Hazardous effects of urban air particulate matter acute exposure on lung and extrapulmonary organs in mice.

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.

Effect of alendronate on the mandible and long bones: an experimental study in vivo.

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.

Prenatal Exposure to Continuous Constant Light Alters Endochondral Ossification of the Tibiae of Rat Pups.

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.

Histomorphometric study and three-dimensional reconstruction of the osteocyte lacuno-canalicular network one hour after applying tensile and compressive forces.

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.

Effects of lead exposure on growth and bone biology in growing rats exposed to simulated high altitude.

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.

Lead bone toxicity in growing rats exposed to chronic intermittent hypoxia.

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.

Effect of strontium ranelate on bone remodeling.

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.

Epithelial rests of Malassez in experimental animals at different ages.

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.

Los restos epiteliales de Malassez (ERMs) son fragmentos de la vaina de Hertwig, en el ligamento periodontal. Se conoce extensamente su rol en la etiología de procesos patológicos y actualmente las evidencias los vinculan al mantenimiento de la homeostasis periodontal. El objetivo del presente trabajo fue evaluar el comportamiento de los ERMs en relación a los cambios de los tejidos periodontales durante el crecimiento en un modelo experimental de ratas Wistar. Se obtuvieron cortes mesio-distales del 1er molar inferior de ratas Wistar de 1 mes (n=7), 3 meses (n=7) y 5 meses (n=6) de edad. En cortes coloreados con H&E, se evalúo: N° de ERMs/mm, tamaño/ área de ERMs (pm2), altura del ligamento periodontal (PL.h) (pm), área de cemento en la zona de furcación (C.Ar) (pm2) y área del hueso relacionado con la zona de los ERMs (BAr/TAr) (%). Los resultados se analizaron estadísticamente mediante ANOVA y Bonferroni post-hoc. El N° de ERMs/mm disminuyó significativamente con la edad (1m: 4.34±1.51, 3m: 1.48±0.89, 5m: 0.27± 0.50, p<0.05), observándose una gran variabilidad de tamaño. Se observó un aumento significativo del CAr (1m: 3418.96 ± 905.88, 3m: 19365.76 ± 5500.52, 5m: 32182.76 ± 7114.51, p< 0.05) y del (BAr/TAr). interradicular (1m: 25.26 ± 2.37, 3m: 44.70 ± 3.95, 5m: 46.81 ± 7.80, p< 0.05: 1 vs 3, 1 vs 5). Además, se halló una disminución significativa en PLh a los 5 meses (1m: 141.42 ± 29.25, 3m: 162.06 ± 28.21, 5m: 124.72 ± 18.67, p< 0.05: 1 vs 3, 3 vs 5). La disminución hallada del número de ERMs conforme aumenta la edad del animal podría vincularse con el aumento significativo del C.Ar y la reducción del PL.h. Resta por dilucidar su relación con el aumento de BAr/TAr. observado. Se requieren más estudios para profundizar sobre el rol de los ERMs y su relación con los tejidos periodontales ante distintos estímulos normales y patológicos.

Evaluation of vascular endothelial growth factor (VEGF) in interradicular bone marrow in olpadronate treated animals.

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.

Chronic exposure to urban air pollution from Buenos Aires: the ocular mucosa as an early biomarker.

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.

Influence of submandibulectomy on alveolar bone loss in rats.

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.

Study of epithelial rests of Malassez in an experimental periodontitis model.

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.

El objetivo de este trabajo ha sido evaluar las alteraciones morfológicas de epithelial cell rests of Malassez (ERMs) y su relación con la reabsorción radicular, en un modelo de experimental periodontitis (EP) a 4 y 11 días. La EP fue inducida en 14 ratas Wistar macho mediante la colocación de una ligadura de hilo de algodón alrededor del cuello del primer molar inferior derecho, a 4 (n=7) y 11 (n=7) días. El molar contralateral (izquierdo) fue usado como control. Tras la eutanasia, se extrajeron los maxilares y se procesaron histológicamente para la obtención de cortes en sentido mesio-distal que se colorearon con H&E y técnica histoquímica de detección de tartrate-resistant acid phosphatase (TRAP). Se tomaron microfotografías y se evaluaron los siguientes parámetros histomorfométricos: Bone area (BAr./TAr)(%), N° de ERMs/mm2, N° de células/ERM, área de ERMs (µm2), y porcentaje de superficies de reabsorción radicular (%RR). Los resultados se analizaron estadísticamente mediante Anova y Bonferroni post hoc (p≤ 0.05). En los molares con PE se observó una pérdida ósea significativa en relación a sus controles. En el grupo EP 4 días no se observaron cambios en los parámetros en relación a los ERMs, sin embargo, en el grupo PE de 11 días se registró reabsorción radicular (%RR) significativa (C: 3.21±3.07, EP-4D: 3.91±3.17, EP-11D: 23.67±11.40; p≤ 0,05) junto con un aumento del área de ERMs (µm2) (C: 455.87±145.42, EP-4D: 577.6±156.1, EP-11D: 1046.3±582.9; p≤ 0,05). No se observaron ERMs TRAP+ en ninguno de los dos grupos. La hipertrofia de los ERMs, podría estar relacionada a la participación de los mismos en mecanismos tendientes a la homeostasis periodontal, inhibiendo dicha reabsorción y contribuyendo a la regeneración periodontal.

Evaluation of apoptosis and osteopontin expression in osteocytes exposed to orthodontic forces of different magnitudes.

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.

Hasta el momento no se ha dilucidado la respuesta temprana in vivo de los osteocitos a la aplicación de fuerzas de diferentes magnitudes sobre el hueso. El objetivo de este estudio fue examinar la respuesta temprana de la aplicación de una fuerza ortodóncica muy liviana (FL: 0,16 N) y muy fuerte (FF: 2,26 N) durante una hora sobre la expresión de apoptosis y osteopontina (OPN) en los osteocitos del hueso alveolar, en ratas. Resultados: FL: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución significativa en el número de osteocitos TUNELpositivos. FF: en comparación con el grupo control, mostraron un aumento significativo en la expresión de OPN y una disminución signi ficativa en el número de osteocitos TUNELpositivos. Nuestros resultados muestran que los osteocitos responden muy temprano a la aplicación de fuerzas de tensión y presión de diferentes magnitudes, y la aplicación de fuerzas disminuye el número de osteocitos apoptóticos y aumenta la expresión de OPN. Estos resultados permiten concluir que los osteocitos se activan rápidamente cuando se los somete a fuerzas aplicadas localmente, ya sean estas fuerzas de presión o tensión, livianas o fuertes.

Vitamin D insufficiency reduces the protective effect of bisphosphonate on ovariectomy-induced bone loss in rats.

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.

Effect of topical administration of monosodium olpadronate on experimental periodontitis in rats.

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.

Effect of lithium carbonate on subchondral bone in sexually mature Wistar rats.

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.

Histomorphometry of the tibia and mandible of healthy female Wistar rats at different stages of growth.

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.

Epithelial rests of Malassez in experimental animals at different ages / Restos epiteliales de Malassez en animales experimentales a diferentes edades

ABSTRACT 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 (μm2), height of periodontal ligament (PL.h) (pm), area of cement in the furcation zone (C.Ar) (μm2) 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.

RESUMEN Los restos epiteliales de Malassez (ERMs) son fragmentos de la vaina de Hertwig, en el ligamento periodontal. Se conoce extensamente su rol en la etiología de procesos patológicos y actualmente las evidencias los vinculan al mantenimiento de la homeostasis periodontal. El objetivo del presente trabajo fue evaluar el comportamiento de los ERMs en relación a los cambios de los tejidos periodontales durante el crecimiento en un modelo experimental de ratas Wistar. Se obtuvieron cortes mesio-distales del 1er molar inferior de ratas Wistar de 1 mes (n=7), 3 meses (n=7) y 5 meses (n=6) de edad. En cortes coloreados con H&E, se evalúo: N° de ERMs/mm, tamaño/ área de ERMs (μm2), altura del ligamento periodontal (PL.h) (μm), área de cemento en la zona de furcación (C.Ar) (μm2) y área del hueso relacionado con la zona de los ERMs (BAr/TAr) (%). Los resultados se analizaron estadísticamente mediante ANOVA y Bonferroni post-hoc. El N° de ERMs/mm disminuyó significativamente con la edad (1m: 4.34±1.51, 3m: 1.48±0.89, 5m: 0.27± 0.50, p<0.05), observándose una gran variabilidad de tamaño. Se observó un aumento significativo del CAr (1m: 3418.96 ± 905.88, 3m: 19365.76 ± 5500.52, 5m: 32182.76 ± 7114.51, p< 0.05) y del (BAr/TAr). interradicular (1m: 25.26 ± 2.37, 3m: 44.70 ± 3.95, 5m: 46.81 ± 7.80, p< 0.05: 1 vs 3, 1 vs 5). Además, se halló una disminución significativa en PLh a los 5 meses (1m: 141.42 ± 29.25, 3m: 162.06 ± 28.21, 5m: 124.72 ± 18.67, p< 0.05: 1 vs 3, 3 vs 5). La disminución hallada del número de ERMs conforme aumenta la edad del animal podría vincularse con el aumento significativo del C.Ar y la reducción del PL.h. Resta por dilucidar su relación con el aumento de BAr/TAr. observado. Se requieren más estudios para profundizar sobre el rol de los ERMs y su relación con los tejidos periodontales ante distintos estímulos normales y patológicos.

Sequential administration of alendronate and strontium ranelate: histomorphometry and bone biomechanics in ovariectomized animals.

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.

Si bien la primera opción terapéutica para el tratamiento farmacológico de la osteoporosis son los bisfosfonatos (BPs), luego de los primeros reportes en 2003 de los casos de osteone crosis de mandíbula asociada al uso de dichas drogas y las fracturas atípicas de fémur, se ha evaluado su seguridad a largo plazo. Además, en aquellos pacientes que no responden al tratamiento con BPs y mantienen elevado el riesgo de fractura, es necesario suspender su administración y alternar con otras drogas. Una de las que se ha utilizado en la clínica luego del tratamiento con BPs es el ranelato de estroncio (SR). Existen varios trabajos clínicos que reportan los efectos de la administra ción secuencial de ambas drogas, aunque estudios experi men tales con un diseño secuencial aun no se han reportado. Por ello el objetivo de este trabajo ha sido evaluar el efecto de la administración secuencial de alendronato, seguido de ranelato de estroncio sobre el tejido óseo de ratas ovariectomizadas. Se utilizaron 48 ratas Wistar hembras de dos meses de edad divididas en 6 grupos de 8 animales cada uno. El día 1 de experiencia todas fueron ovariectomizadas. El día 30 se comenzó con la administración de alendronato (ALN) en una dosis de 0.3 mg/kg/semana o vehículo (VEH) durante 8 semanas. Luego de este período se sacrificaron dos grupos (uno que recibió ALN y su correspondiente control (sólo vehículo). Los cuatro grupos restantes continuaron con ranelato de estroncio (SR) en el agua de bebida durante 4 meses en una dosis de 290 mg/kg/día o sólo agua corriente( TW) Luego de ese período fueron eutanasiados. Así, los grupos experimentales conformados fueron: ALN+SR, ALN+TW, VEH+SR, VEH+TW, ALN y VEH. Para los estudios histomorfométricos se extrajeron ambas tibias y hemimandíbulas; para el estudio biomecánico se utilizó el fémur derecho. Los resultados fueron analizados mediante el test de ANOVA y el test de Bonferroni. Incrementaron significativamente la rigidez diafisaria, la carga elástica límite y la carga de fractura aquellos grupos que recibieron alendronato versus aquellos que no lo recibieron, independientemente del tratamiento posterior con SR. La carga de fractura además fue mayor en el grupo VEH+SR versus el control (VEH+TW). En cuanto al volumen óseo subcondral e interradicular evaluado histomorfométricamente fue significativamente mayor en aquellos animales que recibieron ALN versus aquellos que recibieron vehículo. No se detectaron diferencias entre aquellos grupos que recibieron SR y sus controles. El área y espesor cortical de la tibia no mostraron diferencias entre grupos. Los resultados obtenidos en el modelo estudiado tanto a nivel del volumen óseo subcondral y cortical de la tibia como a nivel del hueso interradicular del maxilar inferior, mostraron que la combinación de ALN con SR y la administración aislada de ALN son igualmente efectivas para prevenir la pérdida ósea causada por la depleción estrogénica de la ovariectomía.

Alveolar bone response in an experimental model of renal failure and periodontal disease: a histomorphometric and histochemical study.

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.