Evaluación de la mineralización en mandíbula y diente de trucha arcoíris (Oncorhynchus mykiss) mediante microscopía electrónica de barrido de presión variable acoplada a detector de espectroscopía de rayos X de energía dispersiva / Evaluation of mineralization in jaw and teeth of rainbow trout (Oncorhynchus mykiss) using variable pressure scanning electron microscopy coupled to energy-dispersive X-ray spectroscopy detector

RESUMEN: En salmonicultura se utilizan imágenes de rayos X, para evaluar la columna vertebral y las aletas, pero estas no permiten cuantificar los minerales que constituyen el hueso, para esto se utilizan otras técnicas que son destructivas. La Microscopía Electrónica de Barrido de presión variable (VP SEM) acoplada al detector espectroscopia de rayos X de energía dispersiva (EDX) acoplado, nos permite analizar la microestructura y a la vez determinar elementos químicos, porcentaje y distribución presentes en puntos específicos en una muestra. Se utilizaron 5 truchas control y 5 con deformidad mandibular, de agua dulce en etapa de smolt, se analizó la estructura y mineralización del hueso, se eligieron en promedio 6 puntos de cuantificación por región de interés y se analizaron mediante Microscopía Electrónica de Barrido (VP SEM-EDX). Los datos obtenidos muestran porcentaje en masa de Ca promedio en hueso articular de truchas fueron de 8.07 % y de 14.48 % en truchas con deformidad mandibular y control respectivamente y el porcentaje en masa promedio de P es de 4.07 % y 7.60 %, en truchas con deformidad mandibular y control respectivamente. Se identificó además otros elementos presentes en la muestra como Mg, Na, C, O, N, S, F, Zn, Al y Fe, con especial interés en el aumento de carbono en las muestras analizadas con deformidad mandibular y la presencia de aluminio en todas las muestras. La Técnica de VP SEM-EDX, permite evaluar de forma directa, sin destrucción de la muestra y con una preparación mínima de la muestra. En el hueso, la aplicación más frecuente de SEM-EDX es la medición del contenido de Ca y P y la relación que existe entre estos elementos Ca/P, en la muestra. Paralelamente la técnica nos permite la detección de otros microelementos provenientes del agua o de la alimentación y que eventualmente pueden provocar alteraciones en los peces, confirmando la hipótesis que el microanálisis elemental tiene utilidad para la salmonicultura.

SUMMARY: In salmon farming systems, X-ray images are used to evaluate the spine and fins, but these do not allow quantifying the minerals that make up the bone, for different techniques that are destructive are used. Variable Pressure Scanning Electron Microscopy (VP SEM) coupled to an Energy Dispersive X-ray spectroscopy detector (EDX), allows us to analyze the microstructure and at the same time determine chemical elements, percentages, and distribution present at specific points in a sample. Five control and five jaw deformity trout, from freshwater and in the smolt stage were used. The structure and mineralization of the bone were analyzed, an average six quantification points were chosen per region of interest (ROI) and then they were analyzed by Scanning Electron Microscopy (VP SEM-EDX). The data obtained have shown the average mass percentage of calcium in trout joint bone was 8.07 % and 14.48 % in jaw deformity and control trout, respectively; and the average mass percentage of phosphorus is 4.07 % and 7.60 %, in jaw deformity and control trout, respectively. Other elements present in the sample were also identified, such as magnesium, sodium, carbon, oxygen, nitrogen, sulphur, fluorine, zinc, aluminium, and iron, with special interest the increase of carbon in the analyzed samples with mandibular deformity and the presence of aluminum in all samples. The VP SEM-EDX Technique allows direct evaluation, without destruction of the sample and with minimal sample preparation. In bone, the most frequent application of SEM-EDX is the measurement of the content of calcium (Ca) and phosphorus (P) and the relationship that exists between these elements, calcium/phosphorus (Ca/P), in the sample. At the same time, the technique allows us to detect other microelements from water or food that can eventually cause alterations in fish, confirming the hypothesis that elemental microanalysis is useful for salmon farming.

Efectos de la administración local de PTH 1-34 a bajas dosis en un modelo experimental de periodontitis: estudio piloto / Local effects of low dose of PTH 1-34 on experimental periodontitis: pilot study

La periodontitis es una patología inflamatoria que aumenta la resorción de hueso alveolar (HA), pérdida de la inserción dentaria y posible exfoliación. Evaluamos el efecto de la administración intermitente de bajas dosis de parathormona (PTH) 1-34 sobre la recuperación de la masa ósea pérdida en un modelo experimental de periodontitis inducida por una ligadura periodontal (LP) con hilo de algodón alrededor de la pieza dentaria. Las ratas fueron divididas luego de 5 días en instaurada la periodontitis en: CT LP sin trata-miento y PTH LP tratados con 0,2 µg/kg PTH 1-34 subcutánea local, tres veces por semana por 17 días. El control absoluto fue un tercer grupo sin LP (CT). Se estudiaron parámetros antropométricos, bioquímicos e histomosfométricos en tibias y hemimandibulas. La calcemia, fosfatemia, CTX sérico, PTHi y vo-lumen óseo (BV/TV%) de tibias fueron similares en los tres grupos. El BV/TV% del HA fue significativamente menor en PTH LP respecto de CT pero mayor que CT LP (p<0.05). La pérdida ósea de HA porcentual fue significativamente mayor en CT LP (p<0.05). La altura del ligamento periodontal fue significativamente menor en PTH LP que en CT (p<0.05) y mayor respecto de CT LP, sin alcanzar diferencias significativas. Los resultados del presente estudio piloto sugieren que la administración intermitente de PTH en bajas dosis y durante un periodo de tiempo corto disminuye la progresión de la enfermedad periodontal sin generar efectos sistémicos. Como no se logró regenerar totalmente el tejido periodontal se requieren estudios adicionales. (AU)

Periodontitis is an inflammatory chronic disease with high prevalence in adults that induces a progressive alveolar bone (AB) loss leading to tooth loss. Experimental periodontitis can be induced in rats by cotton ligature placement (LP) in the gingival sulcus around the molar teeth. The biofilm accumulation and disruption of the gingival epithelium lead to bone resorption. We investigated whether intermittent administration of a low dose of PTH 1-34 may recover the alveolar bone loss in the experimental periodontitis induced in female Wistar rats. Animals were randomly divided in two groups which were subcutaneously injected with: saline solution (CT LP) or 0,2 µg/kg PTH 1-34 (PTH LP) three times per week during 17 days. Unligated rats were taken as healthy controls (CT). Anthropometric, biochemical and histologic analysis of tibia and hemimandible were done. No differences in serum calcium, phosphorus, CTX, PTHi or subchondral tibia bone volume (BV/TV%) were observed between the three groups. AB BV/TV% was significantly lower in PTH LP than in CT but higher than in CT LP (p<0.05). The highest percentage of AB loss was observed in CT LP. The height of periodontal ligament was lower in PTH LP than in CT (p<0.05) but not significantly higher than CT LP.The increase in AB loss by experimental periodontitis appears to be corrected by the intermittent administration of low doses of PTH without systemic effect. As the recovery of periodontal tissue was only partial, additional studies should be done.

Morphological study and mineral analysis of the lower mandible of adult Atlantic salmon (Salmo salar) from Scotland with mandibular deformation / Estudio morfológico y análisis de minerales de la mandíbula inferior de salmones del Atlántico (Salmo salar) adultos con deformación mandibular, provenientes de Escocia

Mandibular deformity is a condition that affects the jaw bone of adult salmon and has been observed in Norway and Chile, causing weight loss, poor quality of farmed fish and increased mortality. The causes range from high temperatures of the state of eggs, to poor nutrition phosphorus or vitamin C. This work aims to analyze this deformity by histochemical and mineral analysis technique during an episode presented in centers of the Scotia Sea. Jaw and spinal segments of 21 Atlantic salmon in Scotland were used. These samples were classified into three groups: Group 1: Severely deformed. Group 2: Mildly affected. Group 3: Normal controls. Four jaws per group were fixed in 10 % formalin and embedded in Paraplast, sections of 5 microns were performed using a Microm® microtome histochemical technique Von Kossa was used for the detection of calcium deposits, which highlights the calcium osteoid black and red color. For proximate analysis, and in order to obtain and compare levels of calcium, phosphorus, zinc and magnesium in total 9 bone jaws (6 affected with DM and 3 controls) and 9 body sections the Mann-Whitney test was used to compare these values between misshapen salmon and controls. To correlate values, jaw and body segment a Spearman corrrelation was applied. Fish group 1 presented a ventral deviation of the alveolar bone body. In fish group 2 prominence of the visible joint on both sides or unilaterally was observed. Comparing the values of % Ca, % P, % Mg and Zn jaws with DM and healthy ones with Mann Witney method it was found that the values of these minerals vary between salmon and controls affected. There was a significant difference in the percentage of P, which indicates that there is less P in affected fish vertebrae. Spearman correlation noted that the percentages of the minerals studied in dental bone and vertebral segments are uncorrelated. Rather, Von Kossa distribution indicates that Ca/P is not homogeneous in the dental bone, as a result of mineral resorption from the skeleton including the operculum, articular bone and dental towards kype. This paper states that Von Kossa histochemical technique showed significant differences between deformed fish and controls and also showed differences between the various segments of the dental bone. The alveolar bone is a dynamic structure adapted to continuous histological changes may be involved in MD, phosphorus deficient diets, coupled with the initial formation of Kippe.

La deformación mandibular es una patología que afecta al hueso dentario de salmones adultos, se observó en Noruega y en Chile, ocasionando disminución de peso, baja calidad de peces cultivados y aumento de la mortalidad. Las causas varían desde temperaturas elevadas al estado de ovas, hasta alimentación deficitaria en fósforo o vitamina C. Este trabajo tiene como propósito analizar esta deformación mediante una técnica histoquímica y de análisis de minerales durante un episodio presentado en centros de mar de Escocia. Se utilizó la mandíbula y segmento vertebral de 21 salmones del Atlántico de Escocia. Estas muestras se clasificaron en tres grupos Grupo 1: Severamente deformes. Grupo 2: Levemente afectados. Grupo 3: Controles normales. Cuatro mandíbulas por grupo fueron fijadas en formalina al 10 % y se incluyeron en paraplast, se realizaron cortes de 5 µm utilizando un micrótomo Microm®. Se utilizó la técnica histoquímica de Von Kossa para la detección de depósitos de calcio la cual destaca al calcio de color negro y el osteoide de color rojo. Para el análisis químico proximal, y con el propósito de obtener y comparar niveles de calcio, fósforo, zinc y magnesio en los huesos se utilizó un total de 9 mandíbulas (6 afectadas con DM y 3 controles) y sus 9 secciones corporales. Para comparar estos valores entre salmones deformes y controles se utilizó la prueba de Mann-Whitney. Para correlacionar los valores de mandíbula y segmento corporal se hizo una correlación por jerarquías de Spearman. Los peces del grupo 1, presentaron una desviación ventral del cuerpo del hueso dentario. En los peces del grupo 2 se observó la prominencia de la articulación visible en ambos lados o unilateralmente. Al comparar los valores de % Ca, % P, % Mg y Zn de las mandíbulas con DM y sanas con el método de Mann Witney se encontró que los valores de estos minerales no varían entre salmones afectados y controles. Hubo una diferencia significativa en el porcentaje de P, lo cual indica que existe menos P en vértebras de peces afectados. La correlación de Spearman señaló que los porcentajes de los minerales estudiados en huesos dentarios y segmentos vertebrales no están correlacionados. Por el contrario, la técnica Von Kossa mostró que la distribución de Ca/ P no es homogénea en el hueso dental producto de la reabsorción mineral desde el esqueleto incluyendo el opérculo, hueso articular y dental hacia la kype. El presente trabajo establece que la técnica histoquímica de Von Kossa fue la que permitió observar diferencias importantes entre peces deformados y controles, además mostró diferencias entre los distintos segmentos del hueso dentario. El hueso dentario es una estructura dinámica adaptada a continuos cambios histológicos pudiendo estar involucrados en la DM, dietas deficientes de fósforo, sumado a la formación inicial del Kippe.

Timing of isotopic integration in marine mammal skull: comparative study between calcified tissues.

RATIONALE: Tissues with different turnover rates have different isotope compositions and reflect the different periods in an animal's life when the isotopes are incorporated during the growth of tissues. Bone is one of the most used tissues for reconstruction of an animal's diet; however, the time of isotope integration remains unknown for many species. METHODS: The δ(15)N and δ(13)C values in tooth dentine and bone tissue from the maxilla and mandible of 21 stranded northern elephant seals, Mirounga angustirostris, collected on the San Benito and Magdalena Islands, Mexico, between 2000 and 2008 were compared. Bone and dentine samples from each growth layer within the tooth were analyzed using a PDZ Europa ANCA-GSL elemental analyzer interfaced with a PDZ Europa 20-20 continuous flow gas source mass spectrometer. RESULTS: The δ(15)N and δ(13)C values were not different between bone structures, indicating similar turnover rates, metabolic activity and amino acid compositions. The differences in the observed δ(13)C values between tissues are probably indicative of differences in their amino acid compositions, although the similarities in δ(15)N values indicated less variation from different amino acids. Correlation of the analyses between isotopic values of tissues suggests that the maxilla and/or mandible of M. angustirostris might reflect the δ(15) N signal incorporated during the last 5 years of life of the individuals. CONCLUSIONS: This study demonstrated the usefulness of the applied approach for providing a best approximation of the timing of isotopic integration into the skull of a marine mammal, thereby reducing uncertainty in exploring historic changes in the species' feeding behavior.

Biomechanical properties of the mandible, as assessed by bending test, in rats fed a low-quality protein.

OBJECTIVE: The present study describes the effects of feeding growing rats with diets containing increasing concentrations of wheat gluten (a low quality protein, G) on both the morphometrical and the biomechanical properties of the mandible. DESIGN: Female rats were fed one of six diets containing different concentrations (5-30%) of G between the 30th and 90th days of life. Control rats were fed a diet containing 20% casein (C), which allows a normal growth and development of the bone. Mandibular growth was estimated directly on excised and cleaned bones by taking measurements between anatomical points. Mechanical properties of the right hemimandibles were determined by using a three-point bending mechanical test to obtain a load/deformation curve and estimate the structural properties of the bone. Bone material properties were calculated from structural and geometric properties. The left hemimandibles were ashed and the ash weight obtained. Calcium content was determined by atomic energy absorption. Results were summarised as means±SEM. Comparisons between parameters were performed by ANOVA and post-test. RESULTS: None of the G-fed groups could achieve a normal growth performance as compared to the C-fed control group. Like body size, age-related increments in mandibular weight, length, height and area (index of mandibular size) were negatively affected by the G diets, as was the posterior part of the bone (posterior to molar III). The cross-sectional geometry of the mandible (cross-sectional area and rectangular moment of inertia) as well as its structural properties (yielding load, fracture load, and stiffness) were also severely affected by the G diets. However, material properties (Young's modulus and maximum elastic stress) and calcium concentration in ashes and the degree of mineralisation were unaffected. CONCLUSIONS: The differences in strength and stiffness between treated and control rats seemed to be the result of an induced loss of gain in bone growth and mass, in the absence of changes in the quality of the bone mineralised material.

Gene expression analysis in microdissected samples from decalcified tissues.

OBJECTIVE: The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples. DESIGN: Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase. RESULTS: Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified. CONCLUSIONS: Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.

Fluorosis in rats exposed to oscillating chronic fluoride doses.

Considering that blood fluoride concentration varies according to fluoride exposure and that dental fluorosis is related to the amount of enamel formed under a given fluoride dose, the present study investigated whether the fluorosis produced by an oscillating chronic fluoride dose would be similar to that caused by exposure to a constant dose, representing the mean of the oscillation during a given time. Rats received during 78 days water with fluoride concentrations of 0, 12.5, 25 or 37.5 microg F/mL, or oscillating concentrations of 12.5 and 37.5 microg F/mL every 72 h (mean exposure=25 microg F/mL). The concentrations of fluoride in the plasma, femur and incisors of the rats were determined at the end of the experimental period. Also, the enamel dental fluorosis index was determined in the incisors using a quantitative method developed by our research group named Dental Fluorosis by Image Analysis (DFIA). Fluoride concentrations in plasma, femur and teeth, and DFIA increased linearly for constant fluoride concentrations in water (p<0.0001, r values=0.87-0.98). The results of the oscillating group and the groups receiving 25 microg F/mL did not differ significantly (p>0.05). The findings of this study suggest that in animals chronically exposed to symmetrically oscillating fluoride doses, the resulting dental fluorosis reflects the metabolic effect of the mean of the oscillating doses.

Fluorosis in rats exposed to oscillating chronic fluoride doses

Considering that blood fluoride concentration varies according to fluoride exposure and that dental fluorosis is related to the amount of enamel formed under a given fluoride dose, the present study investigated whether the fluorosis produced by an oscillating chronic fluoride dose would be similar to that caused by exposure to a constant dose, representing the mean of the oscillation during a given time. Rats received during 78 days water with fluoride concentrations of 0, 12.5, 25 or 37.5 µg F/mL, or oscillating concentrations of 12.5 and 37.5 µg F/mL every 72 h (mean exposure=25 µg F/mL). The concentrations of fluoride in the plasma, femur and incisors of the rats were determined at the end of the experimental period. Also, the enamel dental fluorosis index was determined in the incisors using a quantitative method developed by our research group named Dental Fluorosis by Image Analysis (DFIA). Fluoride concentrations in plasma, femur and teeth, and DFIA increased linearly for constant fluoride concentrations in water (p<0.0001, r values=0.87-0.98). The results of the oscillating group and the groups receiving 25 µg F/mL did not differ significantly (p>0.05). The findings of this study suggest that in animals chronically exposed to symmetrically oscillating fluoride doses, the resulting dental fluorosis reflects the metabolic effect of the mean of the oscillating doses.

Considerando que a concentração de fluoreto no sangue varia de acordo com a exposição ao fluoreto, e que a fluorose dental está relacionada com a quantidade de esmalte formado sob determinada dose de fluoreto, este estudo avaliou se a fluorose resultante da exposição a doses oscilantes de fluoreto seria semelhante àquela causada pela exposição a uma dose constante, representativa da média das oscilações durante um determinado tempo. Durante 78 dias, ratos receberam água com concentrações constantes de fluoreto de 0; 12,5; 25 ou 37,5 µg F/mL, ou concentrações oscilantes de 12,5 e 37,5 µg F/mL alternados a cada 72 h (média de exposição = 25 µg F/mL). Concentrações de fluoreto no plasma, fêmur e dentes incisivos dos ratos foram determinadas após o período experimental. O índice de fluorose, observado nos incisivos dos ratos, foi quantificado usando um método de análise de imagem desenvolvido para essa pesquisa, denominado de índice de fluorose por análise de imagem (DFIA, em Inglês). A concentração de fluoreto no plasma, fêmur e incisivo dos ratos, assim como o DFIA, aumentaram de forma linear para as concentrações constantes de fluoreto na água (p<0,0001, r=0,87-0,98). Não houve diferença significativa entre o grupo que recebeu doses oscilantes e o grupo que recebeu 25 µg F/mL (p>0,05). Os resultados sugerem a fluorose dental decorrente de exposição crônica de animais a doses de fluoreto oscilantes e simétricas reflete o efeito metabólico da média da oscilação.

Reconstruction of a mandibular critical-sized defect using iliac graft in rats.

OBJECTIVE: To evaluate, histologically and by optical densitometry of radiographs, the healing of a critical-sized defect in the rat mandible filled with iliac graft. MATERIALS: The study was conducted on 25 adult Wistar rats. With the rats under general anesthesia, a bicortical critical-sized osseous defect was created in the right mandibular ramus and filled with autogenous iliac crest graft. The animals were divided into 5 groups, with 5 rats in each. They were sacrificed after 1, 7, and 14 days, and 1 and 3 months. The mandibles were removed, fixed in formalin, and radiographed. The right hemi-mandibles were decalcified, and sections were cut and stained with hematoxylin and eosin. RESULTS: Initially, an acute inflammatory process was noted along the graft that was tightly fitted to the defect. Subsequently, intense bone neoformation from external corticals and in the inner spaces of the graft was observed, while medullar spaces were occupied by granulation tissue and osteoblasts. There was remodelation of the receptor site, with a decrease in the graft volume and medullary space, as well as cancellous bone replaced by compact bone. Later, the receptor site was similar to the normal mandible, and only devitalized remnants of corticals of the graft were found. Optical densitometry of radiographs revealed statistically significant differences between experimental and control sites. CONCLUSIONS: This experimental model is valuable in the study of bone healing. The study showed that autogenous iliac graft promoted healing of the critical-sized defect of the mandible with complete bony remodeling.

Bone healing following the use of hydroxyapatite or ionomeric bone substitutes alone or combined with a guided bone regeneration technique: an animal study / Reparação óssea após o uso de hidroxiapatita ou substitutivos de osso ionomérico isolado ou combinados com a técnica de regeneração óssea guiada: estudo em animais

The healing of standardized bone defects grafted with either particulate ionomeric or hydroxyapatite bone substitutes was compared in the mandibular ramus of 30 Sprague-Dawley rats. The possible additional response achieved when combining these materials with a guided bone regeneration (GBR) technique was also evaluated. Three groups of 10 animals received either no implant material or ionomeric or hydroxyapatite bone substitute in defects in the right ramus. The left mandibular defects received the same treatment, except that the operation site was covered by a membrane (GBR technique). Half of the animals were sacrificed at 4 and 10 weeks following surgery, and the inflammatory response at the implant site and the amount of new bone formed in the defects were determined histomorphometrically. Defects implanted with ionometric bone substitute exhibited more bone formation (4 weeks = 3.19 ± 0.38 mm², 10 weeks = 5.35 ± 0.26 mm²) than both defects that received no treatment (4 weeks = 0.88 ± 0.35 mm², 10 weeks = 2.1 ± 0.49 mm²), membrane alone (4 weeks = 1.21 ± 0.005 mm² or hydroxyapatite bone substitute (4 weeks = 1.41 ± 0.46 mm², 10 weeks = 3.34 ± 0,41 mm²) at 4 weeks (P<=.01) and at 10 weeks (P<=.05). The use of a GBR technique did not increase the amount of bone formed, compared to the use of bone substitutes alone. Hydroxyapatite and ionomeric bone substitutes used alone were more effective in inducing repair of the defects than was GBR membrane alone. The use of hydroxyapatite was associated with a greater inflammatory reaction (P<=.01) than was ionomer in this model

Changes in mandible and femur proteoglycans. Physico-chemical properties after hydrocortisone treatment.

Male Wistar rats were injected with 5 mg/kg/day of hydrocortisone during 21 days. Femur and mandible proteoglycans (PG) were isolated and their physico-chemical properties were compared with bone PG from a control and a starved group. The PG molecular weight decreased as a result of the hydrocortisone treatment but to a lower degree than in starvation, whereas the PG density seemed to be more affected by corticosteroid injection. These results allow us to conclude that bone alterations found after glucosteroid treatment may be due--at least partially--to changes in the PG physico-chemical properties of that tissue.