Cone-Beam Computed Tomography to evaluate changes in trabecular lower jawbone microstructure caused by bone loss and antiresorptive treatment / CBCT para evaluar cambios en la microarquitectura ósea de la mandíbula por pérdida ósea y tratamiento antirresortivo

ABSTRACT For decades, conventional histomorphometry has been the gold standard for analyzing trabecular bone microarchitecture. In recent years, micro-computed tomography (μCT) devices have been validated and are now considered the gold standard for quantifying bone microstructure Aim The aim of this preliminary report is to evaluate the usefulness of CBCT to assess trabecular mandible microstructural properties in normal ewes and to compare the quantitative changes associated with ovariectomy and antiresorptive treatment Material and Method Twelve adult Corriedale ewes (n=4/group) aged 3-4 years were divided into 3 groups and studied for 28 months. Eight ewes were ovariectomized (OVX) and divided into OVX and OVX+ZOL groups (n=4/group) which were treated as follows, by jugular injection: OVX received saline solution and OVX+ZOL received zoledronate (Zol) (Gador SA, CABA, Argentina) (4 mg/month). Another four ewes were subjected to sham surgery (SHAM group) and received saline solution Results Densitometry showed that jaw mineral content (BMC) and density (BMD) were significantly lower in OVX than in SHAM and OVX+ZOL ewes; no difference was observed between OVX+ ZOL and SHAM groups. CBCT analysis showed that bone volume (BV/TV%); trabecular thickness (TbTh); connectivity density (CD) and anisotropy degree (AD) were significantly lower, and trabecular spacing (TbSp), significantly higher in OVX than in SHAM ewes. AD was significantly higher and TbSp significantly lower in OVX+ZOL than in OVX groups. BV/TV%, TbTh and CD showed a clear tendency to being higher in OVX+ZOL than in OVX groups. No statistical difference was observed between OVX+ZOL and SHAM ewes. CBCT in a nondestructive, fast, very precise procedure for measuring bone morphometric indices without biopsies, which are not indicated for morphometric evaluation in osteoporosis Conclusions The current study demonstrated the potential of the high-resolution CBCT imaging to assess in vivo quantitative bone morphometry and bone quality of lower jaw cancellous bone under normal conditions and to differentiate changes associated with excessive bone loss induced by estrogen withdrawal and antiresorptive intervention.

RESUMEN Objetivo El presente informe preliminar evaluó la utilidad de Tomografía Computada de Haz Cónico (CBCT) para analizar las propiedades microestructurales trabeculares del maxilar inferior de ovejas y comparar los cambios cuantitativos asociados con la ovariectomía y tratamiento antirresortivo. Se estudiaron dieciséis ovejas Corriedale adultas de 3-4 años Materiales y Método Doce ovejas fueron ovariectomizadas (OVX) y divididas en 2 grupos: OVX y OVX+ZOL (n=4/grupo) cuyo tratamiento por inyección endovenosa en la yugular durante 28 meses fue el siguiente: OVX con solución salina y OVX+ZOL con zoledronato (Gador S.A. CABA. Argentina) (Zol) (4 mg/mes); 4 ovejas fueron sometidas a cirugía simulada (grupo SHAM) Resultados La densitometría (Lunar DPX) mostró que el contenido mineral del hueso maxilar (CMO) y la densidad (DMO) fueron significativamente más bajos en OVX que en SHAM y OVX+ZOL; no se observaron diferencias entre los grupos OVX+ZOL y SHAM. El análisis de las imágenes por CBCT (Planmeca Promax 3D Classic) mostró que el volumen óseo (BV/TV%); el espesor trabecular (TbTh); la densidad de conectividad (CD) y el grado de anisotropía (AD) fueron significativamente menores (p<0.05), y el espaciado trabecular (TbSp), significativamente mayor en OVX que en SHAM (p<0.05). AD fue significativamente mayor (p<0.05) y TbSp, significativamente menor en OVX+ZOL que en OVX (p<0.05). BV/TV%, TbTh y CD mostró una clara tendencia a ser mayor en OVX+ZOL que en OVX. No se observaron diferencias estadísticas entre OVX+ZOL y SHAM Conclusiones En base a nuestros resultados consideramos que CBCT presenta suficiente confiabilidad y validez para evaluar in vivo la morfometría cuantitativa y la calidad del hueso esponjoso del maxilar inferior en condiciones normales, así como para diferenciar los cambios en dichos parámetros asociados a la pérdida ósea excesiva por la caída estrogénica e intervención antirresortiva. Aunque se necesitan estudios futuros, nuestros resultados agregarían una herramienta no invasiva adicional para diferenciar la microestructura del hueso trabecular mandibular en estudios preclínicos , sentando las bases para su futura aplicación en la práctica clínica.

Cone-Beam Computed Tomography to evaluate changes in trabecular lower jawbone microstructure caused by bone loss and antiresorptive treatment.

For decades, conventional histomorphometry has been the gold standard for analyzing trabecular bone microarchitecture. In recent years, micro-computed tomography (µCT) devices have been validated and are now considered the gold standard for quantifying bone microstructure. Aim: The aim of this preliminary report is to evaluate the usefulness of CBCT to assess trabecular mandible microstructural properties in normal ewes and to compare the quantitative changes associated with ovariectomy and antiresorptive treatment. Material and Method: Twelve adult Corriedale ewes (n=4/group) aged 3-4 years were divided into 3 groups and studied for 28 months. Eight ewes were ovariectomized (OVX) and divided into OVX and OVX+ZOL groups (n=4/group) which were treated as follows, by jugular injection: OVX received saline solution and OVX+ZOL received zoledronate (Zol) (Gador SA, CABA, Argentina) (4 mg/month). Another four ewes were subjected to sham surgery (SHAM group) and received saline solution. Results: Densitometry showed that jaw mineral content (BMC) and density (BMD) were significantly lower in OVX than in SHAM and OVX+ZOL ewes; no difference was observed between OVX+ ZOL and SHAM groups. CBCT analysis showed that bone volume (BV/TV%); trabecular thickness (TbTh); connectivity density (CD) and anisotropy degree (AD) were significantly lower, and trabecular spacing (TbSp), significantly higher in OVX than in SHAM ewes. AD was significantly higher and TbSp significantly lower in OVX+ZOL than in OVX groups. BV/TV%, TbTh and CD showed a clear tendency to being higher in OVX+ZOL than in OVX groups. No statistical difference was observed between OVX+ZOL and SHAM ewes. CBCT in a nondestructive, fast, very precise procedure for measuring bone morphometric indices without biopsies, which are not indicated for morphometric evaluation in osteoporosis. Conclusions: The current study demonstrated the potential of the high-resolution CBCT imaging to assess in vivo quantitative bone morphometry and bone quality of lower jaw cancellous bone under normal conditions and to differentiate changes associated with excessive bone loss induced by estrogen withdrawal and antiresorptive intervention.

Objetivo: El presente informe preliminar evaluó la utilidad de Tomografía Computada de Haz Cónico (CBCT) para analizar las propiedades microestructurales trabeculares del maxilar inferior de ovejas y comparar los cambios cuantitativos asociados con la ovariectomía y tratamiento antirresortivo. Se estudiaron dieciséis ovejas Corriedale adultas de 3-4 años. Materiales y Método: Doce ovejas fueron ovariectomizadas (OVX) y divididas en 2 grupos: OVX y OVX+ZOL (n=4/grupo) cuyo tratamiento por inyección endovenosa en la yugular durante 28 meses fue el siguiente: OVX con solución salina y OVX+ZOL con zoledronato (Gador S.A. CABA. Argentina) (Zol) (4 mg/mes); 4 ovejas fueron sometidas a cirugía simulada (grupo SHAM). Resultados: La densitometría (Lunar DPX) mostró que el contenido mineral del hueso maxilar (CMO) y la densidad (DMO) fueron significativamente más bajos en OVX que en SHAM y OVX+ZOL; no se observaron diferencias entre los grupos OVX+ZOL y SHAM. El análisis de las imágenes por CBCT (Planmeca Promax 3D Classic) mostró que el volumen óseo (BV/TV%); el espesor trabecular (TbTh); la densidad de conectividad (CD) y el grado de anisotropía (AD) fueron significativamente menores (p<0.05), y el espaciado trabecular (TbSp), significativamente mayor en OVX que en SHAM (p<0.05). AD fue significativamente mayor (p<0.05) y TbSp, significativamente menor en OVX+ZOL que en OVX (p<0.05). BV/TV%, TbTh y CD mostró una clara tendencia a ser mayor en OVX+ZOL que en OVX. No se observaron diferencias estadísticas entre OVX+ZOL y SHAM. Conclusiones: En base a nuestros resultados consideramos que CBCT presenta suficiente confiabilidad y validez para evaluar in vivo la morfometría cuantitativa y la calidad del hueso esponjoso del maxilar inferior en condiciones normales, así como para diferenciar los cambios en dichos parámetros asociados a la pérdida ósea excesiva por la caída estrogénica e intervención antirresortiva. Aunque se necesitan estudios futuros, nuestros resultados agregarían una herramienta no invasiva adicional para diferenciar la microestructura del hueso trabecular mandibular en estudios preclínicos , sentando las bases para su futura aplicación en la práctica clínica.

Epidermal growth factor prevents thallium(I)- and thallium(III)-mediated rat pheochromocytoma (PC12) cell apoptosis.

We have reported recently that the proliferation of PC12 cells exposed to micromolar concentrations of Tl(I) or Tl(III) has different outcomes, depending on the absence (EGF- cells) or the presence (EGF+ cells) of epidermal growth factor (EGF) added to the media. In the current work, we investigated whether EGF supplementation could also modulate the extent of Tl(I)- or Tl(III)-induced cell apoptosis. Tl(I) and Tl(III) (25-100 µM) decreased cell viability in EGF- but not in EGF+ cells. In EGF- cells, Tl(I) decreased mitochondrial potential, enhanced H2O2 generation, and activated mitochondrial-dependent apoptosis. In addition, Tl(III) increased nitric oxide production and caused a misbalance between the anti- and pro-apoptotic members of Bcl-2 family. Tl(I) increased ERK1/2, JNK, p38, and p53 phosphorylation in EGF- cells. In these cells, Tl(III) did not affect ERK1/2 and JNK phosphorylation but increased p53 phosphorylation that was related to the promotion of cell senescence. In addition, this cation significantly activated p38 in both EGF- and EGF+ cells. The specific inhibition of ERK1/2, JNK, p38, or p53 abolished Tl(I)-mediated EGF- cell apoptosis. Only when p38 activity was inhibited, Tl(III)-mediated apoptosis was prevented in EGF- and EGF+ cells. Together, current results indicate that EGF partially prevents the noxious effects of Tl by preventing the sustained activation of MAPKs signaling cascade that lead cells to apoptosis and point to p38 as a key mediator of Tl(III)-induced PC12 cell apoptosis.

Low dietary calcium and obesity: a comparative study in genetically obese and normal rats during early growth.

PURPOSE: A low calcium intake (LCaI) may predispose to obesity, and excessive fat mass may be detrimental to bone. The impact of Ca inadequacy would be greater in subjects predisposed to obesity. LCaI effect on obesity development during the rapid growth period was compared in two strains of rats: spontaneously obese IIMb/ß (O) and Wistar (W). Pregnant rats were fed 0.5% (N) or 0.2% (L) of Ca (OLCa, ONCa, WLCa and WNCa). Male pups were fed the maternal diet until day 60. METHODS: Body composition, lipid profile, glucose homeostasis, 25 hydroxyvitamin D, Ca-phosphorus, and bone metabolism were evaluated. RESULTS: BW and body fat were higher, whereas body protein was lower in OLCa versus ONCa (p < 0.05). OLCa presented the highest body fat, glucose, non-HDL and total cholesterol, TGL, insulin levels, and HOMA-IR, liver weight, and adipose perigonadal plus retroperitoneal pads (p < 0.05). WLCa did not exhibit an increase BW and only showed a slight change in body composition with minor biochemical alterations compared to WNCa (p < 0.05). Osteocalcin, CTX, and proximal tibia and lumbar spine BMDs were lower in O than in W rats fed the same Ca diet (p < 0.05). Body ash and Ca content, and total skeleton BMC/BW were lower in OLCa and WLCa versus their corresponding NCa groups (p < 0.05). CONCLUSION: The negative effect of a low Ca diet on fat mass accumulation and lipid profile may be more evident in rats predisposed to obesity. Nevertheless, low CaI interferes with the normal glucose homeostasis leading to an increase in insulin resistance. Low CaI during early growth may be an obesogenic factor that may persist into adult life and may account for the development of obesity and some of its co-morbidities.

Especies reactivas de oxígeno y su efecto sobre la actividad de las células óseas / Reactive oxygen species on bone cells activity / Espécies reativas de oxigênio e seu efeito na atividade das células ósseas

Las mitocondrias generan especies reactivas de oxígeno (ERO) que cumplen con una multiplicidad de procesos celulares; cuando se producen en exceso son responsables del estrés oxidativo y de múltiples procesos patológicos, incluyendo osteoporosis. Los factores de transcripción FoxO 1, 3 y 4 actúan como moléculas sensoras de ERO convirtiendo la señal de estrés oxidativo en la inducción de mecanismos de protección o señales apoptóticas. La insulina y los factores de crecimiento insulínicos (IGFs) regulan negativamente a FoxOs en mamíferos. Las ERO están involucradas en el remodelamiento óseo a través del efecto que ejercen sobre osteoblastos y osteoclastos. Los FoxOs controlan la acción de ERO sobre la osteoblastogénesis y la osteoclastogénesis. Con la edad, el aumento del estrés oxidativo acelera la adipogénesis a expensas de la osteoblastogénesis, al mismo tiempo que aumenta la oxidación de ácidos grasos generando compuestos pro-oxidantes que incrementan el estrés oxidativo. Asimismo, la caída estrogénica acelera la osteoclastogénesis por vía genómica o no genómica. Dada la importancia de FoxOs y ERO en la fisiología ósea y durante el envejecimiento, clarificar los eventos celulares y pasos moleculares involucrados en el control del estrés oxidativo sería vital para entender la regulación de la osteoporosis relacionada a la edad.

Reactive oxygen species (ROS) are key players in oxidative stress, and they are generated as by-products of cellular metabolism, primarily in the mitochondria. ROS are well recognised for playing a dual role as both deleterious and beneficial species. FoxOs transcription factors are activated in oxidative stress responses and participate in the regulation of cellular functions, including cell cycle arrest, cell death, and protection from stress stimuli. FoxO activity is inhibited by growth factors and the insulin signaling pathways. They play a fundamental role in skeletal homeostasis by exerting both ROS céludependent and independent effects on bone cells. FoxOs modulate osteoblastogenesis and attenuate osteoclastogenesis through both cell autonomous and indirect mechanisms. With aging there is an inevitable increment in oxidative stress that accelerates adipogenesis at the expense of osteoblastogenesis. There is also an increment in lipid oxidation to form pro-oxidant products that enhance oxidative stress generation. In addition, the estrogen withdrawal accelerates osteoclastogenesis. Given the importance of both FoxOs and ROS in aging and bone biology, understanding the cellular events and molecular pathways that are controlled by FoxOs during aging may be vital to our understanding of the regulation of age-related osteoporosis.

Mitocôndrias geram espécies reativas de oxigênio (ERO) que cumprem uma grande variedade de processos celulares; se produzidas em excesso são responsáveis pelo estresse oxidativo e por múltiplos processos patológicos, incluindo a osteoporose. Os fatores de transcrição FoxO 1.3 e 4 funcionam como moléculas sensoras de ERO transformando o sinal de estresse oxidativo na indução de mecanismos de proteção ou sinais apoptóticos. A insulina e os fatores de crescimento insulínicos (IGFs) regulam em forma negativa Foxos em mamíferos. As ERO estão envolvidos na remodelação óssea através do seu efeito nos osteoblastos e osteoclastos. Os Foxos controlam a ação de ERO na osteoblastogênese e na osteoclastogênese. Com a idade, o aumento do estresse oxidativo acelera a adipogênese à custa de osteoblastogênese; ao mesmo tempo que aumentam a oxidação de ácidos graxos gerando compostos pró-oxidantes que incrementam o estresse oxidativo. Além disso, a queda estrogênica acelera a osteoclastogênese por via genômica ou não genômica. Devido à importância de FoxOs e ERO na fisiologia óssea e durante o envelhecimento, esclarecer os eventos celulares e passos moleculares envolvidos no controle do estresse oxidativo seria vital para a compreensão da regulação da osteoporose relacionada com a idade.

Especies reactivas de oxígeno y su efecto sobre la actividad de las células óseas / Reactive oxygen species on bone cells activity / Espécies reativas de oxigÛnio e seu efeito na atividade das células ósseas

Las mitocondrias generan especies reactivas de oxígeno (ERO) que cumplen con una multiplicidad de procesos celulares; cuando se producen en exceso son responsables del estrés oxidativo y de múltiples procesos patológicos, incluyendo osteoporosis. Los factores de transcripción FoxO 1, 3 y 4 actúan como moléculas sensoras de ERO convirtiendo la señal de estrés oxidativo en la inducción de mecanismos de protección o señales apoptóticas. La insulina y los factores de crecimiento insulínicos (IGFs) regulan negativamente a FoxOs en mamíferos. Las ERO están involucradas en el remodelamiento óseo a través del efecto que ejercen sobre osteoblastos y osteoclastos. Los FoxOs controlan la acción de ERO sobre la osteoblastogénesis y la osteoclastogénesis. Con la edad, el aumento del estrés oxidativo acelera la adipogénesis a expensas de la osteoblastogénesis, al mismo tiempo que aumenta la oxidación de ácidos grasos generando compuestos pro-oxidantes que incrementan el estrés oxidativo. Asimismo, la caída estrogénica acelera la osteoclastogénesis por vía genómica o no genómica. Dada la importancia de FoxOs y ERO en la fisiología ósea y durante el envejecimiento, clarificar los eventos celulares y pasos moleculares involucrados en el control del estrés oxidativo sería vital para entender la regulación de la osteoporosis relacionada a la edad.(AU)

Reactive oxygen species (ROS) are key players in oxidative stress, and they are generated as by-products of cellular metabolism, primarily in the mitochondria. ROS are well recognised for playing a dual role as both deleterious and beneficial species. FoxOs transcription factors are activated in oxidative stress responses and participate in the regulation of cellular functions, including cell cycle arrest, cell death, and protection from stress stimuli. FoxO activity is inhibited by growth factors and the insulin signaling pathways. They play a fundamental role in skeletal homeostasis by exerting both ROS céludependent and independent effects on bone cells. FoxOs modulate osteoblastogenesis and attenuate osteoclastogenesis through both cell autonomous and indirect mechanisms. With aging there is an inevitable increment in oxidative stress that accelerates adipogenesis at the expense of osteoblastogenesis. There is also an increment in lipid oxidation to form pro-oxidant products that enhance oxidative stress generation. In addition, the estrogen withdrawal accelerates osteoclastogenesis. Given the importance of both FoxOs and ROS in aging and bone biology, understanding the cellular events and molecular pathways that are controlled by FoxOs during aging may be vital to our understanding of the regulation of age-related osteoporosis.(AU)

Mitoc¶ndrias geram espécies reativas de oxigÛnio (ERO) que cumprem uma grande variedade de processos celulares; se produzidas em excesso sÒo responsáveis pelo estresse oxidativo e por múltiplos processos patológicos, incluindo a osteoporose. Os fatores de transcriþÒo FoxO 1.3 e 4 funcionam como moléculas sensoras de ERO transformando o sinal de estresse oxidativo na induþÒo de mecanismos de proteþÒo ou sinais apoptóticos. A insulina e os fatores de crescimento insulínicos (IGFs) regulam em forma negativa Foxos em mamíferos. As ERO estÒo envolvidos na remodelaþÒo óssea através do seu efeito nos osteoblastos e osteoclastos. Os Foxos controlam a aþÒo de ERO na osteoblastogÛnese e na osteoclastogÛnese. Com a idade, o aumento do estresse oxidativo acelera a adipogÛnese O custa de osteoblastogÛnese; ao mesmo tempo que aumentam a oxidaþÒo de ácidos graxos gerando compostos pró-oxidantes que incrementam o estresse oxidativo. Além disso, a queda estrogÛnica acelera a osteoclastogÛnese por via gen¶mica ou nÒo gen¶mica. Devido O importÔncia de FoxOs e ERO na fisiologia óssea e durante o envelhecimento, esclarecer os eventos celulares e passos moleculares envolvidos no controle do estresse oxidativo seria vital para a compreensÒo da regulaþÒo da osteoporose relacionada com a idade.(AU)

Low protein intake magnifies detrimental effects of ovariectomy and vitamin D on bone.

Protein-induced changes in bone and calcium homeostasis could potentially be greater in the elderly and in women at risk for osteoporosis. We hypothesize that a low protein intake would magnify the negative changes in bone metabolism seen in vitamin D (vitD) insufficiency and/or estrogen deficiency. The present study was undertaken to better understand how a low protein diet along with vitD insufficiency could affect bone metabolism using a rodent ovariectomized (OVX) model. Rats (n = 60) underwent ovariectomy (OVX) or sham operation. The first 15 days after surgery, all rats were fed a standard rodent diet. Thereafter, rats (n = 10/group) were fed a low protein diet (LP; 2.5 %) or a control diet (NP; 12.5 %) with 100 IU% vitD (+D; cholecalciferol) or without vitD (-D) for 45 days. The groups were as follows: SHAM + NP + D (control); SHAM + LP + D; SHAM + LP - D; OVX + NP + D; OVX + LP + D; OVX + LP - D. Body weight (BW) of control and OVX + NP + D groups increased while those feeding the LP diet, independently of vitD feedings, decreased (p < 0.05). The OVX + LP - D group presented the lowest serum Ca, phosphorus and osteocalcin levels and the highest CTX levels (p < 0.05). At the end of the study, total skeleton bone mineral content, proximal tibia bone mineral density, bone volume and trabecular number levels decreased as follows: SHAM + NP + D (controls) > SHAM + LP + D > OVX + NP + D > SHAM + LP - D > OVX + LP + D > OVX + LP - D (p < 0.05). A low protein diet negatively affected bone mass and magnified the detrimental effects of vitD and/or estrogen deficiencies.

Effect of dietary calcium (Ca) on body composition and Ca metabolism during growth in genetically obese (ß) male rats.

INTRODUCTION: Obese ß rats may be a suitable model to evaluate the association between calcium intake (CaI) and obesity during growth. OBJECTIVE: The present study comparatively evaluated Ca absorption and retention, and changes in body composition in spontaneously genetically obese (ß) male rats fed three different dietary Ca levels: high 0.9% (HCa); normal: 0.5% (NCa); low: 0.2% (LCa). METHODS: Pregnant rats were fed isocaloric diets which varied in Ca content only. Male pups continued feeding the same maternal diet until postnatal day 60. The percentage of Apparent Ca absorption (CaA %), Ca balance (CaB), body composition, glucose, triglycerides (TGL), and insulin levels were evaluated. RESULTS: Food consumption and body weight (BW) were higher in Group LCa than in Groups NCa and HCa (p < 0.01); no differences were observed between the latter two groups. Group LCa presented the highest body fat, liver weight, perigonadal and retroperitoneal fat (p < 0.05); conversely, body ashes and total skeleton bone mineral content were significantly lower compared with animals in both the NCa (p < 0.01) and HCa groups (p < 0.01). CaB (mg/day) reached a plateau at the highest CaI (mg/day) value (r = 0.985, p < 0.001). CaA%, serum glucose, insulin, and TGL levels rose as CaI decreased (p < 0.01). CONCLUSIONS: Although further studies are required, low Ca consumption in this strain of rats could modulate BW inducing changes in several lipid metabolism parameters, which in turn lead to an increase in body fat.