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Epithelial rests of Malassez (ERMs) are fragments of Hertwig's sheath in the periodontal ligament. There is extensive knowledge of their role in the etiology ofpathological processes and current evidence links them to maintenance of periodontal homeostasis. The aim of this study was to assess the behavior of ERMs with relation to the changes in periodontal tissues during growth in an experimental model with Wistar rats. Mesiodistal sections were made of the first lower molars from Wistar rats aged 1 month (n=7), 3 months (n=7) and 5 months (n=6). Sections were stained with H&E to evaluate number of ERMs/ mm, size/area of ERMs (pm2), height of periodontal ligament (PL.h) (pm), area of cement in the furcation zone (C.Ar) (pm2) and bone area related to the ERM zone (BAr/TAr)(%). Posthoc Bonferroni and ANOVA were applied for statistical analysis of results. Number of ERMs/mm declined significantly with age (1m: 4.34±1.51, 3m: 1.48±0.89, 5m: 0.27± 0.50, p<0.05), and there was great variability in size. There was significant increase in C.Ar (1m: 3418.96 ± 905.88, 3m: 19365.76 ± 5500.52, 5m: 32182.76 ± 7114.51, p< 0.05) and interradicular (BAr/TAr) (1m: 25.26 ± 2.37, 3m: 44.70 ± 3.95, 5m: 46.81 ± 7.80, p< 0.05: 1 vs 3, 1 vs 5). There was significant decline in PLh at 5 months (1m: 141.42 ± 29.25, 3m: 162.06 ± 28.21, 5m: 124.72 ± 18.67, p< 0.05: 1 vs. 3, 3 vs. 5). The decline in number of ERMs as animal age increases may be related to the significant increase in C.Ar and reduction in PL.h. It remains to ascertain its relationship with the increase observed in BAr/ TAr. Further studies are needed to learn more about the role of ERMs and their relationship with periodontal tissues when confronted with different normal and pathological stimuli., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article, (Sociedad Argentina de Investigación Odontológica.)

Air particulate matter (PM) can lead to extrapulmonary adverse reactions in organs such as liver and heart either by particle translocation from the lung to the systemic circulation or by the release of lung mediators. Young BALB/c mice were intranasal instilled with 1mg/BW of Urban Air Particles from Buenos Aires or Residual Oil Fly Ash. Histopathology, oxidative metabolism and inflammation on lungs and extrapulmonary organs and the systemic response were evaluated. Lung histophatological analysis supported the rise in the number of inflammatory cells in the bronchoalveolar lavage from PM-exposed animals. Also, both PM caused recruitment of inflammatory cells in the liver and heart parenchyma and IL-6 and transaminases augmentation in serum. We have shown that despite morphochemical differences, both urban air PM altered the lung and extrapulmonary organs. Therefore, exposure to urban air PM may distress body metabolism which, in turn could lead to the development and progression of multifactorial diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Air pollution represents a major health problem in megacities, bringing about 8 million deaths every year. The aim of the study was to evaluate in vivo the ocular and respiratory mucosa biological response after chronic exposure to urban air particles from Buenos Aires (UAP-BA). BALB/c mice were exposed to UAP-BA or filtered air for 1, 6, 9, and 12 months. After exposure, histology, histomorphometry, and IL-6 proinflammatory cytokine level were evaluated in the respiratory and ocular mucosa. Total cell number and differential cell count were determined in the brochoalveolar lavage fluid. In the lung, chronic exposure to UAP-BA induced reduction of the alveolar space, polymorhonuclear cell recruitment, and goblet cell hyperplasia. In the ocular surface, UAP-BA induced an initial mucin positive cells rise followed by a decline through time, while IL-6 level increased at the latest point-time assayed. Our results showed that the respiratory and the ocular mucosas respond differently to UAP-BA. Being that lung and ocular mucosa diseases may be triggered and/or exacerbated by chronic exposure to urban air PM, the inhabitants of Buenos Aires whom are chronically exposed to environmental urban air pollution may be considered a subpopulation at risk. Based on our results, we propose the ocular mucosa as a reliable and more accessible surrogate for pulmonary mucosa environmental toxicity that might also serve as an earlier biomarker for air pollution adverse impact on health.

The presence of metals in the environment is a matter of concern, since human activities are the major cause of pollution and metals can enter the food chain and bioaccumulate in hard and soft tissues/organs, which results in a long half-life of the metal in the body. Metal intoxication has a negative impact on human health and can alter different systems depending on metal type and concentration and duration of metal exposure. The present review focuses on the most common metals found in contaminated areas (cadmium, zinc, copper, nickel, mercury, chromium, lead, aluminum, titanium, and iron, as well as metalloid arsenic) and their effects on bone tissue. Both the lack and excess of these metals in the body can alter bone dynamics. Long term exposure and short exposure to high concentrations induce an imbalance in the bone remodeling process, altering both formation and resorption and leading to the development of different bone pathologies.

The aim of this study was to evaluate the morphological alterations of epithelial cell rests of Malassez (ERMs) and their relationship with root resorption, in an experimental periodontitis (EP) model at 4 and 11 days. EP was induced in 14 male Wistar rats by placing a cotton thread ligature around the neck of the first lower right molar, for 4 (n=7) and 11 (n=7) days. The contralateral molar (left) was used as control. Following euthanasia, jaws were extracted and processed histologically to provide mesio-distal sections which were subject to H&E stain and histochemical detection technique with tartrate-resistant acid phosphatase (TRAP). The following histomorphometricparameters were evaluated on micrographs: bone area (BAr./TAr)(%), number of ERMs/mm2, number of cells/ERM, ERMs area (µm2), and percentage of root resorption surfaces (%RR). The results were analyzed statistically by ANOVA and Bonferronipost hoc (p≤ 0.05). Significant bone loss was observed in molars with EP compared to their controls. In the EP 4-Day group, no change was observed in the parameters with relation to the ERMs; however, in the EP 11-Day group, there was significant root resorption (%RR) (C: 3.21±3.07, EP-4D: 3.91±3.17, EP-11D: 23.67± 11.40; p≤ 0,05) and increase in ERMs area (µm2) (C: 455.87±145.42, EP-4D: 577.6±156.1, EP-11D: 1046.3± 582.9; p≤ 0,05). No TRAP+ ERM was found in either group. ERM hypertrophy may be related to ERMpartici-pation in mechanisms tending to establish periodontal homeostasis, inhibiting resorption and contributing toperiodon-tal regeneration., (Sociedad Argentina de Investigación Odontológica.)

The in vivo response of osteocytes to different force magnitudes soon after they are applied remains to be elucidated. The aim of this study was to examine the early effects of applying a very light (LF: 0,16 N) and a very strong (SF: 2,26 N) orthodontic force during one hour on apoptosis and osteopontin (OPN) expression on alveolar bone osteocytes, in rats. Results: LF: compared to the control group, they showed a significant increase in OPN expression, and a significant decrease in the number of TUNELpositive osteocytes. SF: compared to the control group, they showed a significant increase in OPN expression and a significant decrease in the number of TUNELpositive osteocytes. Our results show that osteocytes respond very early to the application of tension and pressure forces of different magnitudes, and application of forces decreases the number of apoptotic osteocytes and increases OPN expression. These results allow concluding that osteocytes activate rapidly when subjected to locally applied forces, whether these forces be pressure or tension, light or strong forces., (Sociedad Argentina de Investigación Odontológica.)

Bisphosphonates are the first choice therapy for the pharmaco logical treatment of osteoporosis. Following reports of cases of bisphosphonaterelated osteonecrosis of the jaw and atypical femur fracture, the safety of longterm use of bisphosphonates has been evaluated, resulting in the proposal of strontium as an alternative drug. No experimental study using a sequential administration design has been reported to date. Hence, the aim of this study was to evaluate the effect on bone tissue of ovariectomized rats of administration of alendronate followed by strontium ranelate. Fortyeight female Wistar rats were ovariectomized on day 1 of the experiment. Beginning on day 30, they were administered 0.3 mg/kg/week of alendronate (ALN) or vehicle (VEH) for 8 weeks. Two groups (ALN and corresponding control) were euthanized at this time, and the remaining animals were divided into 4 groups and given 290 mg/kg/day of strontium ranelate (SR) in their drinking water (TW) or only water for 4 months. Experimental groups were: ALN+SR, ALN+TW, VEH+SR, VEH+TW, ALN and VEH. The tibiae and hemimandibles were resected for histomorphometric evaluation, and the right femur was used to perform biomechanical studies. ANOVA and Bonferroni test were applied. Diaphyseal stiffness, maximum elastic load and fracture load increased in animals that received alendronate, regardless of whether or not they received subsequent SR treatment. Fracture load also increased in VEH+ SR versus control (VEH+TW). Subchondral and interradicular bone volumes were significantly higher in animals that received ALN than in those that received vehicle. No difference was observed in cortical area or thickness of the tibia among treatments. The results obtained with the model presented here, evaluating tibial and mandibular interradicular bone, showed that the combination of ALN and SR and administration of ALN alone are equally effective in preventing bone loss associated with ovariectomyinduced estrogen depletion., (Sociedad Argentina de Investigación Odontológica.)

Accumulation of cadmium (Cd), an extremely toxic metal, can cause renal failure, decreased vitamin D synthesis, and consequently osteoporosis. The aim of this work was to evaluate the effect of Cd on two types of bone in growing Wistar rats. Sixteen 21-day-old male Wistar rats were assigned to one of two groups. The Cd group subcutaneously received 0.5mg/kg of CdCl2 5 times weekly for 3 months. The control group similarly received bidistilled water. Following euthanasia, the mandibles and tibiae were resected, fixed, decalcified and processed histologically to obtain sections for H&E and tartrate-resistant acid phosphatase (TRAP) staining. Photomicrographs were used to determine bone volume (BV/TV%), total growth cartilage width (GPC.Wi) hypertrophic cartilage width (HpZ.Wi), percentage of yellow bone marrow (%YBM), megakaryocyte number (N.Mks/mm(2)), and TRAP+osteoclast number (N.TRAP+Ocl/mm(2)). Results were statistically analyzed using Student's t test. Cd exposed animals showed a significant decrease in subchondral bone volume and a significant increase in TRAP+ osteoclast number and percentage of yellow bone marrow in the tibia, and an increase in megakaryocyte number in mandibular interradicular bone. No significant differences were observed in the remaining parameters. The results obtained with this experimental design show that Cd would seemingly have a different effect on subchondral and interradicular bone. The decrease in bone volume and increase in tibial yellow bone marrow suggest that cadmium inhibits differentiation of mesenchymal cells to osteoblasts, favoring differentiation into adipocytes. The different effects of Cd on interradicular bone might be due to the protective effect of the mastication forces., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Female Wistar rats are frequently used in experimental models to study hormone and bone pathologies and treatments. Most experimental studies involving histomorphometric evaluation assessed long bones, and few reports also studied mandibular bone. The aim of this work was to clarify and distinguish the age-related histomorphometric changes that occur in the tibia (subchondral bone) and in the mandible (interradicular bone), and thus obtain reference histomorphometric data of healthy female Wistar rats at different growth stages. Three groups of 8 healthy female Wistar rats were euthanized at 6 (GI), 10 (GII), and 14 (GIII) weeks. The tibiae and mandible were resected and histologically processed to obtain H&E stained sections of the tibia and the lower first molar to analyze the following histomorphometric parameters: Bone volume, trabecular width, trabecular number (Th.N)(1/mm), growth cartilage width, hypertrophic cartilage width and number of osteoclasts per area in the tibiae, and bone volume and number of osteoclasts per area N.Oc/mm(2) in the interradicular bone of the first lower molar. A significant decrease in subchondral bone volume as a result of a decrease in trabecular number and growth cartilage width was observed in 14-week-old rats. Conversely, interradicular bone volume was found to increase with age. The results highlight the importance of analyzing both types of bone to better understand the response of two different trabecular bones, contributing in turn to decision making regarding treatment strategies and disease management.

Background: Bisphosphonates are anticatabolic agents that inhibit bone resorption and are widely used to treat osteoporosis and bone metastases in adults. They are also used in young patients with diseases like osteogenesis imperfecta or juvenile osteoporosis. Bone modeling/remodeling is elevated in growing subjects, and inhibition of osteoclastic activity has been shown to interfere with growth. Thus, our objective was to evaluate the effect of alendronate (ALN) on growing animals., Methods: Healthy male Wistar rats, aged 1 mo, received ALN or vehicle for 8 wk. Serum levels (calcemia, phosphatemia, and total alkaline phosphatase) were determined. Morphometric (rat: femur and tibia weight and length and hemimandible growth) and histomorphometric parameters (thickness of tibial epiphyseal cartilage and each cartilage zone, interradicular bone volume in the first lower molar, trabeculae volume, percentage of bone and cartilage, and osteoclast number in mandibular condyles) were assessed., Results: ALN caused a significant decrease in femur and tibia length, tibial cartilage thickness, and longitudinal growth of hemimandibles. It increased interradicular bone volume and mandibular condyle trabeculae volume, increasing the percentage of cartilage and osteoclast number., Conclusion: These findings indicate that administration of ALN to growing animals alters the endochondral ossification process, and thus alters growth.

Unlabelled: Clinical and experimental studies suggest that prenatal exposure to stress can impact the growth and development of offspring. The effect of prenatal exposure to constant light, applied as a chronic stressor, on endochondral ossification of the tibiae of 3-day-old and 15-day-old pups was histomorphometrically evaluated. Pregnant rats were divided into 2 groups: mothers chronically exposed to a 12:12-hour light/light cycle (LL) and control mothers maintained on a 12:12-hour light/dark cycle on days 10-20 of pregnancy. On postnatal days 3 and 15, the pups were weighed and euthanized. The tibiae were resected and histologically processed to obtain sections for hematoxylin and eosin staining (HE) and tartrate-resistant acid phosphatase (TRAP) histochemistry, in order to perform histomorphometric determinations. The data were statistically analyzed. A significant decrease in hypertrophic cartilage thickness was observed in the tibiae of the 3-day-old (LL: 0.134 ± 0.02 vs., Controls: 0.209 ± 0.023 mm; p < 0.01) and 15-day-old (LL: 23.32 ± 3.98 vs., Controls: 22.96 ± 1.93 mm; p < 0.05) prenatally stressed pups. The subchondral bone volume was significantly lower in the tibiae of the 3-day-old LL pups (38.83 ± 6.14%) than in the controls (62.83 ± 10.67%; p < 0.01). The decrease in subchondral bone volume and hypertrophic cartilage thickness shows that the normal growth process of the tibia is impaired in prenatally stressed pups., (© 2015 S. Karger AG, Basel.)

Objective: Tooth eruption is a multifactorial process in which bone tissue plays a prevailing role. In this study we evaluated the bone overlying the developing tooth germ and the degree of tooth eruption of the first mandibular molar in pups born to mothers subjected to constant light during pregnancy., Design: Pregnant rats were divided into two groups: mothers chronically exposed to a 12:12 light/light cycle (LL) from day 10 to 20 of pregnancy and controls (C) maintained on a 12:12 h light/dark cycle. Pups from each group were euthanized at the age 3 or 15 days. Buccolingually oriented sections of mandibles were stained with haematoxylin-eosin or for histochemical detection of tartrate resistant acid phosphatase (TRAP). The histomorphometric parameters evaluated were bone volume, number of osteoclasts, TRAP+ bone surface, number of TRAP+ and TRAP- osteoclasts per mm(2) and degree of tooth eruption (mm)., Results: It was found an increase in bone volume (LL: 58.14±4.24 vs. C: 32.31±2.16; p<0.01) and a decrease in the number of osteoclasts (LL: 3.5±0.65 vs. C: 8.03±1.31; p<0.01) and TRAP+ cells (LL: 0.84±0.53 vs. C: 8.59±1.26; p<0.01) in 3-day-old pups born to LL-exposed mothers. These observations are consistent with the decrease in the degree of tooth eruption observed in 15-day-old experimental pups (LL: -0.605±0.05 vs. C: -0.342±0.02; p<0.0001)., Conclusion: Our results suggest that chronic constant light applied as a pre-natal stressor impairs the resorptive capacity of osteoclasts involved in the formation of the eruption pathway and consequently the degree of tooth eruption., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previously reported studies from this laboratory revealed that rats chronically intoxicated with lead (Pb) under hypoxic conditions (HX) impaired growth parameters and induced damages on femoral and mandibular bones predisposing to fractures. We also described periodontal inflammatory processes under such experimental conditions. Periodontitis is characterised by inflammation of supporting tissues of the teeth that result in alveolar bone loss. The existence of populations living at high altitudes and exposed to lead contamination aimed us to establish the macroscopic, biochemical and histological parameters consistent with a periodontal disease in the same rat model with or without experimental periodontitis (EP). Sixty female rats were divided into: Control; Pb (1000ppm of lead acetate in drinking water); HX (506mbar) and PbHX (both treatments simultaneously). EP was induced by placing ligatures around the molars of half of the rats during the 14 days previous to the autopsy. Hemi-mandibles were extracted to evaluate bone loss by histomorphometrical techniques. TNFα plasmatic concentration was greater (p<0.01) in Pb and HX animals. TBA-RS content was significantly higher in gums of rats with or without EP only by means of Pb. The SMG PGE2 content increased by Pb or HX was higher in PbHX rats (p<0.01). Pb and HX increased EP induced alveolar bone loss, while Pb showed spontaneous bone loss also. In conclusion, these results show that lead intoxication under hypoxic environment enhanced not only alveolar bone loss but also systemic and oral tissues inflammatory parameters, which could aggravate the physiopathological alterations produced by periodontal disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The occurrence of very early morphological changes in the osteocyte lacuno-canalicular network following application of tensile and/or compressive forces remains unknown to date. Thus, the aim of this study was to perform a morphological and morphometric evaluation of the changes in the three-dimensional structure of the lacuno-canalicular network and the osteocyte network of alveolar bone that take place very early after applying tensile and compressive forces in vivo, conducting static histomorphometry on bright-field microscopy and confocal laser scanning microscopy images. Our results showed that both the tensile and compressive forces induced early changes in osteocytes and their lacunae, which manifested as an increase in lacunar volume and changes in lacunar shape and orientation. An increase in canalicular width and a decrease in the width and an increase in the length of cytoplasmic processes were also observed. The morphological changes in the lacuno-canalicular and osteocyte networks that occur in vivo very early after application of tensile and compressive forces would be an indication of an increase in permeability within the system. Thus, both compressive and tensile forces would cause fluid displacement very soon after being applied; the latter would in turn rapidly activate alveolar bone osteocytes, enhancing transmission of the signals to the entire osteocyte network and the effector cells located at the bone surface., (Copyright © 2013 S. Karger AG, Basel.)

Lead chronic intoxication under hypoxic conditions revealed growth retardation in growing rats and damages on femoral and mandibular bones that predispose to fractures. These findings aimed us to investigate if bone material and geometric properties, bone mass in terms of histomorphometry or antioxidant capacity are also impaired in such experimental model. Combined treatments significantly reduced hemimandible cross sectional geometry and intrinsic stiffness (-16% and -34%); tibia and hemimandible bone volume (-45% and -40%) and growth plate cartilage thickness (-19%). These results show a previously unreported toxic effect of lead on mandible however, longer studies should be necessary to evaluate if an adaptation of bone architecture to maintain structural properties may occur and if the oxidative stress can be identified as the primary contributory agent in the pathogenesis of lead poisoning.

Objective: The aim of this study was to assess the short term effect of ethanol administration on periodontal disease in rats., Design: Rats received either ethanol 2g/kg or water by gastric gavage twice a day. On the fifth day ligatures were tied around the molars of half of the rats to induce periodontitis. After 7days gingival tissue was removed and assayed for inflammatory markers. Finally, hemi-mandibles were extracted to evaluate bone loss by histomorphometrical techniques., Results: The experimental periodontitis increased significantly the mRNA expression (p<0.001) and activity (p<0.001) of inducible nitric oxide synthase (iNOS) in the gingival tissue, whilst short time ethanol administration increased iNOS activity (p<0.05) and produced an additive effect on iNOS mRNA expression augmented by periodontitis (p<0.01). The short time ethanol administration also potentiated the periodontitis stimulatory effect on the mRNA expression of interleukin (IL)-1β (p<0.01 and p<0.001, in semi-quantitative and real time PCR, respectively) and on the height of periodontal ligament (p<0.05). However, the ligature-induced periodontitis, but not ethanol administration, increased the prostaglandin E(2) content (p<0.05) and, diminished the alveolar bone volume (p<0.05), as compared to sham rats., Conclusion: The present results suggest that ethanol consumption could represent a risk indicator for periodontal disease since augments the expression of inflammatory markers, in healthy rats, and increases them, at short term, during the illness. However, scale longitudinal investigation and more case-control studies are needed to confirm this statement., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The existence of children living at high altitude suffering from lead (Pb) poisoning prompted us to investigate the long term effects of this pollutant on growth and bone biology in growing rats maintained at simulated high altitude (SHA). Pb and hypoxia (HX) significantly reduced body weight (-9.4 % and -24 %; p < 0.01) and length (-3 % and -8 %; p < 0.01); decreased femoral ultimate load (-16 % and -40 %; p < 0.01) and femoral energy absorption capacity (-18 % and -74 %; p < 0.01). Oral pathologic alterations were observed in experimental groups. Our findings revealed growth retardation and damages on femoral and mandibular bones that predispose to fractures.

Nitrogen-containing bisphosphonates are widely used for treating diverse bone pathologies. They are anticatabolic drugs that act on osteoclasts inhibiting bone resorption. It remains unknown whether the mechanism of action is by decreasing osteoclast number, impairing osteoclast function, or whether they continue to effectively inhibit bone resorption despite the increase in osteoclast number. There is increasing evidence that bisphosphonates also act on bone marrow cells like macrophages and monocytes. The present work sought to evaluate the dynamics of preosteoclast fusion and possible changes in medullary macrophage number in bisphosphonate-treated animals. Healthy female Wistar rats received olpadronate, alendronate, or vehicle during 5 weeks, and 5-bromo-2-deoxyuridine (BrdU) on day 7, 28, or 34 of the experiment. Histomorphometric studies were performed to study femurs and evaluate: number of nuclei per osteoclast (N.Nu/Oc); number of BrdU-positive nuclei (N.Nu BrdU+/Oc); percentage of BrdU-positive nuclei per osteoclast (%Nu.BrdU+/Oc); medullary macrophage number (mac/mm(2)) and correlation between N.Nu/Oc and mac/mm(2). Results showed bisphosphonate-treated animals exhibited increased N.Nu/Oc, caused by an increase in preosteoclast fusion rate and evidenced by higher N.Nu BrdU+/Oc, and significantly decreased mac/mm(2). Considering the common origin of osteoclasts and macrophages, the increased demand for precursors of the osteoclast lineage may occur at the expense of macrophage lineage precursors.

Osteoporosis is a disease in which the microarchitecture of bone tissue deteriorates, with consequent loss of bone mass. Strontium ranelate (SrR) is currently used for treatment of the condition. SrR may have a dual effect: anabolic (stimulating pre-osteoblast replication) and anti-catabolic (reducing osteoclastic activity). However, its mechanism of action has not yet been completely elucidated. The aim of this study is to evaluate the effect of SrR on bone remodeling in healthy Wistar rats. Two-month old female Wistar rats were administered SrR (2 g/L) in drinking water for 30 weeks. Oriented histological sections were prepared from lower jaw and tibia and stained with H&E, and the following histomorphometric parameters were evaluated: a) in interradicular bone: bone volume, and percentages of bone-formation, quiescent and bone-resorption surfaces; and b) in tibia: bone volume, total thickness of growth cartilage, thickness of hypertrophic cartilage zone and number of megakaryocytes. No significant difference was found in the parameters between the control animals and those treated with SrR. The results would therefore show that SrR does not alter the bone parameters studied in this experimental design.

Unlabelled: Distraction osteogenesis (DO) is a surgical technique producing bone lengthening by distraction of the fracture callus. Although a large number of experimental studies on the events associated with DO of craniofacial skeleton have been reported, the few employing rat mandibular bone DO used complicated designs and produced a small volume of newly formed bone. Thus, this study aims to present an original experimental model of mandibular DO in edentulous rats that produces a sufficient quantity and quality of intramembranous bone. Eight male Wistar rats, weighing 75 g, underwent extraction of lower molars. With rats weighing 350 g, right mandibular osteotomy was performed and the distraction device was placed. The distraction device was custom made using micro-implants, expansion screws, and acrylic resin., Study Protocol: latency: 6 days, distraction: ¼ turn (0.175 mm) once a day during 6 d, consolidation: 28 d after distraction phase, sacrifice. DO-treated and contralateral hemimandibles were dissected and compared macroscopically and using radiographic studies. Histological sections were obtained and stained with H&E. A distraction gap filled with newly formed and mature bone tissue was obtained. This model of mandibular DO proved useful to obtain adequate quantity and quality of bone to study bone regeneration.

Arsenic (As) toxicity is a global health problem affecting millions of people, the most toxic forms being Arsenites [As(III)] and Arsenates [As(V)]. Arsenic intoxication can occur through different exposure routes. The aim of the present work was to determine the effect of As on endochondral ossification and bone remodeling in experimental animals, by means of biochemical, histologic, and histomorphometric determinations. Sixteen male Wistar rats, 100g body weight (b.w.), were divided into two groups: experimental group (n=8), treated with 10mg/l of NaAsO(2) in their drinking water, receiving 0.21mg/kgb.w./day during 45 days; and control group (n=8) remained untreated. On day 45, blood samples were obtained by cardiac puncture to perform hematologic blood counts and biochemical determination. The animals were killed, the tibiae, femurs, kidneys and livers were resected, fixed in formalin and processed histologically. Tibia and femur sections were obtained and stained with H&E. The following histomorphometric parameters were determined on tibia and femur sections: bone volume (BV/TV), thickness of growth plate cartilage (GPC.Th) and thickness of hypertrophic zone (HpZ.Th). Biochemical determinations showed that experimental animals exhibited neutrophilia and a decrease in lymphocytes and monocytes. As levels were below 1 microg/dl in both groups. The femur sections of the experimental group showed (1) a statistically significant increase in total growth cartilage plate thickness (p<0.05) at the expense of the hypertrophic zone (p<0.05); (2) subchondral trabecular bone sealed to the growth plate with a non-significant increase in primary spongiosa bone volume. These results suggest that As alters endochondral ossification.

Unlabelled: Vascular endothelial growth factor (VEGF) is a protein that increases vascular permeability and induces the proliferation, migration and survival of endothelial cells. Bisphosphonates (BPs) are antiresorptive drugs that are widely used in the treatment of bone metabolism diseases and bone metastases. Since 2003, cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) have been reported. Few papers explain the mechanisms that induce BRONJ; some authors mention alterations in bone remodelling and a certain antiangiogenic effect of BPs. The aim of this study is to evaluate the expression of VEGF in bone marrow cells and the number of blood vessels and area occupied by them in animals treated with the BP sodium olpadronate (OPD). We used 16 Wistar rats, 60 days old, divided into two groups, experimental (OPD) and control. The OPD group received 0.3 mg/kg/week intraperitoneal OPD for 5 weeks. The control group received an equivalent intraperitoneal volume of physiological saline solution. After euthanasia, hemimandibles were processed and mesio-distal histological sections of the first molar were prepared. Sections were stained with hematoxylin-eosin (HE), immunohistochemical detection of VEGF was performed (sc-7269) and the following histomorphometric parameters were evaluated: In HE-stained sections--number of blood vessels per sq. mm. and percentage (%) of area occupied by blood vessels in relation to total area evaluated; in sections with immunohistochemical detection of VEGF--number of VEGF+ bone marrow cells per sq. mm. Data underwent statistical analysis. Number of blood vessels/mm2 was significantly lower in the OPD group (OPD: 92 +/- 16;, Control: 140 +/- 31; p < 0.05) and % vascular area/total area evaluated showed no significant difference (OPD: 15.6 +/- 6.1;, Control: 10.2 +/- 4.2). Number of VEGF+ cells/mm2 was lower in the OPD group than in the control group, with statistically significant differences (OPD: 7804.8 +/- 597;, Control: 13187.6 +/- 894; p < .001). The results of this study suggest that monosodium olpadronate has an antiangiogenic effect. Further studies are needed to reveal its potential as an antitumor agent and its connection with the onset of BRONJ.

Bisphosphonates (BPs) are widely used to treat several bone pathologies. Their action on bone cells depends on cell lineage, promoting or preventing apoptosis in osteoclastic and osteoblastic lineage respectively. Bone cells and bone marrow (BM) are closely related. Bone marrow megakaryocytes regulate bone turn-over. The objective of this in vivo experimental work was to evaluate the effect of olpadronate (OPD) on osteoclasts (Ocs) and megakaryocytes (Mks) using histomorphometric, histochemical, and immunohistochemical methods. Healthy female Wistar rats were used: experimental and Sham animals received OPD or vehicle during five weeks. After sacrifice, kidneys, liver, spleen, femurs and tibiae were dissected and fixed for histological processing. H and E, histochemical detection of TRAP and immunohistochemical detection of TUNEL and RANKL were performed. Results showed increased bone volume and number of Ocs, larger Ocs with more nuclei, increase in Oc apoptosis, and loss of polarity in OPD-treated animals. Statistically significant association was found between apoptotic morphology and RANKL expression in Ocs. BM and spleen showed a significant increase in Mk number. The number of RANKL+Ocs and MKs per unit area increased. The increase in Oc apoptosis did not counteract the increase in Oc recruitment thus resulting in an increase in Oc number. Ocs recruitment could be associated with RANKL expression in Mks and apoptotic Ocs. The effect of OPD and other BPs on Mks should be investigated further to elucidate the mechanism by which BPs act on the bone-bone marrow functional unit, and understand the therapeutic implications of BPs.

Background: The incidence of dry mouth and its public health impact are increasing as the result of a progressively larger, medicated older population and because chronic diseases, like periodontitis, are prevalent pathologies among elderly patients. Periodontitis and continuous remodeling and rebuilding alveolar processes greatly affect the margin of the alveolar bone, and there is evidence indicating the role of submandibular glands in the regulation of immune/inflammatory reactions. The purpose of this study was to assess the effect of submandibular-sublingual complex ablation (Sx) on alveolar bone loss in rats submitted or not to ligature-induced experimental periodontal disease (EP)., Methods: Wistar male rats were submitted to Sx or sham operations (day 0). Two weeks later, unilateral EP was induced on the right mandibular first molars for 7 days with the contralateral side serving as control. Bone loss at the level of the dental pieces was estimated by bone histomorphometry on mesio-distally oriented sections of the molars and by the determination on lingual and vestibular mandibular surfaces of the distances from the cemento-enamel junction to the alveolar crest., Results: Sx and EP significantly increased lingual and vestibular alveolar bone loss. Molars with EP exhibited greater lingual loss in Sx animals compared to those with the sham operation. EP induced similar interradicular bone loss in sham and Sx rats., Conclusion: Sx has a deleterious effect on the periodontal tissues, particularly marginal alveolar bone, indicating the importance of the submandibular/sublingual glands in maintaining healthy periodontal conditions.

Exposure to uranium is an occupational hazard to workers who continually handle uranium and an environmental risk to the population at large. Since the cellular and molecular pathways of uranium toxicity in osteoblast cells are still unknown, the aim of the present work was to evaluate the adverse effects of uranyl nitrate (UN) on osteoblasts both in vivo and in vitro. Herein we studied the osteoblastic ultrastructural changes induced by UN in vivo and analyzed cell proliferation, generation of reactive oxygen species (ROS), apoptosis, and alkaline phosphatase (APh) activity in osteoblasts exposed to various UN concentrations (0.1, 1, 10, and 100 microM) in vitro. Cell proliferation was quantified by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, ROS was determined using the nitro blue tetrazolium test, apoptosis was morphologically determined using Hoechst 3332 and APh activity was assayed spectrophotometrically. Electron microscopy revealed that the ultrastructure of active and inactive osteoblasts exposed to uranium presented cytoplasmic and nuclear alterations. In vitro, 1-100 microM UN failed to modify cell proliferation ratio and to induce apoptosis. ROS generation increased in a dose-dependent manner in all tested doses. APh activity was found to decrease in 1-100 microM UN-treated cells vs. controls. Our results show that UN modifies osteoblast cell metabolism by increasing ROS generation and reducing APh activity, suggesting that ROS may play a more complex role in cell physiology than simply causing oxidative damage.

Clinical and experimental studies have shown that lithium carbonate causes a number of clinical manifestations such as hyperparathyroidism, hypothyroidism, renal toxicity, and diabetes insipidus. The effect of this drug on the bone biology of experimental animals has not been studied to date. Therefore, the aim of the present experimental work was to study the effect of lithium on bone tissue in healthy sexually mature Wistar rats. Ten female Wistar rats, aged 312-412 months, 210-220 g body weight, were assigned to one of the following groups: untreated control group and experimental group receiving 45 mg/kg body weight/day of lithium carbonate in their drinking water during 3 months. Prior to euthanasia, blood samples were obtained in order to determine plasma phosphorus, calcium alkaline phosphatase, and lithium. After sacrifice, the tibiae were resected, processed, and embedded in paraffin. The following histomorphometric parameters were determined on digital photographs of the histologic sections: BV/TV (%), bone volume; Tb.Th (microm), trabecular thickness; Tb.N (mm(-2)/mm), trabecular number; Tb.Sp (microm), trabecular separation; Ob.S/BS (%), osteoblast surface; ES/BS (%), total erosive surface; Lc.S (%), lining cells surface; and GPC.Th (microm), thickness of growth plate cartilage. The results showed that administration of lithium carbonate cause bone loss in healthy sexually mature Wistar rats. Although the mechanism involved in bone toxicity remains to be clarified, the results obtained in the present study suggest that patients under long-term lithium therapy should be thoroughly evaluated, particularly those presenting other risk factors of osteopenia, such as menopause, low calcium intake, alcohol consumption, and glucocorticoid therapy.

The present study was carried out to obtain an experimental model of vitamin D (vit D) insufficiency and established osteopenia (experiment 1) to then investigate whether vit D status, i.e. normal or insufficient, interferes with bone mass recovery resulting from bisphosphonate therapy (experiment 2). Rats (n = 40) underwent OVX (n = 32) or a sham operation (n = 8). The first 15 days post-surgery, all groups were kept under fluorescent tube lighting and fed a diet containing 200 IU% vit D (+D). They were then assigned during an additional 45 days to receive either +D or a diet lacking vit D (-D) and kept under 12 h light/dark cycles using fluorescent or red lighting. Serum 25HOD was significantly lower in -D rats (P < 0.0001). The type of lighting did not induce differences in 25OHD, calcium (sCa), phosphorus (sP), bone alkaline phosphatase (b-AL), CTX, bone density or histology. No osteoid was observed in undecalcified bone sections. Experiment 2 (105 days): rats were fed either +D or -D according to experiment 1 and were treated with either placebo or 16 mug olpadronate (OPD)/100 g rat/week during the last 45 days. Whereas 25HOD was significantly lower (P < 0.0001) in -D/OPD than in +D/OPD rats, no significant differences in sCa, sP, b-AL or CTX were observed. OPD prevented the loss of lumbar spine (LS) and proximal tibia (PT) BMD and the decrease in bone volume (BV/TV) (P < 0.05) and in the number of trabeculae observed in untreated rats. However, +D/OPD animals presented significantly higher values of LS BMD, PT BMD and BV/TV than -D/OPD rats (P < 0.05). No osteoid was observed in undecalcified sections of bone. In summary, this is the first experimental study to provide evidence that differences in vit D status may affect the anticatabolic response to bisphosphonate treatment. However, the molecular mechanism through which vit D insufficiency reduces the effect of the aminobisphosphonate remains to be defined.

Background: Periodontitis is characterized by gingival inflammation, periodontal pocket formation, and bacterial plaque that lead to alveolar bone destruction. Research studies have recently begun to evaluate the effect of antiresorptive agents using experimental models of periodontitis. Bisphosphonates are the most frequently tested antiresorptive agents; their main effect is inhibition of bone resorption. The aim of this study was to perform a histomorphometric evaluation of the preventive effect of monosodium olpadronate (OPD), an aminobisphosphonate, on experimental periodontitis (EP)., Methods: Twenty male Wistar rats were used in this experiment. The animals were assigned to one of two groups: group I: EP; and group II: EP plus topical administration of OPD (EP + OPD). The contralateral side in both groups served as untreated controls (CI and CII), respectively. Mesio-distally oriented sections of each lower first molar were obtained for histomorphometric evaluation., Results: The treated group (EP + OPD) exhibited marked inhibition of bone loss; interradicular bone volume was significantly greater than that observed in the EP group. The height of the periodontal ligament in the interradicular alveolar bone, which served as an indirect measure of bone loss, was found to be significantly increased in the EP group as compared to the EP + OPD group. Osteoclasts in the OPD treated group were detached from the bone surface, were round in shape, and exhibited a loss of polarity and lack of ruffled borders., Conclusions: The dose used herein was found to inhibit bone loss and to cause marked morphologic changes in osteoclasts. The drug effectively prevented bone loss caused by periodontitis.

Background: Chronic destructive periodontal disease is characterized by gingival inflammation, periodontal pocket formation, and bacterial plaque that lead to alveolar bone destruction. Polymorphonuclear neutrophil leukocytes (PMNs) are the first line of defense against infection caused by dental plaque bacteria. Renal patients present functional abnormalities of PMN, including impaired chemotaxis, phagocytosis, and intracellular killing of bacteria. In view of the above, the aim of this work was to evaluate the effect of renal failure on bone damaged by periodontal disease using histomorphometric and histochemical parameters., Methods: Twenty male Wistar rats weighing 250 g were assigned to one of the following four groups: 1) control (no treatment); 2) renal failure (RF); 3) periodontal disease (PD); and 4) renal failure plus periodontal disease (RF+PD). All the animals were sacrificed 31 days after the onset of the experiment. Mesio-distally oriented sections of the first lower molar were obtained for histomorphometric and histochemical evaluation., Results: Total erosion, active erosion, and total number of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts were found to be increased in the RF+PD group compared with the PD group., Conclusion: Our results demonstrate increased bone resorption in animals with untreated renal failure and periodontal disease, and thus indicate that the release of different factors by inflammatory cells is magnified, accelerating the progression of the disease in this animal model.

Exposure to uranium is a risk for the workers involved in uranium mining, purification, and manufacture, principally by its ingestion or inhalation. It is also a risk for the population at large in case of intake of contaminated water or food. Uranium induces nephropathy that is characteristic of heavy metals, which can lead to death. The toxic effects of uranium can be prevented by a biphosphonate, ethane-1-hydroxy-1,1-bisphosphonate (bisodic etidronate), administered orally or subcutaneously. Employing bisodic etidronate, our laboratory obtained satisfactory results in terms of survival in adult mice, adult rats, and suckling rats. The aim of the present study was to evaluate the efficacy of bisodic etidronate for preventing renal dysfunction induced by a lethal dose of uranyl nitrate, employing serum levels of urea and creatinine as end-points. Two experiments were performed over different time periods, i.e., Experiment A: 48 h, Experiment B: 14 d. Each experiment was performed with 4 groups of 20 male Balb/c mice each, 25 g average body weight. Three of these groups received 350 mg kg(-1) of body weight of uranyl nitrate by gavage (forced oral administration). Two of the three exposed groups were treated with bisodic etidronate either by gavage in a dose of 500 mg kg(-1) body weight or with a subcutaneous injection of 50 mg kg(-1) body weight. The fourth group served as control. Survivors of the experimental groups were sacrificed at the end of the experiment by overdose of inhalation anesthetic (ether). The kidneys were routinely processed for histological analysis. Blood samples were taken by cardiac puncture to assess urea and creatinine serum levels. Urea and creatinine serum levels were markedly lower at 48 h in exposed animals treated with bisodic etidronate than in untreated exposed animals. On day 14 these values in exposed and treated animals did not differ significantly from control values. The renal function of animals treated with orally or subcutaneous bisodic etidronate that survived uranyl nitrate exposure was markedly improved compared to the controls of untreated exposed animals at 48 h. At 14 days, treatment with bisodic etidronate averted renal damage. At this time, the histologic study of kidneys showed images of tissue recovery. These results suggest that the use of EHBP may be of great value in reducing the renal damage.

The risk of oral exposure to uranium potentially involves the population at large. Tooth eruption and development are ongoing processes that begin during fetal development and continue until the age of 18 y. Since one of the mechanisms involved in tooth eruption is bone formation and it is well documented that uranium inhibits bone formation, the aim of the present work was to study the effect of oral administration of uranyl nitrate (UN) on tooth eruption and development. Wistar rats aged 1 and 7 d were orally administered a single dose of 90 mg kg(-1) body weight of uranyl nitrate. Two age matched groups received an equal volume of saline and served as controls. The animals were killed at 7 and 14 d of age, respectively. Mandibles were resected and processed to obtain bucco-lingual sections oriented at the level of the mesial root of the first mandibular molar, and histomorphometric studies were performed. Results showed that an acute high dose of uranyl nitrate delays both tooth eruption and development, probably due to its effect on target cells.

The present study reveals the inhibitory effect of iron intoxication on the process of dentine mineralization. Wistar rats were injected intraperitoneally with iron dextran at 0.88 g/kg body weight per day for 10 days during the period of odontogenesis. An age-matched group was injected intraperitoneally with bisodium etidronate (EHBP) at 20 mg/kg body weight per day for 10 days. Another age-matched group was treated with similar amounts of saline intraperitoneally and considered as control. At the end of the experimental period the animals intoxicated with iron exhibited non-mineralized dentine and mineralized bone. The animals treated with EHBP showed non-mineralized dentine and bone. These findings would suggest the existence of different mineralization mechanisms for bone and dentine.

A dynamic histomorphometric study of bone loss in periodontitis induced by inserting a thread ligature around the neck of the lower first molar of Wistar rats weighing 300 g. was performed. Bone formation fronts were labelled twice by tetracycline injection (day 1 and day 14). On day 16 the animals were divided into 4 groups: Two experimental (ligature in place) and two controls (no ligature). Animals in one experimental and one control groups were killed 72 hours post insertion, and the other two groups 96 hours post-insertion. Grinding sections of the first molar were obtained to perform histomorphometric studies on microphotographs taken under fluorescence microscopy. At 72 hours results showed total loss of the double labeling in the mesial wall, partial loss in the top of the crest and no loss in the distal wall. At 96 hours, loss of the double labeling at the top of the crest was greater, while only one label (the first) could be observed in the distal wall. These results show that in this periodontitis experimental model, bone loss is initiated and is more rapid in the bone remodeling (mesial) wall than in the modeling (distal) wall. This understanding of bone loss dynamics enables the characterization of the model employed herein, contributing to further studies on the course of periodontal disease under different experimental conditions.

The development of bone disease in patients with chronic renal failure is well known. Renal patients frequently suffer anemia and iron oral therapy and/or transfusions are used to treat them. Recent findings show that iron could be a factor that provokes bone lesions but the alterations it causes are not well known. The aim of this work was to study the effect of iron intoxication on endochondral ossification, a recognized model for bone growth evaluation. Male Wistar rats weighing 250-300 g were used. They received 88 mg of dextran-iron per day intraperitoneally during 34 days. The experimental and control groups were killed on day 34. A histomorphometric study of the endochondral plate was performed on histologic sections of tibiae. The results obtained show that iron overloading inhibits endochondral ossification.

The work presented herein, is an experimental study on the effect of an orthodontic appliance with a helicoidal spring designed to exert force toward palatine--i.e. in the opposite direction to the natural vestibular drift--on a bone remodelling surface. The appliance consists of two stainless steel molar bands, with a horizontal bracket tube welded to their palatal aspect through which the arms of the helicoidal spring are passed. Wistar rats, 250 g body weight, were fitted with the device for 48 and 96 hours. One group of rats was administered two doses of tetracycline hydrochloride prior to device placement, in order to label mineralizing fronts. Histomorphometric studies of the periodontal wall of the palatine alveolar bone showed a marked increase of bone resorption at both experimental time points together with an increase in the number of osteoclasts, and no tetracycline labelling after 48 hours. The results show that compressive forces are capable of stimulating resorption, even on bone modelling surfaces. The pressure applied would stimulate osteoblasts to send out signals for osteoclast recruitment and activity.