by western blotting. The levels of three bone-resorptive cytokines [interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α], were measured in gingival tissue extracts and serum by ELISA. RESULTS: Systemic administration of CMC 2.24 to diabetic rats with endotoxin-induced periodontitis significantly inhibited alveolar bone loss and attenuated the severity of local and systemic inflammation. Moreover, this novel tri-ketonic phenylaminocarbonyl curcumin (CMC 2.24) appeared to reduce the pathologically excessive levels of inducible MMPs to near-normal levels, but appeared to have no significant effect on the constitutive MMPs required for physiologic connective tissue turnover. In addition to the beneficial effects on periodontal disease, induced both locally and systemically, CMC 2.24 also favorably affected extra-oral connective tissues, skin and skeletal bone. CONCLUSION: This study supports our hypothesis that CMC 2.24 is a potential therapeutic pleiotropic MMP inhibitor, with both intracellular and extracellular effects, which reduces local and systemic inflammation and prevents hyperglycemia- and bacteria-induced connective tissue destruction.

A Systematic Review on the Implication of Minerals in the Onset, Severity and Treatment of Periodontal Disease.

Periodontal disease is an inflammatory disease with high prevalence in adults that leads to destruction of the teeth-supporting tissues. Periodontal therapy has been traditionally directed at reduction of the bacterial load to a level that encourages health-promoting bacteria and maintenance of oral-hygiene. The role of nutrition in different chronic inflammatory diseases has been the subject of an increasing body of research in the last decades. In this sense, there has been an important increase in the volume of research on role of nutrition in periodontitis since the diet has known effects on the immune system and inflammatory cascades. Minerals play a key role in all these processes due to the multiple pathways where they participate. To clarify the role of the different minerals in the establishment, progression and/or treatment of this pathology, a systemically review of published literature cited in PubMed until May 2016 was conducted, which included research on the relationship of these elements with the onset and progression of periodontal disease. Among all the minerals, calcium dietary intake seems important to maintain alveolar bone. Likewise, dietary proportions of minerals that may influence its metabolism also can be relevant. Lastly, some observations suggest that all those minerals with roles in immune and/or antioxidant systems should be considered in future research.