A new mixed model of periodontitis-induced preeclampsia: A pilot study.

OBJECTIVES/BACKGROUND: Recent studies have shown that periodontal disease is strongly related to gestational complications such as preeclampsia (PE). PE is responsible for 42% of maternal deaths worldwide and kills approximately 76 000 women a year. In addition, children born under PE conditions are at increased risk of hospitalization due to metabolic disorders, epilepsy, and other complications. Numerous reviews and clinical studies on PE have been published, but the mechanisms underlying the relationship between periodontal disease and PE and the way periodontopathogens alter vascular response in pregnant women remain unclear. METHODS: This study aims to verify whether periodontal disease induces PE by using the association of two periodontitis (PD) models: ligature and oral Porphyromonas gingivalis (P. gingivalis) W83 inoculation in Wistar rats. At gestational day 5, the ligature was placed on each mandibular first molar, which was followed by daily oral P. gingivalis inoculation for 15 days. At gestational day 19, urine was collected, and invasive arterial pressure was measured. The animals were euthanized, and plasma and tissues were collected. RESULTS: After 15 days of the association of ligature and P. gingivalis inoculation, the animals presented the characteristic symptoms of PE: altered blood pressure, proteinuria, and change in litter size (number of pups) and pup weight when compared to the control group (p < .005). The PE animals also presented greater bone porosity, trabecular separation, and reduced bone volume in the hemimandibles, as well as altered inflammatory response. The level of cytokine IL-6 was higher in the PE group than in the control group (p < .005). CONCLUSION: The association of two PD models effectively induced PE. To our knowledge, this is the first study on the oral use of P. gingivalis for PE induction. Our results support the importance of PD as a possible cause for PE development, opening an important new avenue to study cause and consequence relationships in inflammation and PE due to exposure to periodontal infection.

Calcium and Phosphorus Levels in Saliva are Influenced by Genetic Polymorphisms in Estrogen Receptor Alpha and Microrna17.

Homeostasis between salivary calcium and phosphorus is important for maintaining oral health. The aim of this study was to evaluate if polymorphisms in ESR1 (Estrogen Receptor Alpha), ESR2 (Estrogen Receptor Beta) and miRNA17 (microRNA17) are associated with calcium and phosphorus levels in saliva. Saliva from 276 12-year-old children were collected by masticatory stimulation and calcium and phosphorus levels were determined by Mass Spectrometry. Genomic DNA was extracted from remaining saliva and genetic polymorphisms in ESR1 (rs12154178, rs1884051, rs9340799 and rs2234693), in ESR2 (rs4986938 and rs1256049) and in miRNA17 (rs4284505) were genotyped using TaqMan chemistry and a real-time PCR equipment. Statistical differences in genotype and allele distributions between 'low' and 'high' calcium and phosphorus levels were determined using chi-square or Fisher´s exact tests. The analysis was also adjusted by sex (alpha of 5%). ESR1 rs9340799 had the less common genotype associated with higher calcium levels (p=0.03). The less common allele of ESR1 rs1884051 was associated with lower phosphorus levels (p=0.005) and there was an excess of heterozygotes for miRNA17 rs4284505 among individuals with lower calcium levels (p=0.002), both adjusted by sex. This study provides evidence that genetic polymorphisms in ESR1 and miRNA17 are involved in determining salivary calcium and phosphorus levels.

Effects of fibrin sealant and bone fragments on defect regeneration performed on rat tibiae: An experimental study.

Fibrin sealant (FS) is a biomaterial that exhibits hemostatic and repairing properties. It has been successfully used as scaffolds and adhesives to improve repair and regeneration of tissues. The objective of this study was to evaluate the effect of FS in the regeneration process of bone defects in male rat tibias through macroscopic, microscopic and mechanical analysis. A bone defect of 2.9 mm was performed on the medial face of the proximal third of the tibia of 40 rats and implanted FS and autologous bone graft (AG). The animals were divided into four groups: animals with bone defect without any treatment (CON), animals treated with fibrin sealant (TFS), animals treated with autologous graft (TAG) and animals treated with fibrin sealant and autologous graft (FSAG). The animals were euthanized 42 days after surgery. Macroscopic analysis showed no difference between the groups (p > 0.05) in relation to tibial weight, but a statistically significant difference (p = 0.005) was observed for their length. Micro-computed tomography (micro-CT) revealed tendentious values regarding bone microarchitecture and FS. Bone mineral densitometry (BMD) showed significance between the FSAG (p = 0.009) and TFS (p = 0.007) groups. The bone mineral content (BMC) presented a significant difference between all groups (p = 0.020). Maximum strength showed a significant difference between the FSAG group (p = 0.007) and the others. The results obtained in relation to the relative stiffness also present a significant difference (p = 0.023). Newly formed bone showed significant differences between groups (p = 0.035). We conclude that bone defect regeneration was directly influenced by the use of FS and AG.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction.

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

A high-fat diet can affect bone healing in growing rats.

A high-fat diet (HFD) can have a negative effect on bone quality in young and old people. Although bone healing in children is normally efficient, there is no evidence that children who have a diet rich in fat have compromised bone fracture regeneration compared with children with recommended dietary fat levels. The purpose of the present study was to evaluate the effects of an HFD on bone healing in growing female rats. Twenty-six postweaning female Wistar rats were divided into two groups (13 animals per group): a standard diet (SD) group and an HFD (with 60% of energy from fat) group. The rats received the assigned diets for 5 weeks, and in the third week they were submitted to an osteotomy procedure of the left tibia. Body mass and feed intake were recorded during the experiment. One day before euthanasia, an insulin tolerance test was performed. After euthanasia, the tibiae were removed and analyzed by densitometry, mechanical testing, histomorphometry, stereology and immunohistochemistry. An HFD caused an adaptive response to maintain energetic balance by decreasing feed intake and causing insulin insensitivity. There was no change in bone mineral density, collagen amount and immunostaining for bone formation, but maximal load and stiffness were decreased in the HFD group. In addition, bone volume had a tendency to be higher in the SD group than in the HFD group. Compared with rats receiving an SD, growing rats receiving an HFD for 5 weeks had similar bone mineral density but altered mechanical properties at the osteotomy defect site.

Genes Involved in the Enamel Development Are Associated with Calcium and Phosphorus Level in Saliva.

Saliva components play a crucial role in the integrity of the dental enamel and in caries susceptibility. The saliva characteristics are controlled by many factors, including genetic factors. Therefore, this study aimed to evaluate the association between the genetic variations in genes expressed in enamel development with calcium and phosphorus levels in saliva. We collected 276 unrelated 12-year-old children from private and public schools. Saliva was collected for DNA extraction from oral cells and for measurement of calcium and phosphorus. Inductively coupled plasma-mass spectrometry determined calcium and phosphorus levels in whole saliva. Fifteen genetic variations in 9 genes were analyzed. The genotype was determined by real-time polymerase chain reactions. Data were analyzed using Plink with an alpha of 5%. Genetic variations in AMELX, AMNB and ESRRB were associated with the calcium level in saliva (p < 0.05). A borderline association was observed in ENAM allele distribution shown with phosphate level in saliva (p = 0.049). In conclusion, our results are the first to report that genetic variations contribute to calcium and phosphorus levels in saliva.

Lead line in rodents: an old sign of lead intoxication turned into a new method for environmental surveillance.

The "lead line" was described by Henry Burton in 1840. Rodents are used as sentinels to monitor environmental pollution, but their teeth have not been used to determine lead. To determine whether lead deposits can be observed in the teeth of lead-exposed animals, since the gingival deposits known as "lead line" would likely have a correlate in the calcified tissue to which the gums are opposed during life. Male Wistar rats were exposed to lead in the drinking water (30 mg/L) since birth until 60 days-old. Molars and the incisors of each hemimandible were analyzed by scanning electron microscopy (SEM) on regular and backscattered electrons (BSE) mode. Elements were determined using electron dispersive spectroscopy (EDS). Clean cervical margins were observed on control teeth, as opposed to the findings of extensive deposits on lead-exposed animals, even in hemimandibles that had been exhumed after being buried for 90 days. BSE/EDS indicated that those deposits were an exogenous material compatible with lead sulfite. Presence of calcium, phosphorus, magnesium, carbon, lead, and oxygen is presented. Lead-exposed animals presented marked root resorption. The lead deposits characterized here for the first time show that the "lead line" seen in gums has a calcified tissue counterpart, that is detectable post-mortem even in animals exposed to a low dose of lead. This is likely a good method to detect undue lead exposure and will likely have wide application for pollution surveillance using sentinels.

Effect of treatment with simvastatin on bone microarchitecture of the femoral head in an osteoporosis animal model.

The objective of this study was to evaluate the microarchitecture and trabecular bone strength at the distal region of the femur, and its biomechanical properties with simvastatin administration with two different doses in ovariectomized (OVX) rats. Ninety rats were divided into six groups to evaluate treatment with the simvastatin drug (n = 15): SH (Sham surgery), SH-5 (5 mg simvastatin), SH-20 (20 mg simvastatin), OVX, OVX-5, and OVX-20. Euthanasia was performed at three different times, five animals per period: 7, 14, and 28 days. The effectiveness of the treatments was evaluated by mechanical testing and histomorphometric analysis of the femurs. The results of analysis by the linear model of mixed effects showed 20 mg of simvastatin results in increased trabecular bone after 14 days (P = 0.039) of ingestion in ovariectomized animals. However, ingestion of 5 mg of simvastatin is able to sensitize the trabecular bone only at 28 days (P = 0.005) of ingestion. In the mechanical tests stiffness improves within 28 days (P = 0.003). Regarding maximum strength, no statistical differences were observed. According to these results, it can be concluded that for a decrease in oral intake, longer treatment times are required. Microsc. Res. Tech. 79:684-690, 2016. © 2016 Wiley Periodicals, Inc.

Lead concentrations in whole blood, serum, saliva and house dust in samples collected at two time points (12 months apart) in Santo Amaro, BA, Brazil.

UNLABELLED: Whole Blood Lead Level (BLL) is the main marker used to verify lead contamination. The present study explores how BLL is associated with lead concentrations in serum, saliva and house dust. Samples were collected twice from Santo Amaro, BA, Brazil, a region that was contaminated by a lead smelter in the past; a time interval of 12 months was allowed between the two collections. It is noteworthy that the following measures have recently been taken to diminish exposure of the population to lead: streets have been paved with asphalt, and educational campaigns have been launched to reduce exposure to contaminated dust. RESULTS: Compared with the first time point, all the samples collected at the second time point contained lower lead concentration (p<0.05), which suggested that the adopted measures effectively reduced exposure of the population to lead present in contaminated soil and dust. Statistically significant correlations only existed between lead in blood collected in the first year and lead in blood collected in the second year (Spearman's r=0.55; p<0.0001; n=62), and lead in house dust collected in the first year and lead in house dust collected in the second year (Spearman's r=0.5; p<0.0001; n=59). CONCLUSIONS: Results support the validity of lead determination in blood and in house dust to assess lead exposure over time. However, lead in blood and lead in dust did not correlate with lead in serum or lead in saliva.

Reduced bone and body mass in young male rats exposed to lead.

The aim of this study was to see whether there would be differences in whole blood versus tibia lead concentrations over time in growing rats prenatally. Lead was given in the drinking water at 30 mg/L from the time the dams were pregnant until offspring was 28- or 60-day-old. Concentrations of lead were measured in whole blood and in tibia after 28 (28D) and 60 days (60D) in control (C) and in lead-exposed animals (Pb). Lead measurements were made by GF-AAS. There was no significant difference (P > 0.05) in the concentration of whole blood lead between Pb-28D (8.0 ± 1.1 µg/dL) and Pb-60D (7.2 ± 0.89 µg/dL), while both significantly varied (P < 0.01) from controls (0.2 µg/dL). Bone lead concentrations significantly varied between the Pb-28D (8.02 ± 1.12 µg/g) and the Pb-60D (43.3 ± 13.26 µg/g) lead-exposed groups (P < 0.01), while those exposed groups were also significantly higher (P < 0.0001) than the 28D and 60D control groups (Pb < 1 µg/g). The Pb-60D group showed a 25% decrease in tibia mass as compared to the respective control. The five times higher amount of lead found in the bone of older animals (Pb-60D versus Pb-28D), which reinforces the importance of using bone lead as an exposure biomarker.

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension.

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.