BackgroundThe AIN-93G reference (REF) diet is used to allow the comparison within and between studies of different research groups but its levels of vitamin D (vit D) and calcium (Ca) may be higher than required for healthy bone structure and bone mineral density (BMD).ObjectiveTo determine if lower dietary levels of Ca (3.5, 3 or 2.5 g Ca/kg diet) at 1 of 2 levels of vit D (100 or 400 IU/kg diet) supports similar development of bone structure and BMD compared to AIN-93G reference (REF) diet in female CD-1 mice at 2 and 4 months of age.MethodsWithin a trial, weanling female mice (n = 12–15/group) were randomized to 1 of 4 diets until necropsy at 4 months of age: Trial 1: 100 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g Ca/kg diet (REF); and Trial 2: 400 IU vit D/kg + 3.5, 3 or 2.5 g Ca/kg diet or 1000 IU vit D/kg + 5 g/kg diet (REF). At age 2 and 4 months, in vivo bone structure and BMD were assessed using micro-computed tomography (μCT) at the proximal and midpoint tibia. At age 4 months, lumbar vertebra 4 (L4) and mandible structure were analyzed ex vivo, femur strength at midpoint and neck was assessed and serum 25(OH)D3 and PTH were quantified.ResultsFor Trial 1 (100 IU vit D/kg), there were no differences in tibia structure at age 2 and 4 months nor L4 or mandible structure or femur strength at the midpoint or neck at 4 months of age despite lower serum 25(OH)D3 among all groups compared to REF. For Trial 2 (400 IU vit D/kg), mice fed 2.5 g Ca/kg diet had lower (p < 0.05) Ct.Ar/Tt.Ar and Ct.Th at the tibia midpoint compared to REF. Furthermore, Ct.Th. was greater in REF and 3.5 g Ca/kg diet compared to 2.5 g Ca/kg diet at age 2 but not 4 months of age. At L4, BV/TV was lower (p < 0.05) in the 3 g Ca/kg diet group compared to REF at age 4 months. There were no differences among groups for serum 25(OH)D3 or femur strength at the midpoint or neck. Serum PTH was not elevated compared to REF in either Trial.ConclusionLowering both dietary vit D (100 IU/kg) and Ca (2.5 g/kg) in AIN-93G diet did not result in differences in bone development of female CD-1 mice at early adulthood. Translational relevance of bone studies conducted using the AIN-93G diet may be affected by its high vit D and Ca content.

Aims Higher intakes of fruits and vegetables, and vitamin C are associated with improved periodontal healing post‐scaling and root planing (SRP). This study determined if this association was sustained at 3–4 years post‐SRP, and if flavonoid intake is associated with periodontal health. Whether reduced probing depth (PD) is sustained and whether PD is correlated with salivary IL‐1β, IL‐6 and CRP at 3–4 years post‐SRP were also studied. Materials and Methods Clinical periodontal outcomes, dietary intakes and salivary markers of inflammation were measured in patients (n = 43, 23 females, 37–93 years) who had undergone SRP 3–4 years earlier and had been part of a periodontal maintenance programme. Results Flavonoid intake was inversely associated with PD (p = .042) and salivary IL‐1β concentration (p = .015) after adjustment for multiple confounders. When changes in PD were considered, the association of flavonoid intake with reduced PD became borderline significant (p = .051) but persisted for IL‐1β (p = .018). PD at 3–4 years and 2–4 months post‐SRP was similar. There was a positive correlation between PD and salivary IL‐1β (p = .005) but not with salivary CRP and IL‐6. Conclusion Higher flavonoid intake is associated with lower IL‐1β. Also, regular supportive periodontal therapy maintained the improved PD at 3–4 years post‐SRP regardless of smoking status.

During pregnancy and lactation, maternal bone mineral density (BMD) is reduced as calcium is mobilized to support offspring bone development. In humans, BMD returns to pre-pregnancy levels shortly after delivery, shifting from a high rate of bone resorption during pregnancy and lactation, into a rapid phase of bone formation post-lactation. This rapid change in bone turnover may provide an opportunity to stimulate a greater gain in BMD and stronger trabecular and cortical structure than present pre-pregnancy. Providing polyphenols present in red rooibos herbal tea may promote such an effect. In vitro, red rooibos polyphenols stimulate osteoblast activity, reduce osteoclastic resorption, and increase mineral production. The study objective was to determine if consuming red rooibos from pre-pregnancy through to 4 months post-lactation resulted in a higher BMD and improved trabecular and cortical bone structure in a commonly used rat model. Female Sprague-Dawley rats (n = 42) were randomized to one of the following groups: PREG TEA (pregnant, received supplemental level of red rooibos in water: ~2.6 g /kg body weight/day in water), PREG WATER (pregnant, received water), or NONPREG CON (age-matched, non-pregnant control, received water) from 2 weeks pre-pregnancy (age 8 weeks) through to 4 months post-lactation. Rats were fed AIN-93G (pre-pregnancy through to the end of lactation) and AIN-93M (post-lactation onwards). BMD and trabecular structure (bone volume fraction, trabecular number, trabecular separation) were improved (p &lt; 0.05) by 1- or 2-months post-lactation when comparing PREG TEA to PREG CON, though neither group recovered to the level of NONPREG CON. Cortical outcomes (cortical area fraction, cortical thickness, tissue mineral density) for PREG TEA and PREG CON were reduced (p &lt; 0.05) following lactation but returned to the level of NONPREG CON by 2-months post-lactation, with the exception of cortical thickness. The lack of recovery of BMD and key outcomes of trabecular bone structure was unexpected. While consumption of red rooibos did not result in stronger bone post-lactation, red rooibos did support the partial recovery of trabecular BMD and bone structure following pregnancy and lactation. The findings also provide insight into the timing and dose of polyphenols to study in future interventions.

While repeated in vivo micro-computed tomography (μCT) allows for longitudinal measurement of bone outcomes in rodent models, it is important to determine that the resulting irradiation – dependent on the frequency and number of scans - does not exceed the effects of the intervention. The objective of this study was to determine whether repeated irradiation exposure from μCT scans at 1-month intervals for a total of four scans would alter trabecular or cortical bone structure outcomes and/or bone mineral density in tibias from both male and female CD-1 mice. The right tibia of male (n = 12) and female (n = 11) CD-1 mice were scanned using μCT at 2, 3, 4, and 5 months of age, while the contralateral left tibia served as a control and was scanned only at 5 months of age. All scans were performed at a resolution of 9 μm using a radiation dose of 460 mGy per scan. Some outcomes of trabecular bone structure were affected by repeated irradiation in both males and females. The bone volume fraction was lower in the irradiated right tibia compared to the non-irradiated left tibia in both males (p < 0.05) and females (p < 0.01) as a result of decreased trabecular number (males p < 0.05; females p < 0.05) and increased trabecular separation (males p < 0.05; females p < 0.01). Some cortical measures were also affected in females but not in males, including lower cortical bone periosteal perimeter (p < 0.05), lower total area (p < 0.01) and lower marrow area (p < 0.05) with repeated irradiation. Exposure to repeated radiation at intervals of 1 month, for a total of four scans, altered trabecular bone in both male and female CD-1 mice while outcomes of cortical bone structure were altered only in females.

BackgroundCalcium (Ca) and vitamin D (vit D) in the AIN-93G diet may be higher than required for healthy bone development, and mask the potential benefit of a dietary intervention.ObjectiveThe objective was to determine if lower levels of Ca and vit D than is present in the AIN-93G diet supports bone development in growing male CD-1 mice.MethodsWeanling male CD-1 mice were randomized to modified AIN-93G diets containing either 100 (Trial 1) or 400 (Trial 2) IU vit D/kg diet within one of two or three Ca levels (0.35, 0.30, or 0.25% Ca diet in Trial 1 or 0.35% or 0.25% in Trial 2) or the AIN-93G diet (1000 IU/kg vit D and 0.5% Ca) from weaning to 4 months of age (n = 13–15/group). At 2 and 4 months of age, BMD and structural properties of the tibia were analyzed in vivo. Structure of lumbar vertebra 4 (L4) and mandible, and femur strength were assessed ex vivo at age 4 months.ResultsThere were no differences in tibia, L4, and mandible structure between the AIN-93G diet and the 0.35% Ca groups at either vit D level. A few structure outcomes were compromised with the 0.25 and/or 0.3% Ca diets but there were no differences in femur biomechanical strength compared to AIN-93G group in either Trial.ConclusionAt 400 or 100 IU vit D/kg diet, Ca can be lowered to 0.35% without detriment to BMD or bone structure while bone strength is not altered at lower Ca (0.25%) compared to CD-1 mice fed AIN-93G diet. Because of genetic variation in CD-1 mice among different breeding facilities, results in CD-1 mice from other facilities may differ from the present study.

Chronic low-grade inflammation has been identified as an underlying cause of many diseases including osteoporosis. Lipopolysaccharide (LPS) is a potent inducer of the inflammatory response that can negatively affect bone outcomes by upregulating bone resorption and inhibiting bone formation. The objective of this study was to assess the longitudinal response of trabecular and cortical bone structure and bone mineral density to LPS continuously administered for 12 weeks in male and female CD-1 mice. Mice were assigned to one of four LPS groups at 8-weeks of age: placebo (0.0 μg/d), low (0.9 μg/d), mid (3.6 μg/d) and high (14.4 μg/d) dose. Trabecular and cortical bone outcomes were measured at 8, 12, 16, and 20 weeks of age using in vivo micro-computed tomography. The anticipated serum LPS dose-dependent response was not observed. Therefore, the low, mid, and high LPS groups were combined for analysis. Compared to the placebo group, endpoint serum LPS was elevated in both males (p < 0.05) and females (p < 0.05) when all LPS treatment groups were combined. However, there was no significant change in trabecular or cortical bone outcomes in the combined LPS groups compared to the placebo following the 12-week LPS intervention for either sex. This suggests that although serum LPS was elevated following the 12-week LPS intervention, the dosages administered using the osmotic pumps was not sufficient to negatively impact trabecular or cortical bone outcomes in either male or female CD-1 mice.

No abstract

body size without altering the balance between bone formation and bone resorption. Interestingly, loss of JAK2 impaired expression of IGF1 in osteoclasts, suggesting an important role for osteoclast-derived IGF1 in determination of body size. We also show that overexpression of circulating IGF1 in Oc-JAK2-KO mice rescued their growth defects, supporting a causal role between JAK2-mediated IGF1 expression in osteoclasts and postnatal growth. Results Generation of Oc-JAK2-KO mice. To validate the osteoclast specificity of the Cathepsin K-Cre (Ctsk-Cre) mouse, we bred Ctsk-Cre + mice to mTmG +/reporter mice whose tissue expressed either fluorescence TdTomato in the absence of Cre-mediated recombination or enhanced GFP (EGFP) in its presence. Sections of the knee joint showed robust expression of EGFP along the surface of the trabecular bone in Ctsk-Cre + mT-mG + mice (Figure 1A). Staining for tartrate resistant alkaline phosphatase (TRAP) on the adjacent serial section confirmed that EGFP was present in the expected distribution for osteoclasts. EGFP was not detected in Crecontrols. Sections of other tissues, including growth plate cartilage, brain, hypothalamus, testis, ovary, pancreas, liver, small intestine, kidney, and thyroid did not express EGFP (Figure 1B). Next, to generate Oc-JAK2-KO mice, Ctsk-Cre + mice were bred to Jak2 floxed mice (Jak2 fl/fl) and resultant Ctsk-Cre +-Jak2 fl/+ mice were crossed to Jak2 fl/fl mice to generate KO mice (Ctsk-Cre + Jak2 fl/fl) and littermate control mice (Jak2 fl/fl and Ctsk-Cre + Jak2 fl/+). To validate osteoclast-specific deficiency of JAK2 in our Oc-JAK2-KO mouse, we measured Jak2 mRNA levels in tissues and in BM-derived osteoclasts from Ctsk-Cre + Jak2 fl/fl and Jak fl/fl mice. JAK2 mRNA expression was reduced by 26% in bone and by 70% in osteoclast cultures from Ctsk-Cre + Jak2 fl/fl mice (Figure 1C). Importantly, Jak2 mRNA expression was unchanged in BM-derived macrophages, liver, testis, ovary, or hypothalamus. There have been previous reports of Ctsk-Cre mediating germline gene deletion of certain floxed alleles (12); however, this did not occur in Ctsk-Cre + Jak2 fl/fl mice, since Jak2 expression was preserved in nonosteoclast cells and tissues. Lastly, we performed IHC for JAK2 on femur sections with TRAP staining on the adjacent serial section to identify TRAP + multinucleated giant cells. In control mice, there was weak to moderate immunostaining for JAK2 in TRAP + multinucleated giant cells (Figure 1D). In Ctsk-Cre + Jak2 fl/fl mice, immunoreactivity for JAK2 was absent in TRAP + multinucleated giant cells, with retained expression in other BM cells. Overall, these results indicate that Ctsk-Cre + Jak2 fl/fl mice represent a rigorous experimental model to study the role of osteoclast JAK2 in vivo. For all experiments, both Jak2 fl/fl and Ctsk-Cre + Jak2 fl/+ mice were used as controls in approximately equal proportions unless otherwise specified. There were no significant differences in any measured outcomes between Jak2 fl/fl and Ctsk-Cre + Jak2 fl/+ mice, in...