In the present study, we investigated whether high dietary Ca and exogenous parathyroid hormone 1–34 fragments (PTH 1–34) have synergistic effects on bone formation in adult mice, and explored the related mechanisms. Adult male mice were fed a normal diet, a high-Ca diet, a PTH-treated diet, or a high-Ca diet combined with subcutaneously injected PTH 1–34 (80 μg/kg per d) for 4 weeks. Bone mineral density, trabecular bone volume, osteoblast number, alkaline phosphatase (ALP)- and type I collagen-positive areas, and the expression levels of osteoblastic bone formation-related genes and proteins were increased significantly in mice fed the high-Ca diet, the PTH-treated diet, and, even more dramatically, the high-Ca diet combined with PTH. Osteoclast number and surface and the ratio of receptor activator for nuclear factor-κB ligand (RANKL):osteoprotegerin (OPG) were decreased in the high-Ca diet treatment group, increased in the PTH treatment group, but not in the combined treatment group. Furthermore, third-passage osteoblasts were treated with high Ca (5 mm), PTH 1–34 (10− 8m) or high Ca combined with PTH 1–34. Osteoblast viability and ALP activity were increased in either the high Ca-treated or PTH-treated cultures and, even more dramatically, in the cultures treated with high Ca plus PTH, with consistent up-regulation of the expression levels of osteoblast proliferation and differentiation-related genes and proteins. These results indicate that dietary Ca and PTH play synergistic roles in promoting osteoblastic bone formation by stimulating osteoblast proliferation and differentiation.

Using a sequential in vitro/in vivo approach, we tested the ability of botanical extracts to influence biomarkers associated with bone resorption and bone formation. Pomegranate fruit and grape seed extracts were found to exhibit anti-resorptive activity by inhibiting receptor activator of nuclear factor-κB ligand (RANKL) expression in MG-63 cells and to reduce IL-1β-stimulated calvarial 45Ca loss. A combination of pomegranate fruit and grape seed extracts were shown to be effective at inhibiting bone loss in ovariectomised rats as demonstrated by standard histomorphometry, biomechanical and bone mineral density measurements. Quercetin and licorice extract exhibited bone formation activity as measured by bone morphogenetic protein-2 (BMP-2) promoter activation, increased expression of BMP-2 mRNA and protein levels, and promotion of bone growth in cultured mouse calvariae. A combination of quercetin and licorice extract demonstrated a potential for increasing bone mineral density in an intact female rat model as compared with controls. The results from this sequential in vitro/in vivo research model yielded botanical extract formulas that demonstrate significant potential benefits for bone health.

Twelve 6-month-old growing female goats and sheep were used in this study. Blood samples were obtained in the morning before goats and sheep were given food and then at 2-h intervals for 24 h (part I). This procedure was repeated 2 weeks later (part II). Concentrations of osteocalcin (OC), activities of total (tAP) and bone-specific alkaline phosphatase (bAP), degradation products of C-terminal telopeptide of type-I collagen (CrossLaps™ CL), and carboxyterminal telopeptide of type-I collagen (ICTP) were measured in serum.

In both parts of the study, all bone marker concentrations were significantly higher in goats than in sheep. The OC concentrations in goats increased in the late afternoon/evening and decreased thereafter to reach values similar to those obtained at the beginning. The ICTP concentrations in goats slowly decreased until 14:00 h, increased, and decreased again. The concentrations in sheep decreased continuously but not significantly, towards the morning sampling. The CL concentrations increased in both sheep and goats during the night but at 06:00 h started to decrease to levels found at the beginning of testing. The bAP activities decreased in goats from 20:00 to 22:00 h. Changes in the concentrations of bone markers were mainly observed in goats of this study. As documented for bone resorption and formation in other species, circadian rhythms were evident for concentrations of ICTP, CL, bAP and OC. The present study indicates that growing goats may have a physiologically higher bone turn-over than growing sheep, because the bone marker concentrations were always higher.

The beneficial effect of bovine whey protein (WP) on bone metabolism has been shown in adult human subjects and ovariectomised rats. However, its effect on bone formation in earlier life, particularly during periods of bone mineral accrual, has not been investigated. Twenty-one male rats (4 weeks old, Wistar strain) were randomised by weight into three groups of seven rats each and fed ad libitum on a semi-purified low-Ca diet (3·0 g Ca/kg diet) containing 0 (control), 10 (diet WP1) or 20 (diet WP2) g bovine WP/kg for 47 d. On day 34 of the dietary intervention, all rats had two gelatine capsules containing demineralised bone powder implanted subcutaneously in the thorax region (a well-established in vivo model of ectopic bone formation). At 14 d after implantation, alkaline phosphatase activity (reflective of bone formation) in the bone implants from animals fed WP1 and -2 diets was almost 2-fold (P<0·01) that of control animals. Insulin-like growth factor (IGF)-I mRNA levels were about 3-fold (P<0·05) higher in implants from animals fed the WP diets compared with those from control animals. Serum- and urine-based biomarkers of bone metabolism and bone mineral composition in intact femora were unaffected by WP supplementation. In conclusion, the present findings suggest that bovine WP can enhance the rate of ectopic bone formation in young growing rats fed a Ca-restricted diet. This effect may be mediated by an increased synthesis of IGF-I in growing bone. The effect of WP on bone formation warrants further investigation.