Consideraciones Metodológicas en el Estudio Histomorfométrico del Hueso Interradicular de los Primeros Molares Inferiores de Ratas Wistar / Methodological Considerations in the Histomorphometric Study of the Interradicular Bone of the First Lower Molars of Wistar Rats

El objetivo fue evaluar el volumen óseo BV/TV (%) del hueso interradicular en ratas Wistar: A) en relación a la edad; B) en relación a la zona de estudio en animales de la misma edad. Se utilizó Grupo A) 15 ratas Wistar hembras de 6 (I), 10 (II) y 14 (III) semanas, Grupo B) 9 ratas Wistar macho de 8 semanas. Tras la eutanasia, se extrajeron los maxilares inferiores y se procesaron histológicamente para obtener cortes mesio-distales del primer molar inferior coloreados con H.E. Sobre microfotografías digitales se evaluó el BV/TV (%). El análisis estadístico se realizó en A) mediante ANOVA y Bonferroni test y en B) se calculó el rango (R). Los resultados en A) el BV/TV (%) aumenta significativamente con la edad de los animales; en B) se encontró que el BV/TV (%) varía hasta un 20% si se considera el volumen total y este rango disminuye a 8.3% al estudiar su mitad coronal. En conclusión, el BV/TV (%) del hueso interradicular del primer molar inferior de ratas Wistar varía considerablemente con la edad de los animales y en animales de una misma edad -según se considere evaluar todo el hueso interradicular del espacio alveolar o la mitad superior del mismo-. Los resultados de este trabajo recomiendan emplear animales de la misma edad y realizar mediciones histomorfométricas empleando la mitad coronal de dicho hueso, especialmente en los diseños de periodontitis experimental (AU)

La singularidad de los maxilares dentro del sistema esquelético / Singularity of the jaws within the skeletal system

En la odontología es frecuente que se describa la peculiaridad de los huesos maxilares en cuanto a la resistencia a las infecciones en comparación con otros huesos de la economía. O que se plantée un desafío cuando es necesario tomar una decisión acerca de aplicar diferentes conductas terapéuticas en pacientes con patologías óseas sistémicas. Por ello, esta actualización tuvo como objetivo realizar una revisión de la bibliografía para integrar y evidenciar las diferencias y similitudes entre los diferentes huesos de la economía haciendo hincapié en los huesos maxilares. Si bien éstos poseen una gran cantidad de similitudes con el resto de los huesos, también presentan diferencias que los hacen entidades únicas dentro del sistema esquelético como el origen embriológico en las células de las crestas neurales, su alta tasa de remodelación, sin olvidar que estos huesos alojan a órganos que poseen una parte de su estructura en el medio interno y otra porción en medio externo de la cavidad bucal: las piezas dentarias (AU)

Impairment of rat tooth eruption in pups born to mothers exposed to chronic stress during pregnancy.

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.

El rol de los restos epiteliales de Malassez en el ligamento periodontal / The role of Malassez epithelial rests in the periodontal ligament

El periodonto de inserción es definido como aquellos tejidos que soportan y rodean a la raíz dentaria y está compuesto opr tres estructuras que constituyen una unidad topográfica y funcional: el cemento radicular, el ligamento periodontal y el hueso alveolar. Estas estructuras evolucionan interrelacionada y coordinadamente durante la vida del diente, adaptándose a las modificaciones que surgen en el medio bucal. El objetivo de este trabajo es transmitir los nuevos conceptos emergentes vinculados al periodonto de inserción, centrándonos fundamentalmente en la estructura y función de los restos epiteliales de Malassez (REM), células presentes en el ligamento periodontal. Si bien es conocida la participación de estas células en procesos patológicos, entre ellos, quistes inflamatorios y tumores odontogénicos, poco se sabe de los mismos. Por ello, para poder llevar a cabo esta actualización sobre los REM, se consideraron las últimas publicaciones al respecto que figuran en la base de datos de Pubmed. A través de esta búsqueda, se obtuvo información referida a su descubrimiento, origen embriológico, características histológicas, productos elaborados por los REM y posibles funciones por ellos desempeñadas. Se puede decir entonces, que los REM desempeñarían un papel importante dentro del ligamento periodontal normal y, posiblemente, durante las terapias periodontales regenerativas. Si bien se requieren más estudios al respecto, es posible por lo tanto, reivindicar a estas células en su función y considerarlas mucho más que simples vestigios de la odontogénesis, presentes en el ligamento periodontal.

El rol de los restos epiteliales de Malassez en el ligamento periodontal / The role of Malassez epithelial rests in the periodontal ligament

El periodonto de inserción es definido como aquellos tejidos que soportan y rodean a la raíz dentaria y está compuesto opr tres estructuras que constituyen una unidad topográfica y funcional: el cemento radicular, el ligamento periodontal y el hueso alveolar. Estas estructuras evolucionan interrelacionada y coordinadamente durante la vida del diente, adaptándose a las modificaciones que surgen en el medio bucal. El objetivo de este trabajo es transmitir los nuevos conceptos emergentes vinculados al periodonto de inserción, centrándonos fundamentalmente en la estructura y función de los restos epiteliales de Malassez (REM), células presentes en el ligamento periodontal. Si bien es conocida la participación de estas células en procesos patológicos, entre ellos, quistes inflamatorios y tumores odontogénicos, poco se sabe de los mismos. Por ello, para poder llevar a cabo esta actualización sobre los REM, se consideraron las últimas publicaciones al respecto que figuran en la base de datos de Pubmed. A través de esta búsqueda, se obtuvo información referida a su descubrimiento, origen embriológico, características histológicas, productos elaborados por los REM y posibles funciones por ellos desempeñadas. Se puede decir entonces, que los REM desempeñarían un papel importante dentro del ligamento periodontal normal y, posiblemente, durante las terapias periodontales regenerativas. Si bien se requieren más estudios al respecto, es posible por lo tanto, reivindicar a estas células en su función y considerarlas mucho más que simples vestigios de la odontogénesis, presentes en el ligamento periodontal.(AU)

Effect of monosodium olpadronate on osteoclasts and megakaryocytes: an in vivo study.

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.

Ultrastructural and metabolic changes in osteoblasts exposed to uranyl nitrate.

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.

Renal function in mice poisoned with oral uranium and treated with ethane-1-hydroxy-1,1-bisphosphonate (EHBP).

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.

Exposure to oral uranyl nitrate delays tooth eruption and development.

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.

Iron overloading inhibits dentine mineralization.

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.

Iron overloading inhibits dentine mineralization

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.

Iron overloading inhibits dentine mineralization.

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.

Dynamics of bone loss in experimental periodontitis.

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.

Iron overloading inhibits endochondral ossification.

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.

Effect of compressive forces on a bone modelling surface.

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.