The response of rat tibiae to incremental bouts of mechanical loading: A quantum concept for bone formation

To investigate the minimum number of loading bouts necessary to produce new lamellar or woven bone formation, and the time required for its initiation, bone formation was measured in 32 retired breeder female Sprague-Dawley rats following one, two, three, or five bouts of applied loading. Bending forces of 54 N were applied to right tibiae using a four-point loading apparatus, and left tibiae served as contralateral controls. Loading was applied as a sine wave with a frequency of 2 Hz for 18 s (36 cycles) per loading bout. Rats were injected with alizarin on day 1 and calcein on days 5 and 12, and were killed on day 19. One bout of loading was sufficient to increase the periosteal woven bone surface (Wb.Pm/B.Pm) from 0% to 40% (p < 0.01), and to 80% after five bouts of loading (p < 0.01), with a dose-response relationship for increases in Wb.Pm/B.Pm (p < 0.0001), mineral apposition rate (Wb.AR; p = 0.002), and bone formation rate (Wb.BFR/BS; p = 0.0001). In the first labeling period (days 1–5), the endocortical lamellar bone forming surface (BS_f/BS) was increased slightly (p < 0.05), but no significant differences were shown for BFR/BS or MAR. From days 5 to 19, right tibiae showed a dose-response increase in BFR/BS (p = 0.002) and BS_f/BS (p = 0.008), but not MAR. These results are consistent with a “quantum” model of bone formation such that a “quantum” of bone cells is activated in response to the loading bout and the strain magnitude dictate the size or microstructural organization of a given packet of new bone. Conversely, the distributed nature of loading may define the recruitment, rather than size, of new packets of bone.     

A comparative study of the bone metabolic response to dried plum supplementation and PTH treatment in adult,osteopenic ovariectomized rat

Dried plum has been reported to have potent effects on bone in osteopenic animal models, but the mechanisms through which bone metabolism is altered in vivo remain unclear. To address this issue, a study comparing the metabolic response of dried plum to the anabolic agent, parathyroid hormone (PTH), was undertaken. Six month-old female Sprague Dawley rats (n = 84) were sham-operated (SHAM) or ovariectomized (OVX) and maintained on a control diet for 6 wks until osteopenia was confirmed. Treatments were initiated consisting of a control diet (AIN-93M) supplemented with dried plum (0, 5, 15 or 25%; w/w) or a positive control group receiving PTH. At the end of 6 wks of treatment, whole body and femoral bone mineral density (BMD) were restored by the two higher doses of dried plum to the level of the SHAM group. Trabecular bone volume and cortical thickness were also improved with these two doses of dried plum. Dried plum suppressed the OVX-induced increase in bone turnover as indicated by systemic biomarkers of bone metabolism, N-terminal procollagen type 1 (P1NP) and deoxypyridinoline (DPD). Dynamic bone histomorphometric analysis of the tibial metaphysis revealed that dried plum restored the OVX-induced increase in cancellous bone formation rate (BFR) and mineralizing surface (MS/BS) to the SHAM group, but some doses of dried plum increased endocortical mineral apposition rate (MAR). As expected, PTH significantly increased endocortical MAR and BFR, periosteal BFR, and trabecular MAR and BFR beyond that of the OVX and maintained the accelerated rate of bone resorption associated with OVX. Dried plum up-regulated bone morphogenetic protein 4 (Bmp4) and insulin-like growth factor 1 (Igf1) while down-regulating nuclear factor T cell activator 1 (Nfatc1). These findings demonstrate that in the adult osteopenic OVX animal, the effects of dried plum differ from that of PTH in that dried plum primarily suppressed bone turnover with the exception of the indices of bone formation at the endocortical surface.     

Trabecular bone response to mechanical and parathyroid hormone stimulation: the role of mechanical microenvironment.

Bone response under combined mechanical and PTH stimuli is important in osteoporosis. A rat tail animal model with computer modeling was used to examine bone response to loading and PTH. PTH enhances and sustains increased bone formation rate, which directly correlates to mechanical microenvironment, suggesting beneficial effects of combined PTH treatment and exercise in preventing osteoporosis. INTRODUCTION: Using an in vivo rat tail vertebra model combined with a specimen-specific, high-resolution microcomputed tomography (microCT)-based finite element analysis (FEA) technique, trabecular bone response to combined dynamic compressive loading and parathyroid hormone (PTH) stimulation was characterized. MATERIALS AND METHODS: Two hundred twenty-four male Sprague-Dawley rats were randomly divided into seven treatment groups: (1) Control, (2) vehicle + 0N, (3) PTH + 0N, (4) vehicle + 50N, (5) PTH + 50N, (6) vehicle + 100N, and (7) PTH + 100N, with three treatment durations (1, 2, or 4 weeks). Rat PTH(1-34) was administered daily in the PTH-stimulated groups approximately 3 h before daily mechanical stimulation with 0, 50, or 100N dynamic compressive loading. microCT-based FEA was performed for each loaded vertebra after death. Bone histomorphometry was performed on trabecular bone with double fluorochrome labeling to assess bone formation. RESULTS: Daily mechanical loading or PTH administration significantly increased bone formation rate (BFR) compared with control or V + 0N with significant increases in both mineral apposition rate (MAR) and labeled bone surface (LS/BS). PTH, when combined with mechanical loading, enhanced BFR mainly through a significant increase in MAR after the first week and through a significant increase in LS/BS after 2 and 4 weeks. Synergistic effects in BFR were present when PTH was combined with mechanical loading, especially after 2 and 4 weeks, where the increase in BFR was sustained. However, when either PTH or mechanical loading was the only stimulus, the bone formation response diminished to the level of Control animals after 4 weeks. Furthermore, significant correlations were observed between the bone formation indices and trabecular bone tissue mechanical microenvironments at 1 and 2 weeks, with PTH administration enhancing and sustaining these correlations into 4 weeks. CONCLUSIONS: The synergistic effects of combined PTH and mechanical stimulation on trabecular bone formation rate suggest a potential benefit for combined PTH administration and exercise in the treatment of osteoporosis.     

Combination use of vitamin K<Subscript>2</Subscript> further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

 Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K₂ treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K₂ leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K₂ with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss. Received: January 30, 2002 / Accepted: November 6, 2002 RID="*" ID="*"  Present address: Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan Acknowledgments. This work was supported in part by a Special Grant for Medical Research from Ministry of Post and Telecommunications, Japan (to M.F.), a grant in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#13671115 to M.F.), and by a grant from the Research Society for Metabolic Bone Disease (to M.F.). We are grateful to Miss Sachiko Suzuki for technical assistance. Offprint requests to: M. Fukagawa     

Mechanical Load Increases in Bone Formation via a Sclerostin‐Independent Pathway

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long‐term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost?/? and wild type mice. Unloading was induced via 0.5U botulinum toxin (BTX) injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200 µe) at 1200 cycles/day, 5 days/week. Another cohort of mice receiving equivalent loads (?9.0 N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost?/? tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost?/? mice over their normal load controls. The increased BV was associated with significantly increased mid‐shaft periosteal mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate/bone surface (BFR/BS), and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost?/? mice than in wild type controls. Thus, long‐term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but it can augment the increase in bone formation with increased load. © 2014 American Society for Bone and Mineral Research.     

Effects of cyclic vs. daily treatment with human parathyroid hormone (1-34) on murine bone structure and cellular activity

Previously, we demonstrated that the human parathyroid hormone (1-34) fragment (hPTH(1-34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1-34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens (n=7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1-34) (40 microg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1-34) (40 microg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1-3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.     

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague–Dawley rats were administered either vehicle, Scl-Ab (3 or 50 mg/kg/week subcutaneously), or human PTH (1–34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50 mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume.Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.     

Effects of combined and separate intermittent administration of low-dose human parathyroid hormone fragment (1–34) and 17β-estradiol on bone histomorphometry in ovariectomized rats with established osteopenia

Summary To evaluate the potential use of a combination of parathyroid hormone (PTH) and estrogen as therapy for osteoporosis, we examined the effects of combined and separate administration of low-dose PTH and estradiol in ovariectomized rats with established osteopenia. Ovariectomized rats were untreated for 5 weeks after surgery and then injected s.c. with vehicle (Ovx+V), 1–34 hPTH (2.5 g/kg/day) (Ovx+P), 17-estradiol (50 g/kg/day) (Ovx+E), or a combination of these (Ovx+P+E), for a further 4 weeks. We found no differences in serum calcium, tubular reabsorption of phosphate, or 25OHD. 1,25(OH)₂D levels were significantly higher in Ovx+P and lower in Ovx+E, when compared with Ovx+V. Though there was no change in bone mineral density (BMD) in the diaphysis region of femurs, reduction of BMD in the distal region of the femurs in Ovx+V was reversed in Ovx+E and Ovx+P+E. Compared with Ovx+V, Ovx+P and Ovx+P+E had significantly higher cancellous bone volume (Cn-BV/TV) whereas Ovx+E showed a nonsignificant increase. When indices of bone turnover were examined, PTH alone showed a small but not significant improvement in bone formation rate (BFR). Increased osteoclast surface (OCS), as the result of ovariectomy, was inhibited in Ovx+E and Ovx+P+E. Estrogen alone (Ovx+E) severely inhibited BFR, but co-administration of PTH and estrogen (Ovx+P+E) showed an impressive reversal of such inhibition. The changes in BFR were mainly derived from changes in double-labeled surface (dLS), except a small increase in mineral apposition rate was also observed in Ovx+P+E. These results suggest that, after extensive cancellous bone loss in the rat tibia, low doses of PTH function anabolically, especially in situations where the bone formation rate is low. A combination of both estrogen and PTH may provide the best treatment for improving bone mass by decreasing resorption and maintaining a high bone formation rate.     

In vivo and in vitro evidence that the high osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is intrinsic to bone cells

Two inbred mouse strains, C3H/HeJ (C3H) and C57BL/6J (B6), displayed a profound difference in femoral peak bone density. We have previously shown that the difference could be attributed to a greater bone formation rate (BFR) that was due to a higher osteoblastic activity [measured by a mineral apposition rate (MAR)] in the C3H (high density) than B6 (low density) mice. The present study sought to determine (1) whether the BFR/MAR differences between the two mouse strains present in weight-loaded endochondral bones are also seen in less weight-loaded membranous bones and (2) whether the difference in osteoblastic activity was seen in vitro in the absence of systemic factors. To address the first objective, we performed histomorphometric measurements on the weakly loaded membranous bones (i.e., parietal bones of the calvaria) to determine if there were similar differences in MAR and BFR of membranous bones as those of highly loaded, endochondral bones. The parietal bones of adult C3H mice showed similar increases in MAR and BFR as the endochondral bones, compared to B6 mice of same age, suggesting that the differences in the MAR and BFR in the two mouse strains are probably not related to differences in mechanical strain. These findings also suggest that the gene(s) responsible for the difference in MAR between strains may not be a mechanical response gene. With respect to the second objective, we isolated osteoblasts from the parietal bones and determined their differentiation status (i.e., ALP-specific activity) and bone-forming ability (i.e., mineralized nodule formation) in vitro. Consistent with the premise that C3H osteoblasts have an intrinsic, higher differentiation status and bone-forming ability than B6 osteoblasts, osteoblasts isolated from C3H mice as compared with those from B6 mice had a significantly greater ALP-specific activity and a greater ability to form mineralized nodules in vitro in the absence of systemic factors. Because differences in ALP activity, bone-forming ability, cortical bone width, and osteoblastic activity were detected at birth, the different MAR/BFR phenotypes develop at very early life and even perhaps during embryogenesis. In conclusion, we have for the first time provided evidence that the genetic differences responsible for the observed MAR/BFR phenotype in the C3H-B6 strains are intrinsic to osteoblasts and might not depend on responses to mechanical loading and/or alterations in systemic factors.     

Loss of the anabolic effect of parathyroid hormone on bone after discontinuation of hormone in rats

We have previously reported that low doses of hPTH 1–34 given daily to rats exert an anabolic effect on bone. The objective of this study was to determine if the anabolic effect of PTH was dependent upon continued daily administration of the hormone. Young, male rats were given daily subcutaneous injections of either vehicle or 8 μg/100g bw hPTH 1–34 for 12, 16, 20, or 24 days. Additional groups were treated with 8 μg/100g bw hPTH for 12 days followed by vehicle for the next 4, 8, or 12 days, or 8 μg/100g bw hPTH 1–34 for 16 days followed by 4 days of vehicle. We measured calcium (Ca), dry weight (DW), and hydroxyproline (Hyp) of the distal femur, percent of osteoblast (Ob.S/BS) and osteoclast (Oc.S/BS) surface, mineral apposition rate (MAR), double label surface (DLS/BS), and bone formation rate (BFR) in the metaphysis of the proximal tibia, and serum calcium and phosphate. Trabecular and cortical bone Ca and DW and the histologic measures of bone formation increased in all PTH-treated rats. Serum calcium and phosphate were comparable in all rats. The PTH-stimulated bone mass was lost 12 days after discontinuation of PTH. Discontinuation of PTH administration for 4, 8, or 12 days, respectively, resulted in a 72%, 68%, or 50% decrease in Ob.S/BS from the 2- to 3-fold increase associated with PTH treatment (p < .05). Oc.S/BS increased compared to controls after 4 days of PTH withdrawal (NS), but was comparable to controls 8 days after withdrawal of PTH. The loss of new bone mass after discontinuation of hormone was due to an inhibition of PTH-stimulated bone formation and an initial transient phase of increased resorption. Thus, the anabolic effect of PTH was dependent upon daily administration of the hormone in our model.     

Regulation of bone volume is different in the metaphyses of the femur and vertebra of C3H/HeJ and C57BL/6J mice

The C3H/HeJ (C3H) mice exhibited a greater bone formation rate (BFR) and a greater mineral apposition rate (MAR) in the cortical bone of the midshafts of the femur and tibia than did C57BL/6J (B6) mice. This study sought to determine if these strain-related differences would also be observed in cancellous bone. Metaphyses of the femur and lumbar vertebra (L5-6) from C3H and B6 mice, 6 and 12 weeks of age, were analyzed by histomorphometry. Similar to cortical bone, the bone volume in the femoral metaphysis of C3H mice was greater (by 54% and 65%, respectively) than that of B6 mice at both 6 and 12 weeks of age. Higher BFR and mineral apposition rate (MAR) contributed to the higher bone volume in the C3H mice compared with the B6 mice. In contrast, bone volume (by 59% and 13%, respectively, p < 0.001) and trabecular number (by 55% and 35%, respectively, p < 0.001) in the vertebrae were lower in the C3H mice than in B6 mice at 6 and 12 weeks of age. At 6 weeks of age, MAR was higher (by 43%, p = 0.004) in C3H mice, but because of a low trabecular number, the BFR (by 37%, p = 0.026) and tetracycline-labeled bone surface (by 52%, p < 0.001) per tissue were lower in the vertebrae of C3H mice than B6 mice. The low bone volume in vertebrae of C3H mice was probably not due to a higher bone resorption, because the osteoclast number (by 55%, p < 0.001) and eroded surface (by 61%, p <0.001) per tissue area in the C3H mice were also lower in B6 mice. At 12 weeks, the trabecular thickness had increased (by 36%, p < 0.001) in the C3H mice and the difference in bone volume between strains was less than that at 6 weeks. These contrasting and apparently opposing strain-related differences in trabecular bone parameters between femur and vertebra in these two mouse strains suggest that the genetic regulation of bone volume in the metaphyses of different skeletal sites is different between C3H and B6 mice.     

New worldwide trends in presentation of renal osteodystrophy and its relationship to parathyroid hormone levels

AIMS: Abnormal mineral metabolism in chronic renal disease is associated with bone disease and extraskeletal calcifications. High turnover, hyperparathyroid bone disease, the most common form of renal osteodystrophy, has been the target for aggressive therapy. More recently, an increasing occurrence of low turnover bone disease has been reported. The present study was undertaken to evaluate the current prevalence of different forms of bone disease in a large population on chronic hemodialysis and its relationship to parathyroid hormone (PTH) levels. METHODS: Ninety-six chronic hemodialysis patients underwent double tetracycline-labeled bone biopsy. Serum PTH levels were obtained in 52 patients at the time of biopsy. Bone formation rate (BFR/BS) was plotted vs. PTH levels in all patients and in subgroups with PTH ranges between 0-150, 150-500 and 500 - 1,200 pg/ml. RESULTS: The histomorphometric data showed that 40% of all patients were affected by osteitis fibrosa cystica (OFC). In the remaining 60%, various forms of low-turnover bone disease were observed. There was no correlation between PTH and BFR/BS in all patients (r = 0.28) and in subgroups whose PTH levels ranged between 150 - 500 and 500 - 1,200 pg/ml (r = 0.027, r = 0.21), respectively. A close correlation between PTH and BFR/BS (r = 0.84, p < 0.05) was found only in the subgroup with a PTH level ranging low-turnover bone disease. The predictive between 0 - 150 pg/ml. CONCLUSIONS: The histomorphometric findings present a wide spectrum of renal osteodystrophy with a shift towardsvalue of PTH is limited as high-turnover osteodystrophy may present with low PTH levels and that with low turnover may occur with high PTH levels. In the latter parathyroidectomy should be avoided. We share the view that bone biopsy remains the "gold standard" diagnostic tool for renal osteodystrophy.     

Biochemical bone turnover markers are useful tools to assess changes in bone metabolism in marmosets

This study was designed to investigate whether biochemical markers of bone resorption and formation could be determined in the serum and urine of marmosets (Callithrix jacchus), using standard laboratory chemistry methods and commercially available human kits. Consequently, the findings from this study will indicate whether the techniques and kits could serve as appropriate tools for assessing changes in bone turnover in this species. Two groups of animals (n = 12/group), consisting of a comparable number of young and old male and female marmosets, were given either isotonic saline or a single dose of the bisphosphonate ibandronate (0.1mg/kg) s.c. in order to suppress bone turnover. Blood and urine were collected at baseline and 5 days after administration. Samples were analyzed for urinary (u) and serum (s) markers of bone formation (serum osteocalcin [sOC], serum N-terminal crosslinks of human pro-collagen type I [sP1NP]) and bone resorption (urinary pyridinoline [uPYD], urinary deoxypyridinoline [uDPD], serum C-terminal crosslinks of human collagen type I (C-telopeptide) [sCTX]), intact serum parathyroid hormone (iPTH) and urinary calcium and creatinine. Levels of all the markers of bone resorption and formation decreased during the study period. As expected, the bone formation markers decreased slightly less relative to the resorption markers. The most sensitive markers were sCTX (–33%; P 0.001) for bone resorption, and sP1NP (–3%; P 0.05) for bone formation. Serum PTH levels increased by 8% (P 0.05), demonstrating a physiological reaction to prevent changes in serum calcium. Although not all variables reached statistical significance within the tested interval, the applied methods and kits were considered suitable for evaluating bone turnover changes in marmosets. Thus, these methods and kits can be utilized not only during the course of pharmacological investigations but also as additional tools to assess the overall bone health of this species.     

Effects of tower climbing exercise on bone mass, strength, and turnover in growing rats.

To determine the effects of tower climbing exercise on mass, strength, and local turnover of bone, 50 Sprague-Dawley rats, 10 weeks of age, were assigned to five groups: a baseline control and two groups of sedentary and exercise rats. Rats voluntarily climbed the 200-cm tower to drink water from the bottle set at the top of it. In 4 weeks, the trabecular bone formation rate (BFR/bone surface [BS]), bone volume (BV/TV), and trabecular thickness (Tb.Th) of both the lumbar vertebra and tibia and the bone mineral density (BMD) of the tibia increased, while the osteoclast surface (Oc.S) decreased. The parameter values in the midfemur, such as the total cross-sectional area, the moment of inertia, the periosteal mineralizing surface (MS/BS), mineral apposition rate (MAR), BFR/BS, and bending load increased, while the endosteal MAR decreased. In 8 weeks, the increases in the bone mineral content (BMC), BMD of the femur and tibia, and the bending load values of the femur were significant, but the climbing exercise did not increase BMC, BMD, or the compression load of the lumbar vertebra. Although the periosteal MS/BS, MAR, and BFR/BS increased, the endosteal MS/BS, MAR, and BFR/BS decreased. These results show that climbing exercise has a beneficial effect on the femoral cortex and tibia trabecular, rather than the vertebral trabecular. In the midfemur, effects on bone formation are site specific, supporting accelerated cortical drift by mechanical stimulation.     

Differential maintenance of cortical and cancellous bone strength following discontinuation of bone‐active agents

Osteoporotic patients treated with antiresorptive or anabolic agents experience an increase in bone mass and a reduction in incident fractures. However, the effects of these medications on bone quality and strength after a prolonged discontinuation of treatment are not known. We evaluated these effects in an osteoporotic rat model. Six‐month‐old ovariectomized (OVX) rats were treated with placebo, alendronate (ALN, 2 µg/kg), parathyroid hormone [PTH(1–34); 20 µg/kg], or raloxifene (RAL, 2 mg/kg) three times a week for 4 months and withdrawn from the treatments for 8 months. Treatment with ALN, PTH, and RAL increased the vertebral trabecular bone volume (BV/TV) by 47%, 53%, and 31%, with corresponding increases in vertebral compression load by 27%, 51%, and 31%, respectively (p < .001). The resulting bone strength was similar to that of the sham‐OVX control group with ALN and RAL and higher (p < .001) with PTH treatment. After 4 months of withdrawal, bone turnover (BFR/BS) remained suppressed in the ALN group versus the OVX controls (p < .001). The vertebral strength was higher than in the OVX group only in ALN‐treated group (p < .05), whereas only the PTH‐treated animals showed a higher maximum load in tibial bending versus the OVX controls (p < .05). The vertebral BV/TV returned to the OVX group level in both the PTH and RAL groups 4 months after withdrawal but remained 25% higher than the OVX controls up to 8 months after withdrawal of ALN (p < .05). Interestingly, cortical bone mineral density increased only with PTH treatment (p < .05) but was not different among the experimental groups after withdrawal. At 8 months after treatment withdrawal, none of the treatment groups was different from the OVX control group for cortical or cancellous bone strength. In summary, both ALN and PTH maintained bone strength (maximum load) 4 months after discontinuation of treatment despite changes in bone mass and bone turnover; however, PTH maintained cortical bone strength, whereas ALN maintained cancellous bone strength. Additional studies on the long‐term effects on bone strength after discontinuation and with combination of osteoporosis medications are needed to improve our treatment of osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Human parathyroid hormone 1-34 reverses bone loss in ovariectomized mice.

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss-Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1-34) [hPTH(1-34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (microCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5-week and 9-week post-OVX, non-PTH-treated animals. PTH induced a approximately 35% recovery of this loss and a approximately 40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein-labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2- and 3-fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss-Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.     

Comparative effects of teriparatide and strontium ranelate in the periosteum of iliac crest biopsies in postmenopausal women with osteoporosis

The periosteum contains osteogenic cells that regulate the outer shape of bone and contribute to determine its cortical thickness, size and position. We assessed the effects of subcutaneous injections of teriparatide (TPTD, 20μg/day) or oral strontium ranelate (SrR, 2g/day) in postmenopausal women with osteoporosis on new bone formation activity at the periosteal and endosteal bone surfaces using dynamic histomorphometric measurements. Evaluable tetracycline-labeled transiliac crest bone biopsies were analyzed from 27 patients in the TPTD group, and 22 in the SrR group after six months of treatment. Measurements were conducted on the thicker and thinner cortices separately, and comparisons between the thicker, thinner and combined cortices were carried out. At the combined periosteal cortex, the mineralization surface as a percent of bone surface (MS/BS%) was greater for TPTD (mean±SE: 8.08±1.22%) than SrR (3.22±1.05%) (p<0.005). The difference in mineral apposition rate (MAR) between TPTD (0.35±0.06μm/day) and SrR (0.14±0.06μm/day) was also significant (p<0.05), while that of bone formation rate per bone surface (BFR/BS) between TPTD (0.014±0.004 mm(3)/mm(2)/year) and SrR (0.004±0.003 mm(3)/mm(2)/year) was not (p=0.057). Statistically significant differences between the two treatments were also observed for MS/BS%, BFR/BS, MAR and the double-labeled perimeter in the periosteum of the thicker, but not thinner, iliac crest cortices. The comparison between the thicker and thinner cortices of both periosteal and endosteal surfaces showed statistically significant differences for MAR and the double-labeled perimeter for TPTD treated women. There were no statistically significant differences in any bone formation dynamic measurements between the two cortices in the SrR group. In conclusion, most of the bone formation and mineralization variables were significantly higher for TPTD- than SrR-treated women at both the periosteal and endosteal combined cortices. The response to TPTD for dynamic bone formation measurements in the periosteal surface was greater for the thicker than thinner cortex, but this difference was not significant in SrR treated patients. This may reflect a greater ability of TPTD to enhance responsiveness of bone to the mechanical loading environment. These effects on bone formation may underlie the improvement in bone quality in patients with osteoporosis treated with TPTD.     

Changes in 3-dimensional bone structure indices in hypoparathyroid patients treated with PTH(1-84): a randomized controlled study

Hypoparathyroidism (hypoPT) is characterized by a state of low bone turnover and high bone mineral density (BMD) despite conventional treatment with calcium supplements and active vitamin D analogues. To assess effects of PTH substitution therapy on 3‐dimensional bone structure, we randomized 62 patients with hypoPT into 24 weeks of treatment with either PTH(1‐84) 100 µg/day subcutaneously or similar placebo as an add‐on therapy. Micro‐computed tomography was performed on 44 iliac crest bone biopsies (23 on PTH treatment) obtained after 24 weeks of treatment. Compared with placebo, PTH caused a 27% lower trabecular thickness (p < 0.01) and 4% lower trabecular bone tissue density (p < 0.01), whereas connectivity density was 34% higher (p < 0.05). Trabecular tunneling was evident in 11 (48%) of the biopsies from the PTH group. Patients with tunneling had significantly higher levels of biochemical markers of bone resorption and formation. At cortical bone, number of Haversian canals per area was 139% higher (p = 0.01) in the PTH group, causing a tendency toward an increased cortical porosity (p = 0.09). At different subregions of the hip, areal BMD (aBMD) and volumetric BMD (vBMD), as assessed by dual‐energy X‐ray absorptiometry (DXA) and quantitative computed tomography (QCT), decreased significantly by 1% to 4% in the PTH group. However, at the lumbar spine, aBMD decreased by 1.8% (p < 0.05), whereas vBMD increased by 12.8% (p = 0.02) in the PTH compared with the placebo group. © 2012 American Society for Bone and Mineral Research.     

Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow

In order to examine the mechanism of the anabolic effect of parathyroid hormone (PTH) on bone formation, human PTH(1-34) [hPTH(1-34)] (30 μg/kg) was injected subcutaneously to 9-week-old rats 5 times a week for 1 or 3 weeks. Trabecular bone volume (BV/TV) in the tibial metaphysis was not significantly different between the PTH- and vehicletreated groups, but the parameters related to bone formation, including osteoid surface (OS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were significantly increased as early as 1 week after PTH treatment. And the parameters related to bone resorption including eroded surface (ES/BS) and osteoclast number (N.Oc/BS) were also significantly increased as early as 1 week after PTH treatment. Treatment with PTH for 1 week induced no significant increase in bone mineral density at the femoral metaphysis, whereas the same treatment for 3 weeks induced a significant increase. When bone marrow cells isolated from femora and tibiae of either PTH- or vehicle-treated rats were cultured at a high density (2 × 10⁷ cells/one well of 24-multiwell plate), cellular alkaline phosphatase (ALP) activity was significantly increased in the cells isolated from PTH-treated rats compared with vehicletreated rats. When bone marrow cells were cultured at a low density (4 × 10⁶ cells/a one well of 6-multiwell plate) to generate colonies (colony forming unit-fibroblastic, CFU-F), PTH induced apparent increases in both the total number of CFU-F and the number of ALP-positive CFU-F. The ratio of the latter to the former was significantly higher in the PTH-treated group than in the vehicle-treated group. These findings suggest that the anabolic effect of PTH is, at least in part, due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow.     

Cancellous bone of aged rats maintains its capacity to respond vigorously to the anabolic effects of prostaglandin E2 by modeling-dependent bone gain

The present study examined the early effects of prostaglandin (PG)E2 on proximal tibial metaphyses of 20-month-old Wistar male rats. PGE, was given to intact rats for 10 and 30 days at 3mg/kg/day. After multiple in vivo fluorochrome labeling, undecalcified longitudinal sections were subjected to analysis of bone histomorphometry and classification of the contour of the cement line in bone formation units. The latter was used to classify bone formation units into modeling, remodeling and uncertain units. After 10 days of treatment, there was a 2% increase in woven bone formation with the appearance of osteoprogenitor cells and increases in the number of osteoblasts (649%) and osteoid (375%) surfaces. Remodeling and modeling units increased by 56% and 429%. respectively. After 30 days of treatment, there was an increase of 212% of total trabecular bone mass, 60% of which was woven bone. In addition, there were increases in labeling surface (147%), mineral apposition rate (760%), bone formation rates tissue area (BFR/T.Ar, 1920%; BFR/B.Pm, 343%), and bone turnover (BFR/B.Ar, 426%). Osteoblasts and osteoid production at 30 days were 29% and 58% less than at 10 days post-treatment. Modeling and remodeling activity did not differ from that seen at 10 days. In addition, PGE2 treatment tended to stimulate the closing of growth plates and decrease the fatty marrow area. We conclude that the aged skeleton was able to respond vigorously to PGE2 treatment. Massive osteoprogenitors cells, and osteoid and osteoblast formations were observed within 10 days. and dramatic woven and lamellar bone formation was seen at 30 days post-treatment. The anabolic effects were driven mainly by modeling.