Examining the Impact of Acute Binge Alcohol Consumption on Trabecular Morphometry and Tensile Strength in Adolescent Sprague Dawley Rat Mandibles / Análisis del Impacto del Consumo Excesivo de Alcohol en la Morfometría Trabecular y la Resistencia a la Tracción en Mandíbulas de Ratas Adolescentes Sprague Dawley

SUMMARY: Underage drinking has become a major public concern having a negative impact on the growth and development of the skeleton. Peak bone mass is attained during adolescence hence the aim of the study was to investigate the effect of acute binge alcohol consumption on trabecular morphometry and tensile strength of the adolescent mandible in the Sprague Dawley (SD) rat. The study comprised of 24 SD rats, aged 7 weeks, placed into either the alcohol-exposed [n=12 (6 males and 6 female)] or pair-fed control group [n=12 (6 male and 6 female)]. The treatment of the groups was as follows; the alcohol exposed group and the pair-fed control were administered a single daily dose of 3 g/kg of 20 % alcohol 3 days a week (alternate days) for 7 days and a caloric equivalent dose of maltose dextrin via oral gavage, respectively. The animals were terminated on day 7 via pentobarbital injection. The mandibles were harvested and scanned using a Nikon XTH 255L 3D-microCT scanner (Nikon Metrology, Leuven, Belgium), and biomechanical tests were done using a Shimadzu universal tensile strength testing machine (China). Following scanning and reconstruction, the trabecular morphometry was assessed using Volume Graphics Studio® software. A 3-point bending test was used to evaluate the tensile strength of the bone. Findings from our study showed changes in some trabecular parameters in the female alcohol-exposed group, while the male groups remained unaffected. No changes in tensile strength were seen when comparing male pair-fed control and alcohol-exposed groups and when comparing female pair-fed control and alcohol-exposed groups. Trabecular and tensile strength differences were observed between the sexes when comparing male pair-fed control and alcohol-exposed groups to female pair-fed control and alcohol-exposed groups. These findings do suggest that acute binge alcohol consumption has detrimental effects on the bone micro-architecture in female alcohol-exposed rats and that differences are seen between the sexes.

El consumo de alcohol entre menores de edad se ha convertido en una importante preocupación pública que tiene un impacto negativo en el crecimiento y desarrollo del esqueleto. La masa ósea máxima se alcanza durante la adolescencia, por lo que el objetivo del estudio fue investigar el efecto del consumo excesivo de alcohol en forma aguda sobre la morfometría trabecular y la resistencia a la tracción de la mandíbula en ratas adolescente Sprague Dawley (SD). El estudio estuvo compuesto por 24 ratas, de 7 semanas de edad, colocadas en el grupo control expuesto al alcohol [n=12 (6 machos y 6 hembras)] y alimentado en parejas [n=12 (6 machos y 6 hembras)]. El tratamiento de los grupos fue el siguiente; al grupo expuesto al alcohol y al control alimentado en parejas se les administró una dosis única diaria de 3 g/kg de alcohol al 20 % 3 días a la semana (días alternos) durante 7 días y una dosis equivalente calórica de maltosa dextrina mediante sonda oral, respectivamente. Los animales fueron sacrificados el día 7 mediante inyección de pentobarbital. Las mandíbulas se recolectaron y se escanearon utilizando un escáner 3D-microCT Nikon XTH 255L (Nikon Metrology, Lovaina, Bélgica), y las pruebas biomecánicas se realizaron utilizando una máquina de prueba de resistencia a la tracción universal Shimadzu (China). Después del escaneo y la reconstrucción, la morfometría trabecular se evaluó utilizando el software Volume Graphics Studio®. Se utilizó una prueba de flexión de 3 puntos para evaluar la resistencia a la tracción del hueso. Los hallazgos de nuestro estudio mostraron cambios en algunos parámetros trabeculares en el grupo de hembras expuestas al alcohol, mientras que los grupos de machos no se vieron afectados. No se observaron cambios en la resistencia a la tracción al comparar los grupos control de machos alimentados en parejas y los grupos expuestos al alcohol y al comparar los grupos control de las hembras alimentadas en parejas y los grupos expuestos al alcohol. Se observaron diferencias trabeculares y de resistencia a la tracción entre los sexos al comparar los grupos control de los machos alimentados en parejas y expuestos al alcohol con los grupos de control de hembras alimentadas en parejas y expuestas al alcohol. Estos hallazgos sugieren que el consumo excesivo de alcohol tiene efectos perjudiciales sobre la microarquitectura ósea en ratas hembras expuestas al alcohol y que se observan diferencias entre los sexos.

The Effects of Acute Binge Alcohol Consumption on the Trabecular Morphometry and Tensile Strength of Adolescent Sprague Dawley Rat Femora

SUMMARY: Excessive alcohol consumption adversely affects bone metabolism, thus resulting in reduced bone length, density, and strength. Moreover, these deficits in bone density and strength are likely to increase the risk of fragility fractures and the early onset of osteoporosis. While excessive alcohol consumption is an established risk factor for osteoporotic fractures, there remains a dearth of information in literature about bone effects of binge alcohol consumption in adolescents. Therefore, our study aimed to examine the effects of acute binge alcohol consumption on the adolescent bone micro-architecture and tensile strength. Twelve male Sprague Dawley rats aged 7 weeks were randomly placed in 2 groups: alcohol (n =6), receiving alcohol (3g/kg) and pair-fed control (n = 6), receiving an isocaloric equivalent of maltose dextrin via oral gavage for 3 days in one week (on alternative days). The femora were dissected and scanned using a Micro-Focus X-ray Computed Tomography (3D-µCT). Following reconstruction, trabecular morphometry was assessed in both the proximal and distal epiphysis, using a Volume Graphics Studio® software. A three-point bending test was employed to examine the effect of alcohol on the tensile strength of the bone. Results showed trabeculae parameters to be affected in the distal epiphysis of the femur, while in the proximal epiphysis it remained unaffected. Tensile strength parameters were also not affected by the consumption of alcohol. These findings may suggest that acute binge alcohol consumption has detrimental effects on the bone micro-architecture specific to the distal epiphysis.

El consumo excesivo de alcohol afecta negativamente al metabolismo óseo, lo que resulta en una reducción de la longitud, densidad y resistencia de los huesos. Además, es probable que estos déficits en la densidad y la fuerza ósea aumenten el riesgo de fracturas por fragilidad y la aparición temprana de osteoporosis. Si bien el consumo excesivo de alcohol es un factor de riesgo establecido para las fracturas osteoporóticas, existe escasa información en la literatura sobre los efectos óseos del consumo excesivo de alcohol en adolescentes. Por lo tanto, nuestro estudio tuvo como objetivo examinar los efectos del consumo excesivo de alcohol en la microarquitectura ósea y la resistencia a la tracción e n ratas adolescentes. Doce ratas macho Sprague Dawley de 7 semanas de edad se colocaron aleatoriamente en 2 grupos: alcohol (n = 6), que recibieron alcohol (3 g/kg) y control (n = 6), que recibieron un equivalente isocalórico de maltosa dextrina mediante sonda oral, durante 3 días en una semana (en días alternos). Los fémures se diseccionaron y escanearon mediante una tomografía computarizada de rayos X con microenfoque (3D-mCT). Después de la reconstrucción, se evaluó la morfometría trabecular tanto en la epífisis proximal como en la distal, utilizando un software Volume Graphics Studio®. Se empleó una prueba de flexión de tres puntos para examinar el efecto del alcohol sobre la resistencia a la tracción del hueso. Los resultados mostraron que los parámetros de las trabéculas se vieron afectados en la epífisis distal del fémur, mientras que en la epífisis proximal no se observaron afectados. Los parámetros de resistencia a la tracción tampoco se vieron afectados por el consumo de alcohol. Estos hallazgos pueden sugerir que el consumo excesivo de alcohol tiene efectos perjudiciales sobre la microarquitectura ósea específica de la epífisis distal del hueso.

Intrauterine alcohol exposure disturbs trabecular morphology in the Sprague Dawley rat humerus epiphysis up to 3- weeks postnatally / La exposición al alcohol intrauterino altera la morfología trabecular en la epífisis del húmero de la rata Sprague Dawley hasta 3 semanas después del nacimiento

SUMMARY: Gestational alcohol exposure inhibits neurological as well as bone growth and development both in fetal and postnatal life. Stunted stature, osteoporosis and fractures in adult life are some of the adverse effects. While the impact of intrauterine alcohol on the brain has been extensively investigated, studies on the effects on bone are relatively few. Therefore, our study aimed to examine the impact of prenatal alcohol exposure on bone microarchitecture in 3-week-old rats using Micro-focus X-Ray Computed Tomography (Micro CT). Time mated pregnant Sprague Dawley dams (13) were randomly placed into 3 groups: ethanol (n=5), saline control (n=5) and untreated control (n=3). The former 2 groups received treatment with 0.015ml/g of 25.2 % ethanol and 0.9 % saline, respectively, for the first 19 days of gestation. The untreated group received no treatment. The pups remained with their dams until termination at 21 days of age. From each dam, 2 pups were collected resulting in: ethanol (n=10), saline controls (n= 10) and untreated controls (n = 6). The humeri of the pups were dissected and scanned using a 3D-μCT scanner (Nikon XTH 225L) at 15μm resolution. Trabecular and cortical parameters were analysed using Volume Graphics Studio® software following reconstruction. Results showed a decrease in trabecular size, spaces, thickness, and volume. There was a decrease in cortical bone area in the ethanol group compared to the controls. These findings may suggest that osteoporosis and fractures seen as gestational alcohol effects may be due to compromised trabecular structure.

RESUMEN: La exposición al alcohol durante la gestación inhibe el crecimiento y desarrollo neurológico y óseo tanto en la vida fetal como posnatal. Algunos de los efectos adversos incluyen la estatura atrofiada, osteoporosis y fracturas en la vida adulta. Si bien se ha estudiado el impacto del alcohol intrauterino en el cerebro, los estudios sobre los efectos en los huesos son escasos. Por lo tanto, nuestro estudio tuvo como objetivo examinar el impacto de la exposición prenatal al alcohol en la microarquitectura ósea en ratas de 3 semanas de edad utilizando Tomografía Computarizada de Rayos X Micro-focus (Micro CT). Las hembras de Sprague Dawley preñadas con apareamiento temporal (13) se colocaron aleatoriamente en 3 grupos: etanol (n = 5), control de solución salina (n = 5) y control sin tratar (n = 3). Los primeros 2 grupos recibieron tratamiento con 0,015 ml /g de etanol al 25,2 % y solución salina al 0,9 %, respectivamente, durante los primeros 19 días de gestación. El grupo no tratado no recibió tratamiento. Las crías permanecieron con sus madres hasta la terminación a los 21 días de edad. De cada madre, se recolectaron 2 crías que dieron como resultado: etanol (n = 10), controles salinos (n = 10) y controles no tratados (n = 6). Se diseccionaron y escanearon los húmero de las crías usando un escáner 3D-μCT (Nikon XTH 225L) a una resolución de 15 μm. Los parámetros trabeculares y corticales se analizaron utilizando el software Volume Graphics Studio® después de la reconstrucción. Los resultados mostraron una disminución en el tamaño trabecular, los espacios, el grosor y el volumen. Hubo una disminución en el área del hueso cortical en el grupo de etanol en comparación con los controles. Estos hallazgos pueden sugerir que la osteoporosis y las fracturas por causa de los efectos del alcohol gestacional se pueden deber a una estructura trabecular comprometida.

To what extent does hyaluronic acid affect healing of xenografts? A histomorphometric study in a rabbit model

ABSTRACT Among the many graft materials that have been used for the treatment of bone defects in oral and maxillofacial regions is xenograft. To improve osteoconductive effects of xenografts, they have been combined with various biocompatible materials, such as hyaluronic acid and bone morphogenetic protein. Objective: To determine bone-healing capacity of high molecular weight hyaluronic acid (HA) combined with xenograft in rabbit calvarial bone defects. Material and methods: Ten adult male New Zealand rabbits (mean weight 3 kg) were included in the study. Three 6-mm-diameter bicortical cranial defects were created on calvarial bone of all rabbits. These defects were filled as follows: a) xenograft; b) HA+xenograft; c) autograft. One month after the first operation, rabbits were sacrificed. Specimens were evaluated histomorphometrically. Results: Considering multiple comparisons, differences regarding new bone were statistically significant between all groups (p<0.05). The volume of residual graft was significantly decreased in HA group compared to xenograft group (p=0.035). Marrow space, trabecular thickness (TbTh), trabecular width (TbWi), trabecular separation (TbSp), and number of node: number of terminus (NNd:NTm) in the autograft group were significantly better than xenograft and HA groups (p<0.05). However, regarding marrow space, TbTh, TbWi, TbSp, and NNd:NTm values, xenograft and HA groups showed similar results and the difference were not significant (p>0.05). Conclusion: These results support that high molecular weight hyaluronic acid could contribute to the healing of xenograft by improving the percentage of new bone formation and reducing the percentage of residual graft. However, HA did not significantly affect the quality of newly formed bone assessed by microarchitectural parameters.

Efeito da terapia de longa duração com ácido zoledrônico no osso esponjoso da mandíbula e fêmur de ratos Wistar / Effect of long-term zoledronic acid treatment on cancellous bone in the mandible and femur of Wistar rats

Os bisfosfonatos (BF) são amplamente utilizados no tratamento de doenças osteolíticas como metástases ósseas e osteoporose. A osteonecrose dos maxilares associada ao uso de BF (OMAB) é caracterizada pela presença de osso exposto ou que pode ser sondado através de uma fístula que persiste por mais de oito semanas em pacientes com história de terapia de BF e sem história de radioterapia na região de cabeça e pescoço e/ou sem doença metastática nos maxilares. A incidência de OMAB aumenta com a potência, duração do tratamento e dose de BF recebida. Até o presente momento, a fisiopatologia da OMAB não está clara, dificultando a prevenção e o tratamento. O objetivo deste estudo foi avaliar o efeito da administração de altas doses Ácido Zoledrônico (AZ) por período prolongado no osso esponjoso da mandíbula e da metáfise proximal do fêmur de ratos Wistar. Para relacionar as descobertas à fisiopatologia da OMAB, o regime de administração de BF de um modelo animal relevante desta lesão foi reproduzido. Seis animais receberam AZ (0,6 mg / kg) e seis receberam solução salina no mesmo volume (Controles). Os compostos foram administrados por via intraperitoneal em cinco doses a cada 28 dias. A eutanásia dos animais ocorreu após 150 dias de início da terapia. As hemimandíbulas e fêmures direitos foram escaneados usando Micro-tomografia computadorizada (Micro-CT) de alta resolução (14 m). Para a primeira análise realizada neste estudo, os dados morfométricos do osso esponjoso foram calculados na região do segundo e primeiro molar na mandíbula e na metáfise do fêmur usando CTAnalyzer (Bruker, Bélgica). Para a segunda análise, cinco amostras de hemimandíbulas de cada grupo foram cortadas em lâminas histológicas (5 m) e coradas com Hematoxilina e Eosina. Para comparar os parâmetros morfométricos na Micro-CT e histologia, as imagens de Micro-CT foram espacialmente alinhadas à histologia. Os dados morfométricos do osso alveolar foram calculados usando o software CTAnalyzer (Bruker, Bélgica) na região entre as raízes mesial e distal do primeiro molar. A densidade da área vascular (área vascular/área total; VA/TA) e os dados histomorfométricos ósseos foram estimados usando Axiovision na mesma região (entre as raízes mesial e distal do primeiro molar). Foi adotada significância estatística de 5% ( = 0,05). Os animais tratados com AZ apresentaram aumento significativo na porcentagem de volume ósseo (p <0,05) com trabéculas mais espessas, osso mais compacto com menor separação trabecular na mandíbula e no fêmur. Na mandíbula, o aumento da densidade óssea e diminuição da separação trabecular foram fortemente correlacionados com a diminuição da área vascular observada no grupo AZ (p <0,05). Em conclusão, o tratamento de longa duração com altas doses de AZ foi significativamente associado ao aumento na densidade óssea e à diminuição dos espaços medulares, canais nutritivos e vasculatura do osso alveolar. A análise com Micro-CT revelou alterações semelhantes na estrutura óssea tanto na mandíbula quanto no fêmur do grupo AZ.(AU)

Bisphosphonates (BFs) are widely used in the treatment of osteolytic diseases such as bone metastases and osteoporosis. The osteonecrosis of the jaws related to BF (ONB) is characterized by the presence of exposed bone or bone that can be probed through a fistula that persists for more than eight weeks in patients with a history of BF therapy and without history of head and neck radiotherapy and / or without metastatic disease in the jaws. The incidence of ONB increases with potency, duration of treatment and dose of BF received. Thus far, the pathophysiology of ONB is unclear, hampering prevention and treatment. The aim of this study was to objectively assess the effect of long-term high-dose Zoledronic Acid (ZA) on cancellous bone in the jaw and femur of Wistar rats. In order to link our findings to the physiopathology of ONB, the therapeutic regiment of a relevant ONB animal model was reproduced. Twelve Wistar rats were randomly divided in two groups: six received Zoledronic acid (ZA; 0.6 mg / kg) and six (Controls) received saline solution in the same volume. The compounds were administrated intraperitoneally in five doses each 28 days. The rats were killed after 150 days of the therapy onset. Mandibles and femurs were scanned using a high-resolution (14m) micro-computerized tomography (Micro-CT). For the first analysis carried in this study, cancellous bone morphometric data were calculates in the region of the second and first molar in the mandible and in the proximal femur using CTAnalyzer (Bruker, Belgium). For the second analysis five samples were cut into histological slices (5m) and stained with Hematoxylin and Eosin. In order to compare the same morphological structures in Micro-CT and histology, the Micro-CT images were aligned to histology. Alveolar bone morphometric data (Micro-CT) was calculated using CTAnalyzer (Bruker, Belgium) in the region between the mesial and distal roots of the first molar. Blood vessels density and bone histomorphometric data were calculated using Axiovision (Carl Zeiss, Germany) in the same region used for Micro-CT evaluation. Statistical significance of 5% (=0.05) was adopted. ZA treated rats presented significant increase in the percentage of bone volume (p<0.05) with thicker trabeculae and more compact bone with smaller marrow spaces in the mandible and femur. In the mandible, the increase in bone density and decrease of marrow spaces size was strongly correlated with the decrease in the vascular area noticed in the ZA group (p<0.05). In conclusion, long-term high-dose ZA treatment was significant associated with the increase of bone density and the diminution of medullary spaces and nutritive canals size as well as decrease in vascularity of the alveolar bone. Micro-CT investigation showed similar changes in bone structure in the mandible and femur in the ZA group.(AU)