Abstract
We report the consequences of prolonged treatment with recombinant human parathyroid hormone (1-34) (PTH) in male and ovariectomized female rats with mature skeletons. Intact male and osteopenic, ovariectomized, female F-344 rats were evaluated after 1 year of treatment with 0, 8, or 40 μg/kg/day s.c. PTH. Males and females were about 6 months of age at study initiation; females were ovariectomized (Ovx) for 5 weeks before initiation of PTH treatment. PTH did not affect the survival of either intact males or ovariectomized females. Qualitative histopathology showed expected changes associated with aging in kidneys and proximal tibiae, with no treatment-related anomalies after 1 year of PTH administration. PTH slightly increased the femoral length of ovariectomized females but not that of males. No significant differences in femoral length were observed between sham and Ovx controls. Proximal femora of the males and ovariectomized females given the high dose of 40 μg/kg showed 211 and 186% greater trabecular bone area, 118 and 94% greater cortical thickness, 170 and 189% greater trabecular number, and 321 and 404% greater connectivity (node-to-node struts) compared with respective vehicle controls. Increased trabecular and endocortical surface apposition coincided with a 78 and 70% loss of marrow space for males and females treated with PTH, respectively. Biomechanical strength (ultimate load) of the femoral neck increased by 73 and 76%, respectively, in males and ovariectomized females. Cortical bone analyses of the femoral midshaft showed 105 and 72% increases in bone mineral content, 67 and 55% increases in bone mineral density, and 22 and 10% increases in cross-sectional area for males and ovariectomized females, respectively, with altered shape of femora. Biomechanical analyses of the midshaft showed substantial increases in strength and stiffness but a reduction in ultimate strain, which was likely due to the altered geometry of the midshaft for PTH groups. Aging effects on strength of vertebra and femoral midshaft were reversed by PTH treatment. In summary, the 1-year treatment duration, which represents about 50% of lifetime, did not affect survival and was not associated with any treatment-related anomalies in the kidney or skeleton. PTH reversed the aging process in bones but not kidneys and substantially increased bone mass and strength to well beyond normally attained levels. However, compared with short-term studies reported previously, there seemed to be no advantages to extending PTH treatment to 12 months in rat bones.
Once daily, subcutaneous administration of human PTH (1-34) stimulates new bone formation in rats, monkeys, and humans (Gasser and Jerome, 1992;Dempster et al., 1993; Cosman and Lindsay, 1998; Hock, 2001; Neer et al., 2001; Rosen and Bilezikian, 2001). In rats, a single administration of PTH was shown to activate bone cells within minutes by regulation of gene expression, and to increase bone-forming surfaces within 6 to 24 h (Hock et al., 1994; Onyia et al., 1995). PTH skeletal efficacy did not seem to be dependent on rat age because 3-, 6-, and 15-month-old ovariectomized females and aged males responded to PTH by increasing bone mass, architecture, and quality in the spine, femoral neck, and long bones after treatment for 12 days to 9 months (Gasser and Jerome, 1992; Dempster et al., 1993; Sato et al., 1997;Kishi et al., 1998; Kneissel et al., 2001).
Despite the many PTH studies in rat models, relatively few studies have evaluated the long-term effects of PTH. For example, only five studies have evaluated the long-term effects of PTH between 6 to 9 months of treatment (Mosekilde et al., 1994a; Sato et al., 1997; Kishi et al., 1998; Stewart et al., 2000; Kneissel et al., 2001). In addition, previous studies have generally lacked sufficient statistical power with which to evaluate the pathology of PTH on target tissues, with the possible exception of one study (Sato et al., 1997).
Rat studies have been remarkably consistent in showing only beneficial effects of PTH on the architecture, quality, and state of mineralization of the skeleton, provided that the doses did not induce sustained hypercalcemia (Dempster et al., 1993; Gunness and Hock, 1995;Hock and Wood, 1995). Nevertheless, one rat study (Neer et al., 2001;Vahle et al., 2002) detected untoward skeletal effects of PTH after near-lifetime treatment. Therefore, one purpose of our study was to ascertain the long-term skeletal effects of PTH in intact male and ovariectomized female Fischer-344 rats after 1 year of treatment. This study was designed to have sufficient statistical power to thoroughly evaluate the biomechanical effects of PTH on the axial and appendicular skeleton. Group sizes of 20 to 30 also permitted analysis of PTH effects on survival and pathology in PTH target tissues, including bone and kidney, because we speculated that prolonged PTH treatment could affect aging processes in target tissues. A preliminary version of these data were presented at the American Society for Bone and Mineral Research (ASBMR) meetings in 2001.
Materials and Methods
Animals and Experimental Protocol.
Virus antibody-free, 5-month-old Fischer-344 intact male and ovariectomized and sham-ovariectomized female rats (Taconic, Germantown, NY) were maintained on a 12-h-light/dark cycle at 22°C with ad libitum access to food (TD 89222 with 0.5% calcium and 0.4% phosphorus; Teklad, Madison, WI) and water. Rats were housed for 5 weeks before treatment to permit bone loss (osteopenia) in ovariectomized females. At about 6 months of age, intact male and ovariectomized rats (20/group) were subcutaneously administered recombinant human PTH (1-34) (LY333334, Lilly Research Laboratories) at 8 or 40 μg/kg/day (Male8, Male40, PTH8, and PTH40) for 12 months. One group of intact male (intact), sham-ovariectomized (sham), or ovariectomized (Ovx) rats (30/group) was treated with vehicle and served as age-matched controls. Ten each of male (baseline) and sham-ovariectomy (ShamB) rats obtained separately were killed at 6 months of age as baseline controls. PTH was prepared in a vehicle of sterile 0.15 M NaCl, 0.001 N HCl, and 2% heat-inactivated rat serum. Doses were administered in a relative volume of 0.5 ml/kg between 8:00 and 10:00 AM each day. Body weight was recorded every other week, and the injection volume was adjusted accordingly. Protocols were approved by the institutional animal care and use committee at Lilly Research Laboratories to ensure compliance with National Institutes of Health guidelines.
At necropsy, rats were assigned a random number identification code to ensure blinding during data acquisition. They were then subjected to cardiac puncture under isoflurane anesthesia and killed by CO2 inhalation. Sera were collected for biochemical analyses. Tibiae, femora, and vertebrae L1 to L6 were removed, cleaned of soft tissue, and preserved in 50% ethanol/saline at 4°C for later analyses.
Serum Biochemistry.
Blood was collected from the orbital plexus of animals using isoflurane anesthesia to measure serum concentrations of immunoreactive PTH. Serum PTH levels were determined using an immunoradiometric assay for samples taken predose and 0.25-h postdose on days 0 (after first dose), 150, and 333 of administration. PTH (1-34) antibodies of goat to rat (Nichols Institute, San Juan Capistrano, CA) and goat to human (DiaSorin, Inc., Stillwater, MN) were used for the assay. Standards (diluted with rat serum) and serum samples (diluted at least 1:5) were incubated with both antibodies and the “sandwiched” complex was measured in a gamma counter at the end of the incubation period (18–24 h). This assay likely measured injected levels of human PTH (1-34) and fragments because predose values and values for vehicle controls were not quantifiable (see Table 1).
Histology of the Proximal Tibia and Kidney.
Excised specimens of proximal tibia were prestained in Villanueva mineralized bone stain for 72 h, dehydrated through graded ethanol into acetone, infiltrated with methyl methacrylate monomer, and then polymerized under vacuum. Histological sections were cut to approximately 10- to 12-μm thickness, using a Polycut-S microtome (Reichert-Jung, NuBlock, Germany) with a carbide-tipped knife. Sections were evaluated by bone histomorphometrists and by a board-certified veterinary pathologist, using brightfield microscopy.
Kidneys were excised from each animal during necropsy and preserved in 10% neutral buffered formalin. Specimens were trimmed, processed through graded alcohol clearing agent, infiltrated, and embedded in paraffin. Specimen blocks were sectioned (Reichert-Jung microtome) and stained with hematoxylin/eosin and evaluated by a veterinary pathologist, using brightfield microscopy. Histologic changes were described, when applicable, according to their distribution, severity, and morphologic character (Bucci, 1991). Description and classification of lesions commonly seen in laboratory animals were noted in accordance with Benirschke et al. (1978), Jubb et al. (1985), and Boorman et al. (1990).
X-Ray Densitometry Analyses of Proximal Tibia and Mid-Femur.
The metaphyses of proximal tibiae were scanned ex vivo by dual energy X-ray absorptiometry (DXA) (Norland, Fort Atkinson, WI) to determine apparent bone mineral density (BMD; grams per centimeter squared), bone mineral content (BMC; grams), and projected area (centimeters squared).
Femoral length was measured using a micrometer (Mitotoyu, Tokyo, Japan). The femoral midshaft was analyzed in cross-section by quantitative computed tomography (QCT) (960A XCT; Norland). Voxel dimensions used were 148 × 148 × 1200 μm. Scan images were analyzed using Dichte software version 5.2 (Norland). Tissue parameters analyzed included volumetric BMD (grams per cubic centimeter), cross-sectional area (millimeters squared), and BMC (milligrams).
Morphometric Analyses.
Proximal femora were scanned using a small-specimen conebeam μCT system from Enhanced Vision Systems (London, Ontario, Canada). Bone morphometry was performed on the transversal and coronal micro-QCT scans of the femoral neck, using isotropic voxel dimensions of 22.6 μm. A digital image analysis system consisting of a digitizing pad (Summagraphic, Fairfield, CT) coupled to a Macintosh 7100/66 computer with a morphometry program (Stereology, KSS Computer Engineers, Magna, UT) was used for the histomorphometric measurements. The mid-cross-section of the femoral neck was analyzed to measure whole cross-sectional area, femoral neck width, marrow area, trabecular area, and perimeter. Also, a 2-mm-long section of the femoral neck was used to analyze indices of trabecular bone connectivity (Garrahan et al., 1986; Compston et al., 1987, 1989). Specifically, trabecular bone spicules were skeletonized to measure the number of trabecular nodes, node-to-node struts, free-end-to-free-end struts, node-to-free-end struts, cortical-to-free-end struts, cortical-to-node struts, and cortical-to-cortical struts. Additional parameters derived, included femoral neck cortical area, mean cortical thickness, percent marrow trabecular area, and trabecular connectivity relative to tissue and bone volume (BV) (total node/BV, free-end-to-free-end/BV, and node-to-node/BV).
Biomechanical Analyses.
Biomechanical testing of bone specimens produces several parameters that describe aspects of bone fragility, as measured from the load-displacement curve (Fig.1). Ultimate force (Fu) represents the strength of the bone, the slope (S) represents bone stiffness, the work to failure (U) represents the energy the bone can absorb before it breaks, and the ultimate displacement (du) is the reciprocal of brittleness. Femoral strength was measured on intact femora using a three-point bending test as described by Turner and Burr (1993). Load displacement curves were recorded at a cross-head speed of 1 mm/s using a servo-hydraulic materials testing machine (MTS Corp., Minneapolis, MN) and an x-y recorder (7090A, Hewlett-Packard Co., Palo Alto, CA). Femoral neck strength was measured by mounting the proximal one-half of the femur vertically in a chuck and applying a downward force at a rate of 1 mm/s on the femoral head until the neck failed (Turner and Burr, 1993; Sato et al., 1997). The bone strength of LV5 vertebrae was measured on the central part after the spinous process, posterior pedicle arch, and both cranial and caudal ends of epiphyses were removed to produce parallel surfaces, using a diamond wafering saw (Buehler Isomet, Evanston, IL).
Statistics.
Data from males and females were analyzed separately and are presented as mean ± S.E.M. Group differences were assessed by analysis of variance with pairwise contrasts examined using Fisher's PLSD, where the significance level for the overall analysis of variance was p < 0.05 (StatView; Abacus Concepts, Berkeley, CA).
Results
Live-Phase Analyses.
PTH was well tolerated by both male and ovariectomized female F-344 rats and had no effect on survival. All animals survived the 1-year treatment phase, with the exception of one male each from the Male8 and Male40 groups, which died on days 254 and 301, respectively. These deaths were consistent with the longevity historical data obtained for F-344 rats in our animal facility and were not treatment related.
Previous studies showed that peak blood levels of PTH are achieved at about 0.25 h after subcutaneous injection of rats (Dobnig and Turner, 1997). Serum levels of immunoreactive PTH at 0.25-h postdose confirmed exposure to PTH depending on dose during the 12 months of treatment. Immunoreactive serum levels of PTH did not seem to change significantly over the duration of the study. There were no significant differences in blood levels between male and female rats for corresponding doses of PTH (Table 1).
Body weight in males treated with PTH decreased by 5 and 8% for Male8 and Male40, respectively, compared with vehicle controls (Table2). PTH did not affect the body weight of ovariectomized rats (Table 2).
Skeletal Analyses.
Femora from control intact males and sham females were 11 and 8% longer after 1 year compared with their respective baseline controls, consistent with anticipated longitudinal growth, which is persistent even in 6-month-old rats. By the end of the study, significant radial growth (endocortical and periosteal) had occurred for control intact males and sham females that were 18 months old because cross-sectional areas of the femoral midshaft were 22 and 16% greater than their respective baseline controls (Fig.2). Both doses of PTH stimulated longitudinal growth in ovariectomized females to increase femoral length by 4 and 5% for PTH8 and PTH40, respectively. PTH did not further modify the femoral length of males (Table 2).
QCT analysis of the femoral midshaft showed significant effects of aging and PTH on cortical bone after 1 year. In control intact males, an 11% reduction in midshaft BMD was observed after 1 year, compared with 6-month-old baseline controls. The altered BMD was attributed to a 22% increase in cross-sectional area (X-area), whereas BMC did not change (Fig. 2). After 1 year of treatment, PTH increased BMD by 46 and 67%, increased BMC by 57 and 105%, and increased X-area by 7 and 22% in Male8 and Male40, respectively, compared with vehicle age-matched controls. Therefore, both doses of PTH increased BMC, BMD, and X-area significantly and substantially beyond the normal range for these cortical bone parameters in male F-344 rats (Fig. 2).
In control sham females that were 18 months of age, there was no change in BMD after 1 year compared with 6-month-old baseline controls because midshaft BMC increased by 22% and cross-sectional area increased by 16%, relative to baselines (Fig. 2). After 1 year of ovariectomy, there was a 10% reduction in BMD that was due to a 7% reduction in BMC and 3% increase in X-area, compared with sham age-matched controls. In ovariectomized females, PTH8 and PTH40 increased BMD by 36 and 55%, increased BMC by 45 and 72%, and increased X-area by 6 and 10%, respectively, compared with Ovx. Therefore, in ovariectomized rats, both doses of PTH increased BMC, BMD, and X-area significantly and substantially beyond baseline, Ovx, and sham levels (Fig. 2). The midshaft of intact males was more responsive to PTH than that of ovariectomized females.
PTH effects on sites enriched with trabecular bone were analyzed by DXA of the proximal tibia and at higher resolution by QCT of the femoral neck. After aging from 6 to 18 months of age, BMC in the proximal tibia of control intact males and sham females increased by 20 and 32% compared with their respective baseline controls (Table 2). In male rats, PTH increased proximal tibia BMC by 83% in Male8 and by 154% in Male40, when compared with age-matched vehicle controls. In female rats, ovariectomy reduced BMC by 20% compared with sham (Table 2). In ovariectomized animals, PTH increased BMC of the proximal tibia by 33 and 67% in PTH8 and by 71 and 115% in PTH40, compared with age-matched sham and Ovx controls, respectively (Table 2). Therefore, both doses of PTH increased bone mass significantly beyond respective age-matched vehicle controls and baseline values. Relative comparisons showed that males responded to a greater degree than ovariectomized females.
Aging effects and PTH effects on trabecular bone architecture were evaluated by micro-QCT of the femoral neck (Table3). Aging effects were not overt because trabecular bone parameters were not significantly different between 18-month-old males and sham females compared with their respective baseline controls that were about 6 months old.
In the femoral neck of males, PTH increased total area (26 and 34%), total bone area (46 and 69%), cortical thickness (46 and 118%), and trabecular bone area (156 and 211%), while reducing marrow area (36 and 78%) for Male8 and Male40, respectively, compared with age-matched vehicle controls (Table 3; Fig. 3). Trabecular connectivity in Male8 and Male40 increased substantially, as evidenced by 108 and 169% increases in node density (node/BV) and 229 and 321% increases in node-to-node struts (node-to-node/BV), respectively (Table 3). QCT images confirmed that both doses of PTH induced substantial apposition of bone onto trabecular, endocortical, and periosteal surfaces, resulting in substantial loss of marrow within the femur and expanded external perimeter, compared with age-matched vehicle controls (Fig. 3).
In the femoral neck of females, ovariectomy for 1 year induced a 52% decrease in trabecular area, 60% loss of node density (node/BV), and 78% loss of node-to-node struts compared with age-matched sham controls (Table 3). These data confirm that, as expected, ovariectomy induced a substantial loss of trabecular bone and connectivity over time. The 6% increase in total area combined with the loss of trabecular bone resulted in a 47% increase in marrow area for Ovx compared with sham (Table 3).
In femoral neck of ovariectomized females, PTH increased total area (8 and 11%), total bone area (40 and 55%), cortical thickness (55 and 94%), and trabecular bone area (151 and 186%), while reducing marrow area (48 and 70%) for PTH8 and PTH40, respectively, compared with age-matched Ovx controls (Table 3). Both doses increased trabecular connectivity, including a 127 and 165% increase in node density and 303 and 404% increase in node-to-node struts for PTH8 and PTH40, respectively, compared with age-matched Ovx controls (Table 3). Therefore, in ovariectomized rats, both doses of PTH induced substantial apposition of bone onto trabecular, endocortical, and periosteal surfaces, resulting in substantial loss of marrow and altered bone geometry, compared with Ovx, sham, and baseline controls. The relative response of trabecular bone to PTH was similar between males and ovariectomized females, when compared with their respective controls.
Long-Term Effects of PTH on Biomechanics of Cortical Bone.
PTH effects on cortical bone quality were evaluated by three-point-bending analyses of the femoral midshaft (Tables4 and 5; Fig. 4). In 18-month-old intact males, failure analysis of the midshaft showed a 12% reduction in cortical bone stiffness with age for intact controls compared with 6-month-old baseline controls (Table 4). PTH increased ultimate load (78, 124%) and stiffness (80, 122%) for Male8 and Male40, respectively, compared with age-matched vehicle controls. PTH increased work to failure (U) compared with intact, to a similar extent in Male8 and Male40 (Table 4). Interestingly, PTH reduced ultimate displacement by 22 and 35% for Male8 and Male40, respectively, indicating an unexpected increase in brittleness.
In 18-month-old sham females, cortical bone fragility increased with age, as evidenced by a 29% loss of work to failure and 29% reduction in ultimate displacement for sham compared with 6-month-old baseline controls (Table 5). Ovariectomy reduced stiffness by 8% but increased ultimate displacement by 12% compared with sham (Table 5). At the midshaft, PTH increased stiffness (54 and 95%), ultimate load (49 and 83%) and work to failure (26 and 11%) for PTH8 and PTH40, respectively, compared with Ovx (Table 5). Similar to males, PTH reduced ultimate displacement by 11 and 30% for PTH8 and PTH40, respectively. These data showed that aging increased cortical bone fragility, and increased brittleness with PTH treatment was shown to occur in the cortical bone of both males and ovariectomized females.
Linear regression analyses were conducted to probe how PTH may alter relationships between bone mass, biomechanical properties, and geometry of the femoral midshaft. Age-matched controls and PTH treatment groups were pooled for males for regression analyses conducted for the midshaft; however, baseline controls were excluded from this analysis because aging over 1 year seemed to have significant effects apart from PTH effects. Strength (ultimate load) of the midshaft was highly correlated with midshaft BMC with a correlation coefficient ofr = 0.95 (P < 0.001). This value was similar to that reported by us for 9-month-old rats treated with PTH for 6 months (Sato et al., 1997). Interestingly, ultimate displacement was negatively correlated with cortical thickness for the midshaft with correlation coefficient of r = 0.78 (P< 0.001), suggesting that thickening of the cortex contributed significantly to the increased brittleness observed with PTH treatment.
Long-Term Effects of PTH on Proximal Femur Biomechanics.
Failure analyses on the femoral neck, a mixed site containing cortical and cancellous bone, were conducted to evaluate the separate effects of aging and PTH on the proximal femur (Tables 4 and 5; Fig. 4). In 18-month-old males, stiffness and ultimate load increased by 253 and 14%, respectively, whereas other parameters for male controls declined including work to failure (33%) and ultimate displacement (49%) compared with 6-month-old baselines (Table 4). PTH increased ultimate load (53 and 73%), work to failure (48 and 38%), and stiffness (11 and 23%) for Male8 and Male40 compared with age-matched vehicle controls but had no effect on ultimate displacement.
In 18-month-old sham females, aging increased femoral neck stiffness (128%) and ultimate load (21%), whereas work to failure (22%) and ultimate displacement (40%) declined compared with 6-month-old baseline controls (Table 5). Ovariectomy reduced femoral neck work to failure still further by 28%, ultimate load by 16%, stiffness by 13%, and ultimate displacement by 10%, compared with age-matched sham controls. Both doses of PTH reversed the detrimental changes induced by ovariectomy, with PTH40 increasing work to failure by 100%, ultimate load by 76%, and stiffness by 53% compared with age-matched Ovx. There was no effect of PTH on ultimate displacement. These data showed that aging increased stiffness and strength of the proximal femur, but reduced work to failure and ultimate displacement for both males and females. Both doses of PTH more than compensated for the detrimental effects of aging and improved the mechanical integrity of the femoral neck in both males and ovariectomized females.
Long-Term Effects of PTH on the Lumbar Vertebra.
Aging and PTH effects on the spine were evaluated by compression testing of excised L6 vertebra (Tables 4 and 5; Fig. 4). In 18-month-old male controls, stiffness and ultimate load declined by 33 and 21%, respectively, with age for lumber vertebra compared with 6-month-old baseline controls (Table 4). Both doses of PTH increased work to failure (163 and 240%), ultimate load (91 and 144%), and stiffness (61 and 83%) for Male8 and Male40, respectively, compared with age-matched vehicle controls (Table 4). PTH increased ultimate displacement by 37% for Male8; Male40 values did not differ from Male8.
In 18-month-old females, lumbar vertebra of sham tended to have lower stiffness (21%) and ultimate load (16%) with age compared with 6-month-old baseline controls; but these differences were not significant (Table 5). Ovariectomy significantly reduced work to failure (26%), ultimate load (25%), and stiffness (17%) for Ovx vertebra, compared with age-matched sham. Both doses of PTH increased work to failure (130 and 213%), ultimate load (119 and 144%), and ultimate displacement (6 and 35%) for PTH8 and PTH40, respectively, compared with age-matched Ovx (Table 5). Interestingly, maximal effects on stiffness (86%) was observed for PTH8 and not PTH40 which was 65% greater than Ovx. These data showed increased bone fragility with aging, which was countered by PTH in the vertebra. Both doses of PTH had largely beneficial effects on the mechanical properties of vertebra after 1 year of treatment, unlike the mixed effects on the midshaft.
Histopathology of the Proximal Tibia and Kidney.
Based on a finding of osteosarcoma in rats treated for nearly a lifetime (Neer et al., 2001; Vahle et al., 2002), histologic evaluations of nondecalcified sections were conducted on the proximal tibia of all groups by bone histomorphometrists and by a veterinary pathologist. Evaluations of the articular cartilage, epiphysis, physis, and primary spongiosa showed no treatment-related anomalies of any kind after 1 year of PTH treatment. Specifically, proliferative lesions were not observed.
To evaluate whether PTH treatment had detrimental effects on the kidney, another important target organ, histologic evaluation of kidneys was performed at study termination. PTH did not cause any morphologic lesions in the kidney (Table6). Progressive glomerulonephrosis and expected morphologic changes in this age of rat were observed in both treated and control males and females. Therefore, histologic evaluations indicated no significant treatment-related anomalies of any kind on bones or kidneys after 1 year of treatment in male or ovariectomized female rats.
Discussion
In a wide variety of rat models (normal or osteopenic, young or mature, female or male), PTH administered once daily by subcutaneous injection reproducibly increased mineral apposition onto trabecular and cortical surfaces in the axial and appendicular skeleton, resulting in increased bone mass, improved architecture, and stronger bones (Gasser and Jerome, 1992; Dempster et al., 1993; Hock, 2001). However, most previous rat studies have been short term in duration (3 months or less) or had insufficient statistical power with which to carefully evaluate PTH effects on pathology of target tissues or on survival long term. Only 1 study had previously evaluated the biomechanical effects of PTH with 26 to 35 animals per group for 6 months (Sato et al., 1997), which was of the longest duration at the time. Therefore, we report for the first time the pharmacological effects of PTH after a prolonged period (1 year) of treatment in intact male and ovariectomized female F-344 rats.
During the live phase, both doses of PTH were well tolerated, had no effect on survival, and at least partially, delayed the aging process in bones, but had no effect in kidneys. PTH slightly increased the femoral length of ovariectomized females, confirming PTH stimulation of longitudinal growth in ovariectomized females (Sato et al., 1997) but had no effect on longitudinal growth after 1 year in males. This PTH effect on longitudinal growth may be attributed to the inhibitory effects of ovariectomy on the closure of growth plates in the femur, as compared with sham animals (Turner et al., 1994). Our 1-year study extended from the time when longitudinal growth had slowed at 6 months of age, although normal growth and development were shown to persist up to at least 12 months of age or longer (Kalu et al., 1984; Nnakwe, 1995; Kimmel, 1996).
Male F-344 rats reportedly attain peak bone mass at about 12 months of age (Kalu et al., 1984). Their bone mass remained stable until about 24 months of age, and then began to decline (Kalu et al., 1984). Female F-344 rats exhibit near lifetime skeletal growth, as evidenced by a continued increase in bone mass measured as ash weight and calcium content with age, up to 29 months—their approximate life span (Nnakwe, 1995). Therefore, rats in our study were treated with PTH for nearly one-half of their life span. In Sprague-Dawley rats, the effects of ovariectomy on bone loss was most pronounced between 3 and 9 months of age, after which age-dependent bone loss in ovary-intact rats lessened the difference in skeletal mass between intact and ovariectomized rats (Wronski et al., 1989). In the femur neck of 3-month-old Sprague-Dawley rats, ovariectomy induced a high turnover state, but cortical osteopenia was not established until 1 year postovariectomy (Li et al., 1997). Our data confirmed dramatic loss of bone in the proximal tibia, femoral neck, vertebra, and femoral midshaft after 1 year postovariectomy.
Aging significantly increased fragility of the vertebra, proximal femur and femoral midshaft. PTH reversed aging effects on vertebra and proximal femur. Our data were largely consistent with skeletal effects reported in 15- to 24-month-old, ovary-intact virgin and multiparous, female Brown Norway, Brown-Norway/Fischer-344, and Sprague-Dawley rats, treated with PTH for shorter periods of 12 to 32 days (Gunness and Hock, 1995; Hock and Wood, 1995). These studies and our data show that the anabolic benefits of PTH to improve bone quality and strengthen rat bones can be induced under widely ranging conditions of treatment duration and age in both intact and ovariectomized females and intact males.
Numerous published studies have shown that in a wide variety of rat models, PTH increases trabecular bone volume, trabecular thickness, trabecular number, and connectivity by stimulating bone formation in the axial and appendicular skeleton (Gasser and Jerome, 1992; Dempster et al., 1993; Mosekilde et al., 1994b; Ejersted et al., 1995; Sato et al., 1997). Previous studies also consistently showed that PTH increased bone mass by stimulating apposition onto endosteal surfaces of trabecular bone, which were accompanied by increased strength at all sites tested (Mosekilde et al., 1991, 1994a,b; Sogaard et al., 1994;Sato et al., 1997). Our DXA, QCT, and biomechanical data largely confirmed other PTH data for cancellous bone sites. As illustrated in Fig. 4, the biomechanical effects of PTH on the vertebra and proximal femur were entirely beneficial after 1 year of treatment.
In rats, PTH was shown to increase cortical width by stimulating mineral apposition onto both endocortical and periosteal surfaces of the diaphysis of long bones (Gasser and Jerome, 1992; Dempster et al., 1993; Ejersted et al., 1993, 1994; Sogaard et al., 1994; Wronski and Yen, 1994; Li and Wronski, 1995; Li et al., 1997; Sato et al., 1997). In the absence of intracortical remodeling, which is observed in species with osteonal skeletons like humans, mineral apposition in response to PTH over time reduced marrow spaces and increased periosteal perimeter, resulting in a thickened cortex and altered geometry.
However, a novel, unexpected result was obtained in biomechanical analyses of the femoral midshaft of males and ovariectomized females after 1 year between 6 and 18 months of age. As shown in Fig. 4, PTH dramatically increased strength and stiffness of the midshaft, but lowered ultimate displacement, indicating increased brittleness. Linear regression analyses indicated that reduced ultimate displacement may be a geometrical consequence of increased cortical width in these rats. Increased brittleness may be a consequence of the inability of intracortical bone in rodents to remodel because cortical bone responses are limited to appositional growth on endosteal and periosteal surfaces. This hypothesis is supported by our 6-month study in mature, Sprague-Dawley, ovariectomized rats that showed a smaller PTH-induced increase in cortical width, with no effect on ultimate strain (Sato et al., 1997). These data argue strongly for the importance of a more thorough biomechanical analysis that includes parameters beyond just strength and stiffness.
Also unexpected was the magnitude of the skeletal effects, compared with short-term treatment of rats with PTH. In a study of 9-month-old Sprague-Dawley ovariectomized rats (Sato et al., 1997), equivalent doses of PTH (8 and 40 μg/kg) increased femoral midshaft BMD (30 and 47%), BMC (36 and 51%), ultimate load (50 and 74%), stiffness (49 and 74%), strength (24 and 33%), and Young's modulus (21 and 30%), respectively, compared with Ovx. In another study, 6-month-old Wistar rats were ovariectomized for 1 month before PTH treatment for 6 months (Kishi et al., 1998). DXA analysis showed about a 25% increase in BMD for the mid-diaphysis of femora after 6 months of PTH treatment (Kishi et al., 1998), which is comparable with our midshaft BMD data (Fig. 2). Finally, a study with 6-month-old Sprague-Dawley rats ovariectomized for 1 month before treatment with 40 μg/kg PTH for 6 months showed increases of 32% BMD and 61% BMC by DXA for whole femora, which translated to biomechanical increases of 52% ultimate load and 50% stiffness (Stewart et al., 2000). The relative magnitude of these PTH effects on the femoral midshaft obtained previously after 6 months is comparable with what we observed for 8 and 40 μg/kg PTH after twice the treatment duration. Therefore, published data (Sato et al., 1997; Kishi et al., 1998; Stewart et al., 2000) compared with data presented here indicate little to no additional benefit during the 6th to 12th month of PTH treatment for the cortical bone of the femoral midshaft in rats.
In vertebra (Sato et al., 1997), 8 or 40 μg/kg PTH for 6 months increased ultimate load (146 and 202%), stiffness (63 and 71%), strength (124 and 167%), and Young's modulus (55 and 66%), respectively. Similarly, vertebra from another ovariectomized rat study with 40 μg/kg PTH for 6 months showed increases of 116% ultimate load, 111% stiffness, and 144% work to failure (Stewart et al., 2000). Thus, efficacy in the spine shown previously after 6 months of PTH treatment was comparable with efficacy observed after 1 year of treatment. The cumulative data taken together indicate that most if not all of the advantageous biomechanical effects of PTH are achieved by 6 months of treatment, for the rat appendicular and axial skeleton.
The cortical bone biomechanical properties suggest that it is possible to treat rats with PTH for too long. Increased brittleness of cortical bone show that one-half a lifetime of PTH treatment is too long for rats. These data taken together with published studies of shorter duration indicate that optimal skeletal efficacy with PTH in rats is achieved by a 6-month duration or less. Rat models that have predicted human efficacy in shorter term studies with PTH and for other bone agents may diverge from clinical relevance when treatment duration is prolonged.
In conclusion, prolonged treatment of intact male and ovariectomized Fischer-344 rats with PTH once daily for 1 year, from 6 to 18 months of age, increased bone mass and strength of the lumbar vertebra, femoral neck, and femoral midshaft. However, improvements in bone mass, strength, and stiffness of the femoral midshaft were partially counter-balanced by increased brittleness and reduction of marrow spaces. Additional studies are required to elucidate cell and molecular mechanisms responsible for these effects and their relevance to humans who have a cortical bone physiology (osteonal remodeling) that is very different from that of rats.
Acknowledgments
We gratefully thank R. Cain, P. Francis, C. Frolik, and S. J. Smith (all from Lilly Research Laboratories) and A. Tashjian (Harvard University, Cambridge, MA) for their assistance and helpful suggestions.
Footnotes
-
This study was funded by Lilly Research Laboratories.
- Abbreviations:
- PTH
- parathyroid hormone
- Ovx
- ovariectomized
- BMC
- bone mineral content
- BMD
- bone mineral density
- QCT
- quantitative computed tomography
- DXA
- dual energy X-ray absorptiometry
- PLSD
- protected least significant difference
- X-area
- cross-sectional area
- Fu
- ultimate force
- Received January 14, 2002.
- Accepted March 1, 2002.
- The American Society for Pharmacology and Experimental Therapeutics