Thomas J. Wronski

Temporal relationship between bone loss and increased bone turnover in ovariectomized rats

SummaryTo characterize osteopenic changes in ovariectomized (OVX) rats as a function of time, female Sprague Dawley rats (240 g body weight, 90 days old) were subjected to bilateral ovariectomy or sham surgery and killed at various times from 14–180 days postovariectomy. The proximal tibial metaphysis was processed undecalcified for quantitative bone histomorphometry. Osteopenia and increased indices of bone resorption and formation were detected in OVX rats as early as 14 days. Longitudinal bone growth was also significantly increased by ovariectomy at 14 days, but returned to control levels at all later times. In OVX rats, osteopenia became progressively more pronounced with time up to 100 days postovariectomy, after which trabecular bone volume appeared to stabilize at the markedly reduced level of 5%. Changes in osteoclast surface, osteoblast surface, and fluoro-chrome-based indices of bone formation in OVX rats followed a similar time course. The maximal increase in these parameters occurred during the first several months postovariectomy followed by a gradual decline toward control levels. Our results indicate that the initial rapid phase of bone loss in OVX rats is coincident with the maximal increase in bone turnover. At later times postovariectomy, bone loss and bone turnover both subside. These findings emphasize the close temporal association between the development of osteopenia and increased bone turnover in OVX rats.

Long-term effects of ovariectomy and aging on the rat skeleton

SummaryThe long-term skeletal effects of ovariectomy and aging were studied in female Sprague-Dawley rats sacrificed at 270, 370, and 540 days after bilateral ovariectomy (OVX) or sham surgery at 90 days of age. The proximal tibia was processed undecalcified for quantitative bone histomorphometry. For continuity, data from these late time points were combined with previously published data from earlier time points (0–180 days). A biphasic pattern of cancellous bone loss was detected in the proximal tibial metaphysis of OVX rats. An initial, rapid phase of bone loss out to 100 days was followed by an intermediate period of relative stabilization of cancellous bone volume at the markedly osteopenic level of 5–7%. After 270 days, a slow phase of bone loss occurred during which cancellous bone volume declined to 1–2%. Both the initial, rapid phase and the late, slow phase of bone loss in OVX rats were associated with increased bone turnover. In control rats, cancellous bone volume remained constant at 25–30% out to 270 days (12 months of age), then decreased to ∼10% by 540 days (21 months of age). This age-related bone loss was also associated with increased bone turnover. It is interesting to note that the proximal tibial growth plates were closed in approximately a quarter of the control rats by 15–21 months of age. Our data indicate that a slow rate of bone loss and increased bone turnover persist in OVX rats during the later stages of estrogen deficiency. Therefore, the development of osteopenia is coincident with increased bone turnover in OVX rats as well as in aged, control rats.

Skeletal alterations in ovariectomized rats

SummaryFemale Sprague Dawley rats were subjected to either bilateral ovariectomy or sham surgery. Tetracycline derivatives were administered to each rat on two separate occasions to label sites of bone formation. All rats were sacrificed at 5 weeks postovariectomy and their proximal tibiae were processed undecalcified for quantitative bone histomorphometry. A twofold decrease in trabecular bone volume was noted in the proximal tibial metaphysis of ovariectomized rats. This bone loss was associated with elevated histomorphometric indices of bone resorption and formation. Ovariectomy increased osteoclast surface and numbers as well as osteoblast surface and numbers. Elevations in calcification rate and fractional trabecular bone surface with double tetracycline labels also suggest that bone formation was stimulated in ovariectomized rats. In addition, ovariectomized rats exhibited a greater rate of longitudinal bone growth relative to sham-operated control rats. These histomorphometric data indicate that ovariectomy induces marked bone loss and accelerated skeletal metabolism in rats.

Histologic evidence for osteopenia and increased bone turnover in ovariectomized rats

Bone histomorphometry was performed in the proximal tibia and lumbar vertebra of female Sprague-Dawley rats subjected to either bilateral ovariectomy or sham surgery. Ovariectomized rats killed 10 weeks after surgery were characterized by osteopenia and histomorphometric indices of increased bone turnover at both skeletal sites. Osteoclast surface and number as well as osteoblast surface and number were significantly increased in ovariectomized rats. Tetracycline-based data were consistent with accelerated bone formation in response to ovariectomy. Active formation surface (double-labeled surface), calcification rate, and bone formation rate (tissue level, total surface referent) were all significantly elevated in the proximal tibia of ovariectomized rats. Ovariectomy also increased active formation surface and bone formation rate in the lumbar vertebra, but the vertebral calcification rate was nearly identical in control and ovariectomized rats. Osteopenia and dynamic tetracycline-based estimates of increased bone turnover in ovariectomized animals were more pronounced in the proximal tibia relative to the lumbar vertebra. Longitudinal bone growth in the proximal tibia and mineralization lag time at the tibial cortical endosteal surface were not affected by ovariectomy. These histomorphometric data indicate that ovariectomy induces osteopenia and accelerated skeletal metabolism in rats.

Time course of vertebral osteopenia in ovariectomized rats

Osteopenic changes in cancellous bone tissue of the first lumbar vertebral body were characterized in ovariectomized (OVX) rats as a function of time. Female Sprague Dawley rats (240 g body weight, 90 days old) were subjected to bilateral ovariectomy or sham surgery and sacrificed at various times from 0-540 days postovariectomy. The first lumbar vertebra was processed undecalcified for quantitative bone histomorphometry. Cancellous bone volume remained relatively constant in control rats at approximately 40% throughout the duration of the study. In contrast, cancellous bone volume was moderately decreased to 30-35% in OVX rats out to 180 days postovariectomy. Vertebral osteopenia became more pronounced in OVX rats at later times as cancellous bone volume declined to approximately 20% between 180 and 270 days and remained at that osteopenic level for the duration of the study. Osteoblast and osteoclast surface were highly elevated in OVX rats at 35 days, declined gradually toward control levels out to 180 days, then increased markedly at 270 days. Mineralizing surface and bone formation rate (tissue level, total surface referent) were maximally increased in OVX rats at 35-70 days before declining toward control levels at later times. However, these parameters remained significantly increased in OVX rats relative to control rats between 270 and 540 days. Mineral apposition rate was nearly identical in control and OVX rats at all time points and declined linearly with age in both groups. Our results indicate that osteopenia and increased bone turnover occur in the lumbar vertebral bodies of OVX rats, as had been previously observed in the proximal tibial metaphyses of these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

FGF23 neutralization improves chronic kidney disease–associated hyperparathyroidism yet increases mortality

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is associated with secondary hyperparathyroidism (HPT) and serum elevations in the phosphaturic hormone FGF23, which may be maladaptive and lead to increased morbidity and mortality. To determine the role of FGF23 in the pathogenesis of CKD-MBD and development of secondary HPT, we developed a monoclonal FGF23 antibody to evaluate the impact of chronic FGF23 neutralization on CKD-MBD, secondary HPT, and associated comorbidities in a rat model of CKD-MBD. CKD-MBD rats fed a high-phosphate diet were treated with low or high doses of FGF23-Ab or an isotype control antibody. Neutralization of FGF23 led to sustained reductions in secondary HPT, including decreased parathyroid hormone, increased vitamin D, increased serum calcium, and normalization of bone markers such as cancellous bone volume, trabecular number, osteoblast surface, osteoid surface, and bone-formation rate. In addition, we observed dose-dependent increases in serum phosphate and aortic calcification associated with increased risk of mortality in CKD-MBD rats treated with FGF23-Ab. Thus, mineral disturbances caused by neutralization of FGF23 limited the efficacy of FGF23-Ab and likely contributed to the increased mortality observed in this CKD-MBD rat model.

Histomorphometric study of alveolar bone turnover in orthodontic tooth movement

Selected histomorphometric parameters were measured in alveolar bone adjacent to rat molars treated with a 40-gram tipping force designed to tip the molar mesially and with a sham procedure. Undecalcified parasagittal sections of teeth and surrounding tissues were prepared for static and dynamic histomorphometry at 1, 3, 5, 7, 10, and 14 days. Initial tooth displacement was assessed by measuring differences between groups in the widths of the day-1 periodontal ligaments (PDL) at various vertical locations and correlating these using linear regression analysis. All parameters were measured in the alveolar bone adjacent to four quadrants around the teeth (mesial-distal; occlusalapical). Means and standard errors for each parameter in each group were calculated and compared for time- and group-specific differences using ANOVA and pairwise comparisons with Scheffes multiple comparison tests. In sham-treated rats, bone resorption predominated on the distal alveolar surface, but significant surface-related differences between mesial and distal surfaces in bone formation were not demonstrated. Time-specific effects in bone resorption were not evident on either surface in the shams. These findings suggested that molar distal drift in the sham rat is facilitated by resorption in the distal alveolar bone. PDL width changes in orthodontically-treated rats were greatest in the mesial occlusal half of the root and decayed linearly toward the apex indicating that the greater initial tooth displacement occurred in the occlusal half of the root surface. Histomorphometric parameters of alveolar bone turnover were also seen to be greater in these locations, suggesting that these processes were sensitive to the increased tooth displacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabolic effects of parathyroid hormone on cortical bone in ovariectomized rats

The purpose of this study was to determine whether PTH has anabolic effects on cortical bone in ovariectomized (OVX) rats similar to those previously observed in cancellous bone. Groups of OVX rats were untreated for the first 4 weeks postovariectomy followed by treatment for 5-, 10-, or 15-week periods with vehicle, estrogen, the bisphosphonate rise-dronate (NE-58095), PTH, PTH+estrogen or PTH+NE-58095. A group of sham-operated control rats was treated with vehicle alone. Cross sections of the tibial diaphysis immediately proximal to the tibiofibular junction were subjected to quantitative bone histomorphometry. In comparison to vehicle-treated control rats, the following structural changes were detected in cortical bone of OVX rats after 15 weeks of vehicle treatment: increased cortical bone tissue area, increased cortical bone area, and a trend for increased bone marrow area. Indices of periosteal bone formation were increased in vehicle-treated OVX rats at 5 weeks, but declined to control levels at the later time points. In addition, vehicle-treated OVX rats exhibited a nonsignificant trend for increased endocortical bone formation relative to vehicle-treated control rats throughout most of the study. Estrogen, but not NE-58095, prevented most of the changes in cortical bone structure associated with ovariectomy. In comparison to vehicle treatment of OVX rats, estrogen decreased both periosteal and endocortical bone formation whereas NE-58095 decreased endocortical, but not periosteal, bone formation. Treatment of OVX rats with PTH alone increased cortical bone area and width as well as decreased bone marrow area compared to vehicle-treated OVX rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone and hormonal changes induced by skeletal unloading in the mature male rat

To determine whether the rat hindlimb elevation model can be used to study the effects of spaceflight and loss of gravitational loading on bone in the adult animal, and to examine the effects of age on bone responsiveness to mechanical loading, we studied 6-mo-old rats subjected to hindlimb elevation for up to 5 wk. Loss of weight bearing in the adult induced a mild hypercalcemia, diminished serum 1,25-dihydroxyvitamin D, decreased vertebral bone mass, and blunted the otherwise normal increase in femoral mass associated with bone maturation. Unloading decreased osteoblast numbers and reduced periosteal and cancellous bone formation but had no effect on bone resorption. Mineralizing surface, mineral apposition rate, and bone formation rate decreased during unloading. Our results demonstrate the utility of the adult rat hindlimb elevation model as a means of simulating the loss of gravitational loading on the skeleton, and they show that the effects of nonweight bearing are prolonged and have a greater relative effect on bone formation in the adult than in the young growing animal.

Aging Increases Stromal/Osteoblastic Cell-Induced Osteoclastogenesis and Alters the Osteoclast Precursor Pool in the Mouse†

Stromal/osteoblastic cell expression of RANKL and M‐CSF regulates osteoclastogenesis. We show that aging is accompanied by increased RANKL and M‐CSF expression, increased stromal/osteoblastic cell‐induced osteoclastogenesis, and expansion of the osteoclast precursor pool. These changes correlate with age‐related alterations in the relationship between osteoblasts and osteoclasts in cancellous bone.