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Endocrinology Vol. 143, No. 9 3230-3242
Copyright © 2002 by The Endocrine Society

ARTICLE

Abnormal Bone Architecture and Biomechanical Properties with Near-Lifetime Treatment of Rats with PTH

M. Sato, J. Vahle, A. Schmidt, M. Westmore, S. Smith, E. Rowley and L. Y. Ma

Lilly Research Laboratories, Indianapolis, Indiana 46285

Address all correspondence and requests for reprints to: Dr. Masahiko Sato, MC 86N, Lilly Research Laboratories, Indianapolis, Indiana 46285. E-mail: Sato_Masahiko{at}Lilly.Com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Skeletal effects are described for near-lifetime treatment of young, female rats with recombinant human PTH (1–34) (PTH). Rats (5–8 wk of age) were administered 0, 5, 30, or 75 µg/kg·d sc PTH for up to 2 yr, as part of an oncogenicity evaluation, which is required by regulatory agencies for potential chronic therapies. Proliferative lesions were observed in the skeleton as described in Vahle et al. (1 ); in this paper, we describe the quantitative bone data for this study. In the appendicular skeleton, PTH stimulated trabecular and endocortical mineral apposition to the near exclusion of marrow spaces at 5 µg/kg, with some periosteal apposition at 30 µg/kg, followed by considerable periosteal apposition and altered geometry at 75 µg/kg. Increased bone mass was observed for all treatment groups that substantially exceeded normal levels attained by vehicle controls and exceeded skeletal efficacy reported previously for similar doses in shorter-term studies. Dose-dependent increases in osteocalcin levels and a linear increase in wet weight of femora were observed for the entire treatment duration, suggesting nearly continuous PTH stimulation of osteoblasts and skeletal growth throughout life. Histology showed many osteocytes and prominent osteoblasts, but a conspicuous absence of osteoclasts. Morphometry showed a lack of distinction between trabecular and cortical bone. Biomechanics of vehicle controls showed that optimal mechanical integrity for the normal skeleton is observed at about 11 months of age. PTH greatly strengthened and stiffened vertebra and femora; however, the midshaft showed reduced toughness and increased brittleness with treatment, which was not the case for vertebra. Related studies of 6 and 9 months duration showed that the optimal duration for PTH skeletal efficacy was about 6 months in rats, based on toughness, strength, ultimate displacement, and architecture, especially for cortical bone. Therefore, treatment duration is an under appreciated aspect of PTH pharmacology; and PTH skeletal effects are a complex function of dose and duration. Comparative analyses showed that short-term treatment (6 months or less) is more advantageous than near-lifetime treatment, because PTH stimulates skeletal growth throughout life, resulting in abnormal architecture and untoward biomechanical properties in rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CURRENTLY, THE TREATMENT of osteoporosis is limited to agents that decrease the rate of bone turnover, by directly or indirectly inhibiting the resorption activity of osteoclasts and secondarily suppressing the formation activity of osteoblasts. These therapies include estrogen, calcitonin, bisphosphonates, and selective estrogen receptor modulators (2, 3, 4). Alternatively, a new potential therapy currently under clinical evaluation (2, 5) is the sc, once-a-day administration of the amino-terminal (1–34) fragment of human PTH. Clinically relevant doses of PTH have been shown to increase the rate of bone turnover in vivo by stimulating new bone formation in rats, monkeys, and humans, with little to no stimulation of bone resorption activity (5, 6, 7, 8, 9). Therefore, significant net gains in bone mass can be realized with once daily administration of PTH because mineral apposition proceeds at a greater rate than resorption activity.

Extensive biomechanical analyses have shown impressive effects of PTH on the axial and appendicular skeleton of rats (10). Load-to-failure analyses in rats showed that the femoral neck, vertebra, and diaphysis of long bones were stronger, stiffer, and tougher as a result of PTH treatment (10, 11, 12, 13, 14, 15). Unfortunately, biomechanics of rat bones has uncertain relevance to humans, because of physiological differences between rat and primate bones and because doses tend to be considerably higher in rats than used clinically. Nevertheless, PTH was shown recently to significantly reduce the incidence of vertebral and nonvertebral fractures in postmenopausal women with osteoporosis (2). At present, PTH is the only known clinical agent capable of increasing bone formation rate, trabecular bone volume, and trabecular connectivity (16, 17, 18, 19), while reducing fracture incidence in osteoporotic humans (2, 20).

Previous studies showed that PTH stimulates mineral apposition onto cancellous and cortical bone surfaces in rats, resulting in substantial gains in bone mass (8, 9, 10, 21). Similar cancellous bone data have been obtained for primate vertebra (2, 5, 7, 22, 23). However, by contrast, the effects of PTH on the cortical bone of primates have been controversial. Early clinical data suggested that the trabecular bone benefits occurred at the expense of cortical bone (17, 24). More recent studies in monkeys (22, 25) and in humans (2, 26, 27) have shown no effect or loss of bone mineral density (BMD) at cortical bone sites. Therefore, not all of the skeletal effects of PTH in rodents may be predictive of efficacy in primates.

Despite the long history and extensive literature describing PTH skeletal efficacy in rats (8, 9, 21), the cell and molecular basis to the appositional effects of PTH are not completely understood. At the tissue level, formation of new bone with PTH occurs by the activation of bone lining cells, the stimulation of osteoblast differentiation from precursors, and by reducing osteoblast apoptosis (28, 29, 30). In vivo studies conducted in rats showed that once daily PTH treatment increases osteoblast number and activity without stimulating osteoblast proliferation (29, 30); however, at least one in vitro study has reported a proliferative effect on osteoprogenitor cells (31). Because of the complexity of the histological findings, a focused description of the proliferative lesions has been presented separately by Vahle et al. (1). We present here quantitative bone data to clarify the pharmacodynamic effects of a near-lifetime (2 yr) of PTH treatment in the same rats, which may help to elucidate aspects of the oncogenicity data (1). The 2-yr study included both Fischer 344 males and females; however, because complementary analyses in two related studies contained only females, the female rat will be the primary focus of this publication.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rat groups and dosing regimens
Study 1 was conducted with intact F344 female rats (Taconic Laboratory Animals and Services, Germantown, NY), approximately 5–7 wk of age. Animals were group housed (approximately 4 per cage) and maintained on a 12-h light/dark cycle at 22 C with ad libitum access to Certified Rodent Diet 5002 (PMI Nutrition International, Inc., St. Louis, MO) and water. Except for baseline controls, rats were administered 0, 5, 30, or 75 µg/kg·d recombinant human PTH (1–34) (LY333334, Lilly) for 9 months. PTH was administered sc into the dorsal region of the animal at the indicated doses in 1 µl/g body weight of the injection vehicle consisting of 20 mM NaH2PO4, 0.9% NaCl, and mannitol (2.13 g/liter). Each group consisted of eight animals.

Study 2 was conducted with intact F344 female rats (Taconic Laboratory Animals and Services), approximately 7–8 wk of age. Animals were individually housed and maintained on a 12-h light/dark cycle at 22 C with ad libitum access to Certified Rodent Diet 5002 (PMI Nutrition International, Inc.) and water. Rats were administered 0, 5, or 30 µg/kg·d recombinant human parathyroid hormone PTH (1–34) (LY333334, Lilly) for 6 months. PTH was administered sc into the dorsal region of the animal at the indicated doses in 1 µl/g body weight of the injection vehicle consisting of 20 mM NaH2PO4, 0.9% NaCl, and mannitol (2.13 g/liter). Each group consisted of 30 animals.

Study 3 was an oncogenicity bioassay conducted with intact F344 female and male rats (Taconic Laboratory Animals and Services), approximately 7–8 wk of age. Animals were individually housed and maintained on a 12-h light, 12-h dark cycle at 22 C with ad libitum access to Certified Rodent Diet 5002 (PMI Nutrition International, Inc.) and water. Rats were administered 0, 5, 30, or 75 µg/kg·d recombinant human PTH (1–34) (LY333334, Lilly) for 2 yr. PTH was administered sc into the dorsal region of the animal at the indicated doses in 1 µl/g body weight of the injection vehicle consisting of 20 mM NaH2PO4, 0.9% NaCl, and mannitol (2.13 g/liter). Each group consisted of 60 animals.

Animal procedures were reviewed before implementation by an internal animal welfare committee to ensure compliance with NIH guidelines. Longitudinal analyses in vivo were conducted under anesthesia with isoflurane, whereas ex vivo analyses were conducted postnecropsy for animals surviving until study termination.

Dual energy x-ray absorptiometry (DXA)
In study 1, whole animals including the head were scanned longitudinally from baseline by DXA with an Eclipse (Norland, Ft. Atkinson, WI) using scan steps of 1 x 1 mm, scan speed of 20 mm/second, and histogram average width of 0.04 mg/cm2. Whole body bone mineral content (BMC, g) was measured. In addition, the DXA reported whole body BMD, whole body lean mass, projected area, and fat mass; however, because precise positioning of anesthetized rats was problematic (especially with baby rats), variance precluded meaningful interpretation of all parameters.

Serum and tissue collection
Baseline controls were euthanized by CO2 inhalation at the initiation of each study. One day after the last dose, rats were anesthetized with isoflurane, subjected to cardiac puncture, and euthanized by CO2 inhalation. Blood samples were allowed to clot at 4 C for 2 h before centrifugation at 2000 x g for 10 min. Sera were stored at -20 C for possible future analysis. Osteocalcin levels in rat sera were measured by RIA using a kit from Biomedical Technologies, Inc. (Stoughton, MA). Additional serum parameters were analyzed with an automated clinical blood analyzer (i-STAT, Sensor Devices, Inc.).

Lumbar vertebra, tibia, and femora were excised at necropsy, partially cleaned of soft tissue, preserved in 50% ethanol/saline, and stored at 4 C. For studies 1 and 3, length, lateral-medial width, and wet weight were measured for all undamaged left femora using calipers (Mitotoyo, Japan) and a balance (Mettler, New York, NY).

Quantitative computed tomography (QCT)
The proximal tibial metaphysis of anesthetized rats was scanned longitudinally at monthly intervals from baseline for study 1, as described previously (32, 33). Briefly, the proximal tibia was scanned about 2 mm below the growth plate, using a 960A pQCT loaded with Dichte software version 5.2 (Norland/Stratec, Ft. Atkinson, WI). Volumetric BMD (mg/cc), cross-sectional area (X-area, mm2), and BMC (mg) were quantitated for the whole cross-section of the metaphysis. Excised bones in 50% ethanol/saline at room temperature were analyzed at higher resolution using the 960A pQCT or microXCT loaded with Dichte software version XCT540 (Norland/Stratec). The midshaft of left femora were scanned with a 960A pQCT loaded with Dichte software version 5.2 (Norland/Stratec), using pixels of 150 x 150 µm and slice thickness of 1200 µm. L-6 vertebrae were scanned in cross-section with a microXCT loaded with Dichte software version XCT540 (Norland/Stratec), using voxels of 150 x 150 x 150 µm. In addition, high resolution images of the proximal femur were analyzed at 20-µm resolution (isotropic) using an EVS microCT (EVS Co., London, Ontario, Canada). Morphometric features of these images were analyzed with a semiautomatic digitizing system (KSS Image Analysis, KSS Scientific Consultants, Magna, UT) coupled to a PowerPC 7100/66 (Apple Computer, Cupertino, CA), using the image capture functions of NIH Image 1.59 (NIH, Bethesda, MD). Parameters analyzed included marrow area, cortical bone area, trabecular bone area, mean cortical thickness, and trabecular number.

As a comparison, femoral midshafts from four intact, female cynomolgus monkeys were scanned with the same 960A pQCT loaded with Dichte software version 5.2 (Norland/Stratec), using pixels of 150 x 150 µm and slice thickness of 1200 µm. Segmentation algorithms were used to measure BMD for the whole midshaft and for only the cortical bone. Additionally, a rod (6 mm diameter) was machined from the outer cortical diaphysis of a steer femur and scanned with the same 960A pQCT loaded with Dichte software version 5.2 (Norland/Stratec), using pixels of 150 x 150 µm and slice thickness of 1200 µm.

Biomechanical analyses
The biomechanical properties of the femoral diaphysis, femoral neck, and L-6 vertebra were measured for surviving animals, postnecropsy. Mechanical properties of the midshaft were measured for intact left femora using 3-point bending (34). Load was applied midway between two supports that were 15 mm apart. Femora were positioned so the loading point was about 7.5 mm proximal from the distal popliteal space and bending occurred about the medial-lateral axis. Specimens were tested in a saline bath at 37 C. Each specimen was submerged in the saline bath for about 3 min before testing to allow equilibration of temperature. Load-displacement curves were recorded at a cross-head speed of about 1 mm/sec using a materials testing machine (model 661.18c-01, MTS Corp., Minneapolis, MN) and analyzed using TestWorks 4 software (MTS Corp.). Parameters analyzed included ultimate load (Fu), strength (ultimate stress), stiffness, Young’s modulus (E), work to failure (energy), modulus of toughness, ultimate displacement, and ultimate strain, as described previously (10, 34).

Fu for the femoral neck was measured by mounting the proximal half of the femur vertically in a chuck at room temperature and applying a downward force on the femoral head until failure (34). The Fu was measured as the maximum force sustained by the femoral neck and was considered to be an estimate of femoral neck strength. All tests were conducted using the materials testing machine and analyzed using TestWorks 4 software (MTS Corp.).

Mechanical properties of L-6 vertebrae were analyzed after the posterior processes were removed and the ends of the centrum were made parallel using a diamond wafering saw (Buehler Isomet, Evanston, IL). Vertebral specimens were loaded in compression, using the materials testing device and analyzed using TestWorks 4 software (MTS Corp.). The compressive load was applied through a pivoting platen to correct for possible nonparallel alignment of the faces of the vertebral body (34). Specimens were tested in a saline solution at 37 C, after equilibration. Parameters measured included Fu, strength (ultimate stress), stiffness, E, work to failure (energy), modulus of toughness, and ultimate strain (10, 34). Briefly, ultimate stress was estimated as the maximum load divided by the gross cross-sectional area {pi}AB/4, where A and B are the vertebral widths in the anterior-posterior and medial lateral directions. Stiffness was calculated as the maximum slope of the load-displacement curve. Young’s modulus (E) was calculated by multiplying stiffness times 4T/{pi}AB, where T is the specimen thickness. Toughness was calculated as the area under the load-displacement curve divided by {pi}ABT/4.

Statistics
Group differences were assessed by ANOVA with pairwise contrasts examined using Fisher’s protected least significant difference (PLSD) where the significance level for the overall ANOVA was P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Longitudinal analysis of PTH effects on the skeleton of young F344 females
The proximal tibial metaphysis was analyzed longitudinally for study 1 because this site was shown previously to be highly responsive to PTH (9, 21) and because proliferative lesions were prevalent at this site in study 3 (1) (Fig. 1AGo). Vehicle controls showed a 100% increase in volumetric BMD compared with baselines over 9 months as rats matured. Most of the increase in proximal tibia BMD occurred during the first 5 months (or by about 7 months of age) with BMD of about 700 mg/cc, followed by a slower rate of increase thereafter. Compared with vehicle controls, little effect of PTH on BMD was observed during the first 3 months of treatment; however, 5, 30, and 75 µg/kg PTH groups significantly increased BMD by 4 months of treatment, with increasing separation from vehicle controls thereafter. Regression analysis showed that the 30, and 75 µg/kg groups could be characterized by linear BMD rates of 61.4 mg/cc/month (r2=0.945) and 75.0 mg/cc/month (r2=0.960), respectively.



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  		Figure 1. Longitudinal analysis of PTH effects on the proximal tibial metaphysis and whole body bone mass of young, intact, F344 females. In panel A, the proximal tibial metaphysis from F344 females were analyzed in vivo by QCT along the transverse plane. In panel B, whole body BMC were evaluated by DXA in vivo for 9 months. Animals were 5–7 wk old at study initiation. Data are mean ± SE for group sizes of n = 8, with significant differences from the respective vehicle control at each time point indicated by * (P < 0.05, Fisher’s PLSD).

 
PTH effects on the total mass of the rat skeleton were evaluated by DXA analysis of whole body BMC for study 1 (Fig. 1BGo). Whole body BMC for vehicle controls increased dramatically by 400% compared with baseline over 9 months, as rats matured. During the first month of treatment, a rapid 50–65% increase in whole body BMC was observed for 5, 30, and 75 µg/kg PTH groups compared with vehicle control, with no difference in efficacy between 5, 30, and 75 µg/kg. During the second month, whole body BMC for vehicle controls increased such that only the 30 and 75 µg/kg PTH groups remained significantly different from controls. From 4–9 months of treatment, 30 and 75 µg/kg groups continued to steadily increase whole body BMC compared with vehicle controls. These data showed that whole body BMC was a more sensitive assessment of the early skeletal effects of PTH than proximal tibia BMD, and confirmed that PTH dose-dependently increases the rate of bone gain in young rats. Additionally, the longitudinal plots showed that PTH effects on the skeleton do not actually level off dose-dependently after several months, but rather continue to increase although at a slower rate than initially.

To quantitate further the effects of age and PTH treatment on longitudinal skeletal growth, we evaluated femoral length and wet weight for studies 1, 2, and 3 (Fig. 2Go). Examination of femoral length after 9 months of treatment showed a 26% increase for vehicle control compared with baseline, indicating substantial longitudinal bone growth over the 9 months (about 11 months of age) (Fig. 2AGo). After 24 months, a further 1.9% increase in femoral length was observed for vehicle controls, indicating a much slower rate of longitudinal growth during the second year of life for normal femora (Fig. 2AGo). After 9 months, PTH dose-dependently increased femoral length, with a maximal 2.8% elongation obtained for 75 µg/kg relative to vehicle controls. Further elongation was observed between 9 and 24 months of PTH treatment, as 5, 30, and 75 µg/kg were 2.4%, 5.2%, and 6.0% longer than vehicle, respectively, after 2 yr. These data show that PTH increases longitudinal growth of femora over the entire 2 yr.



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  		Figure 2. Femoral length and wet weight. Femoral length (A) and wet weight (B) were measured for femora from animals from studies 1, 2, and 3 treated with 0, 5, 30, or 75 µg/kg PTH. Data are plotted together to simplify presentation. Plotted are mean ± SE with significant differences with respect to corresponding vehicle controls at each time point indicated by * (P < 0.05, Fisher’s PLSD).

 
Regression analysis of vehicle controls showed a linear increase in wet weight for the entire 2-yr period at a rate of 0.0218 g/month (r2 = 0.999) (Fig. 2BGo). At 9 months, femoral weight increased by 35% for vehicle control compared with baseline, with an additional 46% increase observed after 2 yr compared with 9-month controls. Dose-dependent increases of gain in femoral weight were observed for 5, 30, and 75 µg/kg PTH, which was 13%, 32%, and 51% greater than vehicle controls after 2 yr, respectively. These data showed dose-dependent effects of PTH on increasing gain of wet weight of whole femora that persisted for the entire 2-yr duration of treatment.

Cortical bone effects of PTH in F344 females
In an effort to understand the cortical bone effects of long-term PTH treatment, we analyzed the femoral midshaft along the transverse plane (Figs. 3Go and 4Go). Volumetric BMD appeared to peak around 1100 mg/cc between 8 and 11 months of age (6–9 months treatment) for vehicle controls, with some loss in BMD after 2 yr (35). After 9 months of treatment, PTH induced a dose-dependent elevation of BMD, with a 10% increase in BMD for 75 µg/kg PTH compared with vehicle controls. In data not shown, the increase in BMD resulted from a larger, dose-dependent, 18% increase in BMC and 7% increase in X-area for 75 µg/kg PTH, compared with vehicle controls after 9 months. By 24 months, PTH induced a dose-dependent elevation of BMD with a 43% increase at 75 µg/kg compared with vehicle controls (Fig. 3Go). Large, dose-dependent increases in BMC of 107% and X-area of 46% were observed at 75 µg/kg compared with vehicle controls (see Fig. 4Go).



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  		Figure 3. QCT analysis of the femoral midshaft after 6, 9, and 24 months of treatment. Volumetric BMD of the whole midshaft were measured along the transverse plane. Data are mean ± SE, with significant differences with respect to corresponding vehicle controls indicated by * (P < 0.05, Fisher’s PLSD).

 


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  		Figure 4. QCT images of the femoral midshaft after 2 yr. The midshaft of left femora were analyzed along the transverse plane by QCT, using voxel dimensions of 150 x 150 x 1200 µm. Images shown include vehicle control (A), 5 µg/kg (B), 30 µg/kg (C), and 75 µg/kg (C). Images show reduction of the marrow cavity at 5 µg/kg, loss of marrow space and expansion of bone at 30 µg/kg, and further expansion of bone area with altered geometry at 75 µg/kg.

 
QCT images of the midshaft showed distinct marrow and cortical bone compartments for only the vehicle controls, after 2 yr (Fig. 4AGo). Reduction of the marrow cavity was apparent at 5 µg/kg, with essentially no marrow space and periosteal expansion of cortical bone at 30 µg/kg. Further periosteal expansion of bone area was observed with considerable altered geometry at 75 µg/kg (Fig. 4DGo). The QCT scans showed that PTH stimulated endocortical bone apposition to the near exclusion of marrow at 5 µg/kg, that was followed by periosteal expansion and altered geometry at the higher doses. BMD for the 30 and 75 µg/kg groups were both about 1400 mg/cc after 2 yr (Fig. 3Go). These femora data showed that PTH treatment induced massive gains in bone mass that were realized primarily through endocortical apposition to the exclusion of marrow spaces, followed by periosteal expansion with minor effects on femoral length.

Biomechanical properties of the whole femoral diaphysis were ascertained by 3-point bending of the midshaft for studies 1, 2, and 3 (Table 1Go; Fig. 5Go). Vehicle controls showed a 28% increase in Fu and 24% increase in stiffness between 6 and 24 months (Table 1Go). Dose-dependent elevation of Fu with PTH was observed at 6, 9, and 24 months of treatment with substantial increases of 154% observed with 75 µg/kg PTH after 2 yr, compared with controls. Dose-dependent elevation of stiffness was also observed at each time point, with substantial increases of 143% with 75 µg/kg PTH after 24 months, compared with controls. The low dose of 5 µg/kg increased stiffness by 59%, compared with controls after 2 yr. Vehicle controls showed no change in ultimate displacement after 6, 9, and 24 months. Similarly, PTH had no effect on ultimate displacement at 6 and 9 months of treatment; however, significant reductions in ultimate displacement were observed for all groups after 2 yr, with a maximal reduction of 34% observed for the 5 µg/kg group (Table 1Go and Fig. 5Go). The treatment-induced reduction in ultimate displacement indicated increased brittleness for the midshaft after 24 months treatment compared with controls, but not at 6 and 9 months. These QCT plus stiffness and displacement data showed a surprising level of efficacy in the midshaft with 5 µg/kg that was achieved primarily between 9 and 24 months of treatment.


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  		Table 1. Biomechanical properties of the femoral midshaft

 


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  		Figure 5. Material properties of the femoral midshaft after 6, 9 and 24 months of PTH treatment. Material properties were approximated by loading femora to failure by 3-point bending and normalizing values by the cross-sectional moment of inertia. Plotted are ultimate stress (strength, panel A), ultimate displacement (B) and Young’s modulus (C). Data are mean ± SE, with significant differences with respect to corresponding vehicle controls indicated by * (P < 0.05, Fisher’s PLSD).

 
Material properties of the femoral diaphysis were approximated by normalization of the midshaft properties by the radial distribution of bone mass [cross-sectional moment of inertia (Fig. 5Go, Table 1Go)]. In this study, vehicle controls achieved peak levels of strength and E after 9 months (or at about 11 months of age), whereas toughness was not observed to differ between 6 and 24 months. Therefore, some loss of bone quality with age was observed during the second year for normal rats. PTH increased strength compared with controls at each time point; but maximal strength for 5, 30, and 75 µg/kg PTH was achieved between 6 and 9 months, with significant loss observed for all treated groups at termination compared with 9-month groups. Treatment had little effect on E at 6 and 9 months compared with controls. After 2 yr, E was greater than controls for the 5 µg/kg group, but 75 µg/kg significantly reduced E to below controls and other treatment groups. Treatment had complex effects on toughness. At 6 months, PTH induced a dose-dependent increase in toughness, consistent with previous studies (10); however, toughness was no longer greater than controls, at 9 months. After 2 yr, toughness decreased below control with 5 µg/kg, whereas toughness was not different for other treatment groups with respect to control.

The midshaft biomechanical data showed unfavorable effects of aging and long-term treatment on cortical bone properties, especially between 9 months and 2 yr of treatment. Substantial, dose-dependent increases in stiffness were seen at 6, 9, and 24 months; but reductions in ultimate displacement for all doses, and some reduction in E and toughness at 75 or 5 µg/kg indicate undesirable effects on the material quality of rat cortical bone after 2 yr. The optimal duration for PTH cortical bone efficacy appeared to be about 6 months treatment in rats, based on toughness, strength, and ultimate displacement. These data indicate that duration of treatment is an under appreciated aspect of PTH efficacy and that 6 months treatment has advantages over near-lifetime treatment for rat cortical bone.

Near-lifetime treatment effects of PTH on the lumbar vertebra
Vertebra were evaluated as another site associated with proliferative lesions in rats after 2 yr treatment (1). QCT showed dose-dependent increases in BMC, X-area, and BMD for L-6 after 6 and 24 months, that exceeded normally attained levels for each parameter (Figs. 6Go and 7Go). Images of 2-yr vertebra showed pronounced alteration in architecture, including considerable periosteal expansion, thickening of processes, and loss of marrow spaces, especially in the 75 µg/kg group (Fig. 6Go, C and D). Dose-dependent elevations of 140% BMC, 67% X-area, and 38% BMD were observed with 75 µg/kg PTH after 2 yr compared with controls. The images showed, however, that BMC, X-area, and BMD do not adequately describe the geometric alterations induced by long-term PTH treatment.



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  		Figure 6. QCT images of L-6 vertebra after 2 yr of PTH treatment. L-6 were scanned along the transverse plane by QCT, using voxel dimensions of 150 x 150 x 150 µm. Panels A and B are images from two vehicle controls, and panels C and D are from the 75 µg/kg group. Considerable loss of marrow and alteration of geometry were observed with 2 yr treatment.

 


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  		Figure 7. QCT analysis of lumbar vertebra after 6 and 24 months of treatment. L-6 vertebra were excised and scanned along the mid-transverse plane, using 150 x 150 x 150 µm voxels. Parameters analyzed included volumetric BMD (A), BMC (B), and X-area (C). Note that study 2 lacked a 75 µg/kg PTH group. Data are mean ± SE, with significant differences with respect to corresponding vehicle controls indicated by * (P < 0.05, Fisher’s PLSD).

 
Whole bone properties for L-6 vertebrae were ascertained by compression testing of specimens to failure (Table 2Go). Substantial, dose-dependent elevations of 135% Fu, 71% stiffness, and 243% work-to-failure (energy) were observed for the 75 µg/kg group, compared with vehicle controls after 2 yr. Interestingly, Fu increased by 89% for the 5 µg/kg group, showing that efficacy at the low dose was more similar to the high dose group than controls, after 2 yr. PTH increased strain by a maximum of 24% compared with controls, indicating reduction of brittleness for vertebra after 2 yr.


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  		Table 2. Biomechanical properties of L-6 vertebrae

 
Material properties were approximated for vertebra by normalizing the above parameters by the X-area. Considerable, dose-dependent elevations of 86% vertebral strength, 56% E, and 127% toughness were observed for 75 µg/kg PTH compared with vehicle controls after 2 yr (Fig. 8Go, Table 2Go). The vertebra biomechanical data taken together show maximal or near maximal efficacy for stiffness, strain, toughness, strength, and E for the 5 µg/kg group, confirming substantial efficacy for this low dose of PTH, after 2 yr.



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  		Figure 8. Biomechanical properties of vertebra after 2 yr of PTH treatment. L-6 vertebrae from rats in study 3 were loaded to failure in compression. Parameters shown include ultimate stress (strength, panel A), E (B), modulus of toughness (C), and ultimate strain (D). Data are mean ± SE, with significant differences with respect to vehicle controls indicated by * (P < 0.05, Fisher’s PLSD).

 
Serum and histological evaluations
Osteocalcin levels in sera were analyzed in an effort to assess osteoblast activity in bone formation, during the live phase of studies 1 and 3 (Fig. 9Go). Control levels were similar between 9 and 24 months, showing that bone formation activity did not change appreciably during the last 15 months of rat life (Fig. 9Go). At 9 months, the highest osteocalcin levels were observed as a 120% increase for 75 µg/kg, compared with vehicle controls. At 24 months, the highest levels were now observed as a 76% increase for 30 µg/kg, with 30 not different from 75 µg/kg (Fig. 9Go). The dose-dependent elevations in osteocalcin seen between 9 and 24 months of treatment were consistent with a PTH stimulation of osteoblastic activity. However, not shown in Fig. 9Go were baseline levels of 278 ± 33 ng/ml for 5- to 7-wk-old rats, which were off scale, indicating that bone formation during the initial 5–7 wk of age was considerably higher than that of the last 15 months of rat life. In data not shown, hypercalcemia was not observed after 6, 9, or 24 months of PTH treatment. These data show a significant and persistent stimulation of osteoblasts for the entire duration of PTH treatment. PTH effects on osteocalcin were consistent with the marked effects on bone mass observed in the axial and appendicular skeleton.



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  		Figure 9. Serum osteocalcin. Osteocalcin levels were measured for sera from studies 1 and 3. Data are mean ± SE with significant differences with the respective vehicle controls indicated by * (P < 0.05, Fisher’s PLSD). Baseline values (278 ± 33 ng/ml) were measured for 5- to 7-wk-old rats, but were not plotted because they were off scale.

 
Qualitative evaluation of histological sections of femora, tibia and vertebra showed prominent osteoblasts on trabecular and endocortical surfaces, but osteoclasts were rarely seen after 2 yr of PTH treatment. Major changes included substantial increases in the number of osteocytes which were associated with the massive increases in lamellar bone (see Figs. 4Go and 6Go) and substantial reductions in hematopoietic cells, as hematopoiesis migrated from the marrow to the spleen (1).

A semiquantitative morphometric analysis of the femoral neck was attempted after 2 yr of PTH treatment (Table 3Go). Most of the treated animals lacked discernible marrow spaces, including six of six from the 5 µg/kg group. Approximations of cortical and trabecular bone parameters are presented in Table 3Go; however, many parameters were not measurable because of difficulty in distinguishing between cortical and trabecular bone. Nevertheless, these limited data were consistent with the femoral midshaft and vertebral analyses showing exaggerated skeletal effects beyond normally attained levels in all groups after 2 yr of PTH treatment, even at 5 µg/kg.


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  		Table 3. Morphometry of the femoral neck from F344 females after 2 yr of PTH treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data from three studies conducted with young, intact female F344 rats were pooled to permit a longitudinal assessment of the skeletal effects of 1–24 months of PTH treatment. These studies were conducted within a 3-yr period with animals from the same breeding colony. Because more than half of the vehicle control rats were lost during the 2-yr study, we feel justified in describing this treatment period as "near-lifetime" in duration. Assessment of femoral length and whole body BMC by DXA confirmed that longitudinal skeletal growth normally slows at about 11 months of age for intact F344 females (35, 36); however, a small but significant increase in femoral length was observed between 11 and 26 months of age, which was further increased with PTH treatment during the second year of life. All three doses of PTH significantly increased the longitudinal growth of femora at every time point evaluated during the entire duration of treatment, including during the second year of life. However, evaluation of femoral length and midshaft showed that most of the bone gain observed with PTH was the result of cortical apposition of mineral.

Prevalent sites for proliferative lesions observed in study 3 included tibia, femora, and vertebra (1). Profound, dose-dependent effects of PTH on BMC, BMD, and strength were observed, confirming that these skeletal sites were highly responsive to treatment. A dose-dependent linear increase in the wet weight of femora was observed that showed no signs of abating for the entire 2 yr. Analyses of the femoral midshaft and neck showed that 5 µg/kg stimulated trabecular and endocortical apposition to the exclusion of marrow, with some periosteal apposition at 30 µg/kg, followed by considerable periosteal apposition and altered femoral geometry (shape) at 75 µg/kg. The collective data showed that rat femora have an amazing capacity to accrue bone mass, largely through cortical apposition of mineral even after filling in the marrow spaces. Osteocalcin measurements confirmed stimulation of osteoblast activity in bone formation during the entire duration of treatment. Not surprisingly, spleens were enlarged in treated animals, indicating migration of hematopoietic function from marrow to the spleen (1).

An interesting tendency of the femoral midshaft to level off at about BMD =1400 mg/cc was observed with 30 to 75 µg/kg PTH after 2 yr. Previously, no change or a reduction in BMD in response to subcutaneous PTH treatment was observed in cortical bone sites of osteoporotic women and men (2, 17, 26, 27). Therefore, in an effort to understand the relevance of these rodent findings to primates, we scanned the femoral midshaft from four adult, intact, female, cynomolgus monkeys (Macaca fascicularis) with the same QCT. The volumetric BMD for the whole monkey midshaft was 894 ± 13 mg/cc, whereas the cortex had a cortical bone BMD = 1176 ± 7 mg/cc. Therefore, the whole rat midshaft, and presumably the whole rat femur, attained a BMD after 2 yr of PTH treatment that was significantly greater than normal monkey cortical bone. Whole rat femur BMD also compared favorably with a rod of pure cortical bone that was machined from a bovine femur which had BMD = 1393 ± 5 mg/cc.

Cortical bone analyses in cynomolgus macaques treated with 1 or 5 µg/kg PTH for 18 months showed no effect of PTH on the BMD of the radius midshaft, as measured by QCT (22). BMD for the femoral midshaft from our rat 5 µg/kg group was 1269 ± 10 mg/cc after 2 yr compared with BMD = 915 ± 18 mg/cc for the radius midshaft of monkeys treated with 5 µg/kg for 18 months (Sato, M., unpublished data). The former was 31% greater than age-matched rat vehicle controls, whereas the latter was not different from monkey vehicle controls.

The extreme density attained by rat femora is likely due to apposition onto endocortical and periosteal surfaces (14, 37) with limited intracortical bone remodeling (38, 39, 40, 41). Previous studies showed porosity of only about 0.1–1.5% in rat long bones (35, 38, 42), compared with about 8% in 40-yr-old and 20% in 80-yr-old human long bones (43, 44). As a result, rats are largely unable to replace (turnover) cortical bone by osteonal remodeling. By contrast, primate bones are characterized by Howship’s lacunae, and PTH treatment was shown to increase osteonal remodeling in monkeys and humans, resulting in cortical porosity (7, 17, 24, 25). However, despite increased cortical porosity, measures of cortical bone strength actually increased with PTH, because PTH stimulated endocortical apposition resulting in greater cortical thickness, cortical area, and moment of inertia (25). In addition, the preferential localization of porosities toward the endocortical region tended to minimize their effect because the endocortical region bears less stress during bending than does the periosteal region (25). Interestingly, recent clinical data from osteoporotic women suggests similar effects of PTH on human cortical bone (27, 45). Therefore, these data attest to an important difference in skeletal physiology between rats and primates, and shows that all PTH skeletal effects observed in rodents are not entirely relevant to primates, especially cortical bone effects.

Dramatic skeletal efficacy was observed with 5 µg/kg PTH after 2 yr of treatment in rats. Previous studies with similarly low doses (10, 46, 47) showed significant but comparatively modest effects at cancellous and cortical bone sites after 6 months in rats. However, in our study, maximal or near maximal efficacy for strain, toughness, strength and E for vertebra, displacement and E for the midshaft, and histomorphometry of the femoral neck were observed for 5 µg/kg PTH after 2 yr. Previous data (10, 46, 47) showed that PTH effects were dose dependent but were limited in duration to a maximum of 6 months PTH treatment. Our 2-yr data clearly show that treatment duration in the rat is an under appreciated aspect of PTH skeletal efficacy; and that PTH skeletal effects are a complex function of dose and duration.

Interestingly, untoward effects of PTH on rat cortical bone quality were measured after near-lifetime treatment. All previous biomechanical analyses of rat femora showed only beneficial effects on the mechanical integrity of the midshaft (10, 13, 14). However, the femoral midshaft from the 5 µg/kg group was shown to be more brittle and less tough than controls after 2 yr. Linear regression analyses suggested that reduced ultimate displacement may be a geometrical consequence of increased cortical width and occlusion of marrow in these animals.

By contrast, vertebra from all time points showed only beneficial effects on mechanical integrity, as the 2-yr vertebra were stronger, stiffer, tougher, and less brittle than controls. However, images showed disfigured vertebra after 2 yr indicating that current QCT and biomechanical parameters may not fully characterize the architectural effects of treatment. In considering the 6-, 9-, and 24-month biomechanical data together with the published literature, the collective data indicate that short-term PTH treatment of 6 months (or less) is preferable for improving bone quality and architecture for rats, especially for cortical bone. An optimal treatment duration for rats suggests the existence of an optimal PTH treatment duration for humans; however, additional clinical studies are required to clarify what this duration might be for osteoporotic humans.

Because PTH is a potential therapy to treat osteoporosis, the possible relevance of these rat findings to humans must be considered. We would suggest that the exaggerated skeletal effects observed after 2 yr of PTH have little relevance to the proposed clinical utility of PTH for several reasons. First, young rats were treated for 2 yr, which constitutes 70–90% of their normal life-span. The anticipated duration of PTH therapy in osteoporosis is up to 2 yr, or about 2–3% of a normal human life span. Second, there exist important physiological differences between rat and human skeletons. Bone growth slows substantially after 1 yr of age but proceeds at some skeletal sites in rats for most of their lives (48, 49), whereas skeletal growth ceases between 18 and 30 yr of age in humans (50). PTH stimulated radial (cortical apposition) and longitudinal skeletal growth in rats; but these data are not likely to be relevant to elderly humans who have ceased growing. Additionally, rats lack haversian canals to remodel cortical bone. The lack of osteonal remodeling helps to explain the massive accumulation of bone observed for the midshaft, which does not occur in primates (25). PTH stimulates mineral apposition onto both endocortical and periosteal surfaces resulting in essentially a solid skeleton after a lifetime of treatment in rats. In primates, PTH stimulates intracortical remodeling, which results in increased porosity, with most of the porosity preferentially localized toward the endocortical surface (25). Intracortical remodeling helps to explain why BMD for cortical bone sites does not change or actually decreases in monkeys and people treated with PTH (2, 22, 25, 26). Finally, the clinical intent is to restore bone in osteoporotic patients so that they will be less likely to fracture. Previous studies showed about a 10–15% gain in vertebral BMD by DXA over about 2 yr in osteoporotic women, who remained osteopenic after treatment but less likely to fracture (2, 26). By contrast, rats with normal bone levels were dosed with PTH, resulting in 140% greater bone mass (BMC) than normal peak levels for vertebra after 2 yr. Clearly, excessive bone beyond normally attained levels is not a desirable outcome for rats or people.

In summary, near-lifetime stimulation of osteoblast activity with PTH in the rat had untoward effects on skeletal architecture and bone material properties, indicating that PTH skeletal efficacy is a complex function of dose and duration. The optimal treatment duration for PTH skeletal efficacy was shown to be about 6 months (or less) in rats, based on toughness, strength, ultimate displacement, and architecture, especially for cortical bone.


   Acknowledgments
 


   Footnotes
 
Abbreviations: BMC, Bone mineral content; BMD, bone mineral density; DXA, dual energy x-ray absorptiometry; E, Young’s modulus; Fu, ultimate load; PLSD, protected least significant difference; QCT, quantitative computed tomography; X-area, cross-sectional area.

Received February 7, 2002.

Accepted for publication May 1, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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