Does Vitamin D Insufficiency Influence Prebiotic Effect on Calcium Absorption and Bone Retention?

Higher calcium (Ca) absorption would partially compensate for Ca intake below requirements for bone health. Previously, we found that GOS/FOS prebiotic mixture (PM) increases Ca absorption in the colon and retention in bone. Ca absorption and retention are regulated by vitamin D (VD). Hence, it is relevant to explore whether VD insufficiency influences the effect of the PM in the colon. The effect of the PM on Ca, phosphate (IP), and magnesium (Mg) absorption and retention under conditions of VD sufficiency and insufficiency (VDInsuff) was compared using a preclinical model of VDInsuff and low bone mass. Ovariectomized rats were fed isocaloric semisynthetic diets according to AIN-93 M. The diets varied in Ca (0.5% or 0.3%), VD [100 IU% (+ D) or 0 IU% (- D)], and PM (2.5% or 0%) content. The following eight groups were studied: + D0.5; + D0.3; + DPM0.5; + DPM0.3; - D0.5; - D0.3; - DPM0.5; and - DPM0.3. Irrespective of Ca content, VDInsuff did not affect the prebiotic effect of the PM on caecum pH, lactobacillus colony growth, or Mg absorption but significantly decreased its effect on colonic crypt length and cell/crypt and Ca and IP absorption. The PM failed to counterbalance the pro-inflammatory effect of VDInsuff. Moreover, bone retention i.e., bone mineral content and density, bone volume, and bone quality parameters were significantly lower (p < 0.05) and bone turnover significantly was higher (p < 0.05). Although the PM is a useful tool to improve mineral absorption and bone retention, it would seem important to monitor VD nutritional status to ensure the full prebiotic effect in the large intestine.

Effect of Adding a Galacto-Oligosaccharides/Fructo-Oligosaccharides (GOS/FOS®) Mixture to a Normal and Low Calcium Diet, on Calcium Absorption and Bone Health in Ovariectomy-Induced Osteopenic Rats.

Menopause is associated with bone loss. Prebiotics increase Ca, inorganic phosphorus (Pi), and Mg absorption, improving bone health. These increases would supply an extra amount of minerals, decreasing bone resorption and possibly reversing ovariectomy-induced bone loss. The present experimental study sought to evaluate the effect of adding a prebiotic GOS/FOS® mixture to a normal or a low Ca diet on Ca, Pi, and Mg absorption, in osteopenic rats. Four groups of n = 8 rats each were OVX, and 8 rats were SHAM operated. All rats were fed a commercial diet for 45 days. They were then fed one of the following diet for 45 days: C-0.5%: SHAM fed AIN 93 M containing 0.5%Ca; O-0.5% and O-0.3%: OVX rats fed AIN 93 M, containing 0.5% or 0.3%Ca, respectively; GF-0.5% and GF-0.3%: OVX rats fed AIN 93 M, containing 0.5% or 0.3%Ca+ 2.5% GOS/FOS®, respectively. At the end of the experimental time point, Ca, P, and MgAbs% was significantly higher in GF-0.5% and GF-0.3% as compared to the remaining groups (p < 0.01). Irrespective of diet Ca content, CTX decreased whereas femur Ca and P content, tibia BV/TV and GPC.Th, lumbar spine and proximal tibia BMD, bone strength, bone stiffness, and elastic modulus increased in the GF-0.5% and GF-0.3% groups as compared to O-0.5% and O-0.3%, respectively (p < 0.05). This prebiotic mixture would be a useful tool to prevent the increase in bone loss associated with menopause and aging.

Effect of a mixture of GOS/FOS® on calcium absorption and retention during recovery from protein malnutrition: experimental model in growing rats.

INTRODUCTION: During growth, protein deprivation impairs epiphyseal growth plate (EGP) height, bone volume (BV) and endochondral ossification. During catch-up growth, Ca availability becomes essential to ensure the extra amount needed to achieve optimal peak bone mass and strength. GOS and FOS improve mineral absorption in the colon. PURPOSE: The effect of a mixture of GOS/FOS® 9:1 added to a 0.5 %Ca (NCa) and a 0.3 %Ca (LCa) diets on Ca, P and Mg absorptions and bone mineralization, density and structure using an experimental model of growing rats recovering from early protein malnutrition was investigated. METHODS: To induce protein malnutrition, rats were fed a low protein diet: 4 % (LPD) during 1 week and then were randomly assigned to recovery groups (R) until day 50 (T = 50) as follows: R0.5 %: NCa; RP0.5 %: NCa + 5.3 % GOS/FOS®; R0.3 %: LCa and RP0.3 %: LCa + 5.3 % GOS/FOS®. Control groups received the 0.5 %Ca or 0.3 %Ca diet from weaning until day 40 or 50. RESULTS: Body weight and length increased in C groups throughout the study; both were arrested in all R during LPD consumption and increased immediately after re-feeding. Independently of dietary Ca content, LS counts, ß-glucosidase and Ca, P and Mg absorption increased, whereas cecum pH, ß-glucuronidase, urease and tryptophanase decreased in RP0.5 %: and RP0.3 %: as compared to the other studied groups (p < 0.01). Prebiotic consumption decreased CTX levels and increased femur Ca, Mg and P contents, total skeleton bone mineral content, proximal tibia and spine BMD, BV, EGP height and hypertrophic zone thickness, stiffness and elastic modulus as compared to recovery groups fed the prebiotic-free diets. CONCLUSION: Under the present experimental conditions, GOS/FOS® mixture induced colonic positive effects, which increased Ca, P and Mg absorption. Thus, consuming the prebiotic-containing diet resulted in an extra amount of minerals that improved bone development in growing rats recovering from protein malnutrition.

Effect of a combination GOS/FOS® prebiotic mixture and interaction with calcium intake on mineral absorption and bone parameters in growing rats.

AIM: Increasing calcium intake is the most effective strategy for avoiding Ca deficit. However, if intake remains inadequate, improving Ca absorption becomes an important tool to optimize Ca homeostasis and bone health. PURPOSE: The effect of a mixture of GOS/FOS(®) 9:1 added to a normal- or low-Ca diets on Ca absorption and bone mineralization, density and structure was investigated, in a model of growing rats. Several colonic parameters to help support the findings were also evaluated. RESULTS: Weanling Wistar rats received one of the four experimental AIN-93G diets: C5: 0.5% Ca; C3: 0.3% Ca; P5: 0.5% Ca + 5.3% GOS/FOS(®); P3: 0.3% Ca + 5.3% GOS/FOS(®) until 50 days (T = 50). At T = 50, lactobacillus and cecum weights were higher, whereas cecum pH was lower in P5 and P3 versus C5 and C3 (p < 0.001). At T = 50, fecal Ca, Mg and P were lower and their absorptions (mg/dL) were higher in P5 and P3 versus C5 and C3, respectively (p < 0.05). Ca, Mg and P absorption % was higher in P5 and P3 versus C5 and C3 (p < 0.001). Femur Ca and P content, bone mineral content, trabecular bone mineral density, tibia length, bone volume, osteoblast surface, stiffness and elastic modulus were higher in P5 and P3 versus C5 and C3 (p < 0.05). Despite the lower Ca content, P3 group reached similar values than C5 in all these latter parameters. CONCLUSIONS: Supplementing diets with the GOS/FOS(®) mixture increased bone mineralization, density and structure due to an increase in Ca, P and Mg absorptions. Thus, this prebiotic mixture may help to improve bone development in a period of high calcium requirements.

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.

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.

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.

High-fat diets affect energy and bone metabolism in growing rats.

BACKGROUND: High-fat diets are usually associated with greater weight (W) gain and body fat (BF). However, it is still unclear whether the type and amount of fat consumed influence BF. Additionally, dietary fat intake may also have consequences on skeletal health. OBJECTIVE: To evaluate in healthy growing rats the effects of high-fat diets and type of dietary fat intake (saturated or vegetable oils) on energy and bone metabolism. METHODS: At weaning, male Wistar rats (n = 50) were fed either a control diet (C; fat = 7% w/w) or a high-fat diet (20% w/w) containing either: soybean oil, corn oil (CO), linseed oil (LO), or beef tallow (BT) for 8 weeks. Zoometric parameters, BF, food intake and digestibility, and total and bone alkaline phosphatase (b-AP) were assessed. Total skeleton bone mineral density (BMD) and content (BMC), BMC/W, spine BMD, and bone volume (static-histomorphometry) were measured. RESULTS: Animals fed BT diet achieved lower W versus C. Rats fed high-fat vegetable oil diets showed similar effects on the zoometric parameters but differed in BF. BT showed the lowest lipid digestibility and BMC. In contrast, high vegetable oil diets produced no significant differences in BMC, BMC/W, BMD, spine BMD, and bone volume. Marked differences were observed for LO and BT groups in b-AP and CO and BT groups in bone volume. CONCLUSION: BT diet rich in saturated fatty acids had decreased digestibility and adversely affected energy and bone metabolisms, in growing healthy male rats. There were no changes in zoometric and bone parameters among rats fed high vegetable oil diets.

Salivary bone turnover markers in healthy pre- and postmenopausal women: daily and seasonal rhythm.

No studies had investigated circadian and circannual rhythms of bone biomarkers in whole saliva. We evaluated the salivary daily and seasonal rhythm of carboxy-terminal telopeptide of type I collagen (CTX) and bone alkaline phosphatase (b-ALP). Forty clinical and oral healthy ambulatory pre- and postmenopausal women from two southern Argentine cities: Comodoro Rivadavia (latitude 45º S) and Ushuaia (latitude 54º S) were included in the study. CTX levels were evaluated in serum, urine, and saliva, and b-ALP levels were measured in serum and saliva. In both groups of women, salivary CTX showed a maximum percentage of change early in the morning (80%) and a minimum in the late afternoon (45%), similarly to the pattern observed in urinary samples. No daily rhythm was observed in serum or salivary b-ALP. 25-Hydroxyvitamin D levels decreased in winter vs. summer (p < 0.01) without differences between the two studied groups. Conversely, parathormone reached higher levels in winter (p < 0.05) which induced a slight non-significant increment in salivary CTX and b-ALP levels. The results showed that, as in serum and urinary samples, salivary CTX exhibits daily and a slight seasonal rhythmicity. Whole non-stimulated saliva is a useful tool to detect several oral and systemic diseases because it has important advantages compared to serum and urinary samples. Then, it may also be a promising sample to test changes in bone metabolism contributing to diagnose and to monitor the therapy of several metabolic bone diseases.

Aminobisphosphonate-treated ewes as a model of osteonecrosis of the jaw and of dental implant failure.

BACKGROUND: Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) and dental implant failure are two negative side effects of chronic aminoBP treatment. METHODS: Eleven ovariectomized (OVX) ewes and four ewes subjected to sham surgery (SHAM) were treated as follows: OVX (n = 5): OVX plus saline solution; zoledronic acid-treated group (ZOL) (n = 6): OVX plus ZOL; SHAM (n = 4): SHAM plus saline solution. Extraction of the first upper molar was performed at 1 year, dental implant placement at 2 years, and sacrifice at 28 months. RESULTS: Implants remained in place in SHAM and OVX ewes but were lost in all ZOL ewes. ZOL sheep (2/6) showed inflammation and necrotic bone at mandibular region. No differences in serum calcium (Ca), inorganic phosphate (Pi) or 25-hydroxyvitamin D were observed, whereas bone alkaline phosphatase levels decreased in the three studied groups (P < 0.05). The significantly lowest levels of carboxy-terminal telopeptide of type I collagen were observed in ZOL (P < 0.05), and showed no differences between SHAM and OVX. OVX showed the lowest and ZOL the highest Ca and Pi contents in femur and maxilla (P < 0.05). Bone volume (BV/TV%) and iliac crest were similar at baseline and at month 4. At the end of the study, BV/TV%, proximal femur and hemi-mandible bone mineral content and bone mineral density, and trabeculae number were similar in SHAM and ZOL, and lower in OVX (P < 0.05). CONCLUSION: All ZOL-treated ewes on a schedule similar to that used in cancer patients showed extensive suppression of bone remodeling and implant failure. Some of the ZOL ewes developed BRONJ.

Correlation between salivary and serum markers of bone turnover in osteopenic rats.

BACKGROUND: Previously, we measured bone alkaline phosphatase (b-ALP) and terminal C-telopeptide of collagen type I (CTX) in saliva. The present longitudinal experimental study sought to determine whether salivary concentrations of b-ALP and CTX have the same response as in serum samples under different conditions: normal, increased, and reduced bone remodeling. METHODS: Thirty rats were ovariectomized (OVX) to induce osteopenia 60 days after surgery, and 10 rats were sham operated. Then, the rats were divided into four groups and treated as follows for 45 days: group 1 (G1) = SHAM + vehicle; group 2 (G2) = OVX + 8 microg olpadronate (OPD)/100 g of body weight; group 3 (G3) = OVX + 4 microg OPD/100 g of body weight; and group 4 (G4) = OVX + vehicle. Saliva and serum CTX and b-ALP were determined at 60 days (baseline) and at 75 days (T(75)). Lumbar spine and proximal tibia bone mineral density (BMD) was determined using dual-energy x-ray absorptiometry at baseline and at 105 days. RESULTS: SHAM baseline and T(75) salivary b-ALP and CTX levels correlated with serum concentrations (P <0.01 and P <0.004, respectively). A correlation was observed between saliva and serum concentrations of b-ALP and CTX in OVX at baseline (P <0.0001 and P <0.004, respectively). Baseline salivary b-ALP and CTX levels were lower in SHAM animals compared to OVX groups (P <0.01). After treatment, T(75) saliva and serum CTX remained higher in G4 compared to G1 (P <0.05), was lower in G2 than in G1 (P <0.01) and G3 (P <0.01), and was similar in G1 and G3. Changes in BMD were the result of variations in salivary CTX levels due to OPD treatment (P <0.05). CONCLUSIONS: Saliva determinations may prove to be practical and reliable for the detection of systemic signs of increased bone remodeling, particularly in cases involving pediatric, obese, and elderly patients, and in screening large populations. Moreover, saliva CTX may be one of the best candidate markers to detect the activity and severity of periodontal disease.

MicroARN circulantes y su potencialidad como futuros biomarcadores óseos / Circulating microRNAs and their potential use as future bone biomarkers / MicroRNAs circulantes e a sua potencialidade como futuros biomarcadores ósseos

Resumen El remodelamiento óseo es ejercido por la actividad de osteoblastos y osteoclastos y puede evaluarse por marcadores bioquímicos de formación y resorción ósea. Sin embargo, el nivel de los marcadores óseos está sometido a una enorme cantidad de variables y, además, carece o presenta escaso valor pronóstico. Los microARN (miARN) fueron recientemente estudiados como una alternativa potencial para ser utilizados como nuevos marcadores óseos. Los miARN son pequeñas moléculas de ARN no codificantes (15-25 nucleótidos) que, a través de la inhibición o degradación de ARN mensajeros, modifican una serie de funciones biológicas. Los miARN específicos de hueso ejercen funciones reguladoras sobre factores transcripcionales involucrados en la osteoblastogénesis y osteoclastogénesis, modificando el remodelamiento óseo. La mayoría de los miARN permanecen dentro de la célula, pero algunos son liberados a la circulación donde pueden ser dosados. Los miARN circulantes presentan gran estabilidad en fluidos biológicos, lo que los hace potenciales candidatos a ser utilizados como nuevos biomarcadores óseos. Cambios en el patrón normal de miARN circulantes específicos de hueso reflejarán modificaciones en el metabolismo óseo y señalan el posible inicio o progresión de enfermedades óseas, como la osteoporosis. Si bien es promisorio, el uso en la práctica clínica de los miARN específicos circulantes como nuevos biomarcadores óseos, ello implica primeramente cumplir con una serie de requisitos que permitan estandarizar las condiciones preanalíticas, analíticas y posanalíticas de estas moléculas. La presente revisión brinda información reciente sobre los estudios clínicos tendientes a determinar el posible uso de los miARN circulantes como nuevos biomarcadores óseos, ya que cuentan con elevada sensibilidad y especificidad diagnósticas, valor predictivo positivo y valor predictivo negativo.

Abstract Osteoblasts and osteoclasts activity determines the level of the bone remodelling process which can be assessed by biochemical markers of bone formation and resorption. However, bone marker levels are subject to a series of variables resand, furthermore, they lack or have little prognostic values. MicroRNAs (miRNAs) were recently studied as a potential alternative to be used as new bone biomarkers. The miRNAs are endogenous small noncoding RNA molecules (15-25 nucleotides) that regulate many biological functions by inhibiting or degrading specific messenger RNAs. Bone-specific miRNAs exert regulatory functions on key transcriptional factors involved in osteoblastogenesis and osteoclastogenesis, modifying the bone remodelling process. Most miRNAs remain within the cell, but some of them are released into circulation. Circulating miRNAs show great stability in biological fluids, which makes them excellent candidates to be used as new bone biomarkers. Modifications in the normal pattern of bone-specific circulating miRNA might reflect changes in bone metabolism, signalling the possible onset or progression of bone diseases, such as osteoporosis. Although promising, the use of specific circulating miRNAs as new bone biomarkers in clinical practice implies fulfilling a series of requirements that lead to standardising the pre-analytical, analytical and post-analytical conditions of these molecules. The present review gives an overview on the clinical studies related to the possible use of specific circulating miRNAs as new bone biomarkers.

Resumo A remodelação óssea é exercida pela atividade dos osteoblastos e osteoclastos e pode ser avaliada para a medição dos marcadores bioquímicos de formação e reabsorção óssea. No entanto, o nível dos marcadores ósseos está sujeito a grande quantidade de variáveis e, além disso, carece ou tem pouco valor prognóstico. Os microARN (miARN) foram estudados recentemente como uma alternativa potencial para serem utilizados como novos marcadores ósseos. Os MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificantes (15-25 nucleotídeos) que, através da inibição ou degradação de RNA mensageiros modificam uma série de funções biológicas. Os miRNAs específicos de osso exercem funções reguladoras sobre fatores transcricionais envolvidos na osteoblastogênese e na osteoclastogênese, modificando o processo de remodelação óssea. A maioria dos miRNAs permanece dentro da célula, mas de RNA mensageiros alguns são liberados na circulação, onde podem ser determinados bioquimicamente. Os miRNAs circulantes apresentam grande estabilidade em fluidos biológicos, o que os torna excelentes candidatos para serem usados como novos biomarcadores ósseos. Mudanças no padrão normal de miRNA circulantes específicos do osso mostrarão mudanças no metabolismo ósseo, sinalizando o possível início ou progressão de doenças ósseas, como osteoporose. Embora promissor, o uso de miRNAs específicas circulantes na prática clínica como novos biomarcadores ósseos, implica primeiramente, atender uma série de requisitos que permitem padronizar as condições pré-analíticas, analíticas e pós-analíticas dessas moléculas. A presente revisão fornece informações recentes sobre estudos clínicos destinados a determinar o possível uso dos miRNAs circulantes como novos biomarcadores ósseos, visto que contam com elevada sensibilidade e especificidade diagnósticas, valor preditivo positivo e valor preditivo negativo.

Vitamina D y embarazo: una experiencia piloto / Vitamin D and pregnancy: a pilot experience

La insuficiencia de vitamina D (VD) en el embarazo se relaciona con una mayor incidencia de cesáreas, preeclampsia y partos prematuros. Objetivo: evaluar si el grado de insuficiencia de VD se asocia a mayor número de cesáreas y evaluar la correlación entre la 25 hidroxivitamina D (25OHD) materna y en sangre del cordón del recién nacido. Las mujeres (n=127) se dividieron según sus niveles de 25OHD (ng/mL):G1:<20 (deficiencia), G2:20-30 (insuficiencia), G3:>30 (suficiencia). Se registraron edad; edad gestacional (EG); índice de masa corporal (IMC); tensión arterial sistólica y diastólica; tipo de parto y la estación del año en que se tomó la muestra. Se determinaron calcemia (ng/mL); 25OHD; parathormona intacta (pg/mL); fosfatasa alcalina ósea (UI/L) y crosslaps (pg/mL). La edad media fue de 26±6 años y la EG de 35,8±2,7 semanas, sin diferencias entre grupos. El porcentaje de cesáreas fue mayor en G1 que en G2 y G3 (31,3%, 21,4% y 25%, respectivamente; p<0,05). El mayor porcentaje de muestras se tomó en primavera (p<0,05). No se observaron diferencias en las demás variables maternas estudiadas. La 25OHD materna correlacionó positivamente con los valores de la sangre de cordón de sus respectivos recién nacidos (r= 0,67; p<0,0001). Independientemente de la época del año y del IMC, se observó que un porcentaje significativo de las mujeres embarazadas estudiadas tenía niveles de 25OHD inferiores a 30 ng/mL. Conclusión: evidenciamos que la deficiencia de VD materna se asoció al número de cesáreas. Asimismo, los niveles séricos de 25OHD en sangre de cordón umbilical correlacionaron significativamente con los maternos. (AU)

Vitamin D (VD) insufficiency in pregnancy is associated with a higher incidence of cesarean section, preeclampsia, and preterm delivery. Objective: to evaluate if the degree of VD insufficiency is associated with the incidence of cesarean section and to determine the correlation between maternal and newborn cord blood 25-hydroxy VS (25OHD). Women (n=127) were divided according to their 25OHD levels (ng/mL): G1:<20 (deficiency), G2:20-30 (insufficiency), G3:>30 (sufficiency). Age; gestational age (GA); body mass index (BMI); systolic and diastolic blood pressure (mmHg); type of delivery and the season of the year in which the sample was taken were recorded. Calcemia (ng/mL); 25OHD; intact parathormone (pg/mL); bone alkaline phosphatase (IU/L) and Crosslaps (pg/mL) levels were determined. Mean age was 26±6 years and GA was 35.8±2.7 weeks with no differences among groups. The % of cesarean sections was higher in G1 than in G2 and G3 (31.3%, 21.4% and 25%; p<0.05). The highest % of samples were taken in spring (p<0.05). No differences were observed in the other maternal variables studied. Maternal serum 25OHD levels correlated positively with those of cord blood from their respective newborns (r=0.67; p<0.0001). Regardless the season of the year and BMI, a high % of the studied pregnant women presented 25OHD levels lower than 30 ng/ml. Conclusion: we found that maternal VD deficiency is associated with the number of cesarean sections. In addition, 25OHD levels in the newborn significantly correlate with maternal serum levels. (AU)

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.

Absorción mineral y retención ósea en ratas normales en crecimiento por el consumo de un yogur experimental reducido en lactosa que contiene galactooligosacáridos (GOS) / Mineral absorption and bone retention in normal growing rats by consumption of an experimental lactose-reduced yogurt containing galactooligosaccharides (GOS)

Introducción: Los GOS son prebióticos naturales presentes en la leche materna que pue-den obtenerse enzimáticamente a partir de la lactosa de leche de vaca durante la fabricación de yogur. El producto lácteo resultante será reducido en lactosa y contendrá prebióticos y bacterias potencialmente probióticas. Sin embargo, mantendrá la baja relación Ca/Pi que aporta la leche de vaca, lo que podría alterar el remodelamiento óseo y la mineralización. Objetivo: comparar si un yogur reducido en lactosa que contiene GOS (YE) ofrece ventajas adicionales respecto de un yogur regular sin GOS (YR) sobre las absorciones (Abs) de Ca y Pi, retención y calidad ósea durante el crecimiento normal. Al destete, ratas machos fueron divididas en 3 grupos alimentados con AIN ́93-G (C), YE o YR durante 28 días. Resultados: YE mostró el mayor aumento de lactobacilos fecales; producción de ácidos grasos de cadena corta especialmente p, profundidad de las criptas colónicas y menor pH cecal. El %AbsCa y %AbsPi aumentó en el siguiente órden: YE> YR> C (p < 0,05). El contenido de Ca y Pi en fémur, la densidad y contenido mineral óseos y los parámetros biomecánicos fueron similares en YE y C, mientras que YR mostró valores significativa-mente menores (p < 0,05). Conclusiones: YE aumentó las Abs y biodisponibilidad de minerales, alcanzando la retención y calidad ósea de C. El aumento en las Abs observado en YR no logró obtener la retención y calidad ósea de C. Conclusión: YE habría contrarrestado el efecto negativo del mayor aporte de Pi de la leche de vaca y sería una buena estrategia para lograr el pico de masa ósea y calidad del hueso adecuados, especialmente en individuos intolerantes a la lactosa. (AU)

Breast milk contains an optimal calcium/phosphate (Ca/Pi) ratio and GOS. These natural prebiotics can be enzymatically produced via cow's milk lactose inyogurt manufacture. This milk product is low in lactose and contains prebiotics and potentially probiotic bacteria but maintains a low Ca/Pi ratio that could alter bone remodeling and mineralization. We evaluated if a lactose-reduced yogurt containing GOS (YE) offers additional advantages over regular yogurt without GOS (YR) on Ca and Pi absorption (Abs), bone retention and quality during normal growth. Weaning male rats were divided into 3 groups fed AIN'93-G (C), YE or YR for 28 days. Results: YE showed the highest increase in fecal lactobacilli; short-chain fatty acids production, especially propionate and butyrate; intestine crypt depth, and the lowest cecal pH. AbsCa% and AbsPi% increased in this order: YE> YR> C (p <0.05). Ca and Pi content in femur, bone density and mineral content, and biomechanical parameters were similar in YE and C, while YR showed the significantly lowest value (p < 0.05). Conclusions: YE increased mineral Abs reaching the retention and bone quality of C. Although YR increased Abs, bone retention and quality did not achieve C values. Seemingly, YE compensated for the negative effect of the higher Pi supply and would be a good strategy to achieve adequate peak bone mass and bone quality, especially in lactose intolerant individuals. (AU)

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.

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

The present study was carried out to obtain an experimental model of vitamin D (vit D) insufficiency and established osteopenia (experiment 1) to then investigate whether vit D status, i.e. normal or insufficient, interferes with bone mass recovery resulting from bisphosphonate therapy (experiment 2). Rats (n = 40) underwent OVX (n = 32) or a sham operation (n = 8). The first 15 days post-surgery, all groups were kept under fluorescent tube lighting and fed a diet containing 200 IU% vit D (+D). They were then assigned during an additional 45 days to receive either +D or a diet lacking vit D (-D) and kept under 12 h light/dark cycles using fluorescent or red lighting. Serum 25HOD was significantly lower in -D rats (P < 0.0001). The type of lighting did not induce differences in 25OHD, calcium (sCa), phosphorus (sP), bone alkaline phosphatase (b-AL), CTX, bone density or histology. No osteoid was observed in undecalcified bone sections. Experiment 2 (105 days): rats were fed either +D or -D according to experiment 1 and were treated with either placebo or 16 mug olpadronate (OPD)/100 g rat/week during the last 45 days. Whereas 25HOD was significantly lower (P < 0.0001) in -D/OPD than in +D/OPD rats, no significant differences in sCa, sP, b-AL or CTX were observed. OPD prevented the loss of lumbar spine (LS) and proximal tibia (PT) BMD and the decrease in bone volume (BV/TV) (P < 0.05) and in the number of trabeculae observed in untreated rats. However, +D/OPD animals presented significantly higher values of LS BMD, PT BMD and BV/TV than -D/OPD rats (P < 0.05). No osteoid was observed in undecalcified sections of bone. In summary, this is the first experimental study to provide evidence that differences in vit D status may affect the anticatabolic response to bisphosphonate treatment. However, the molecular mechanism through which vit D insufficiency reduces the effect of the aminobisphosphonate remains to be defined.

Influence of maternal dietary calcium levels on milk zinc, calcium and phosphorus contents and milk production in rats.

The aim of this study was to analyze zinc (Zn), calcium (Ca) and phosphorus (P) contents in milk and the lactational performance in rats fed different Ca levels. Female Wistar rats were fed during pregnancy and lactation with experimental diets containing 20% protein and high (0.90%, HCa), normal (0.60%, NCa) or low (0.20%, LCa) Ca levels. Milk samples were collected after 15 days to determine the milk mineral composition. Pup weight was recorded from birth to weaning (litter size: 6-8 pups) to determine weight gain and calculate milk production. At delivery there were no significant differences in the body weight of the pups between the groups, but at day 15, the LCa group showed lower values than both NCa and HCa groups (p<0.05). The weight gain of the LCa group was significantly lower than of the HCa and NCa groups, between delivery and day 5 (p<0.05). This reduced rate of weight gain led to the LCa group reaching weaning weight later than the other groups. Milk production (g/pup/day) was significantly lower when dams were fed the LCa than the NCa and HCa diets (p<0.05). There were no significant differences among the groups in milk Ca, P and Zn levels and Ca/P ratio. The body mineral composition of the pups at birth did not differ between the groups; at weaning, however, both LCa and HCa groups had lower element contents than the NCa group (p<0.05). In conclusion, dams fed with a diet containing low Ca levels produced smaller volumes of milk and their pups reached weaning weights later than the other groups. As the milk mineral composition was not affected, it can be hypothesized that in dams fed low dietary Ca, the smaller milk yield might have been a way of maintaining milk quality. High Ca levels affected neither pregnancy outcome nor lactational performance.

[Bone remodeling markers in saliva as compared to serum in rats]. / Marcadores del remodelamiento oseo en saliva y su correlación con los niveles sanguíneos en ratas.

Bone markers are useful tools to measure bone remodeling; currently they are assessed in serum and urinary samples; however there is little information concerning their measurement in saliva. The present experimental study evaluates the possibility to measure collagen type I carboxiterminal telopeptide (CTX) and bone alkaline phosphatase (b-AP) in saliva, its correlation with serum samples in normal conditions and in the increase of the bone remodeling due to estrogen deficiency. Twenty four normal adult Wistar rats (300 +/- 20 g) [12 SHAM and 12 rats after 1 week of bilateral ovariectomy (OVX)] were studied. Fasting serum and total saliva after stimulation with pilocarpine were collected. In both samples were measured: CTX (ng/ml) by ELISA (RatLabs, Osteometer Bio Tech, Denmark) and b-AP (IU/L) (Wiener, colorimetrically). Both CTX and b-AL in serum samples were significantly higher in OVX than in SHAM rats (15.3 +/- 4.0 vs. 21.8 +/- 6.4, p < 0.05 y 71 +/- 29 vs. 104 +/- 23; p < 0.01, respectively). Saliva presented the same behaviour (3.6 +/- 0.5 vs. 6.4 +/- 2.9; p < 0.02 y 73 +/- 29 vs. 90 +/- 8; p < 0.003, respectively). When saliva CTX and b-AP were plotted against serum concentration significant positive correlations were obtained: r = 0.58, p < 0.05 and r = 0.59; p < 0.05, respectively. In conclusion, the present results are promisory in the sense of the potential use of a salivary-based test for evaluating bone remodeling. However, the use of this methodology for clinical practice needs extensive additional investigations.