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Endocrinology Vol. 138, No. 10 4330-4337
Copyright © 1997 by The Endocrine Society

ARTICLES

Biosynthetic Human Parathyroid Hormone (1–34) Effects on Bone Quality in Aged Ovariectomized Rats

Masahiko Sato, George Q. Zeng and Charles H. Turner

Department of Endocrine Research (M.S., G.Q.Z.), Lilly Research Laboratories, Indianapolis, Indiana 46285; and Department of Orthopaedic Surgery (C.H.T.), and The Biomechanics and Biomaterials Research Center, Indiana University Medical Center, Indianapolis, Indiana 46202

Address all correspondence and requests for reprints to: Dr. Masahiko Sato, MC 797, Department of Endocrine Research, Lilly Corporate Center, Indianapolis, Indiana 46285. E-mail: sato-masahiko{at}lilly.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
For the first time, PTH (1–34) was found to significantly affect bone quality, femora length, and body weight of aged, ovariectomized rats. Specifically, we examined the effects of biosynthetic human PTH (1–34) in 9 month-old rats that were ovariectomized and dosed for the ensuing 6 months with 8 or 40 µg/kg PTH. Bone content, architecture, and quality of axial and appendicular skeletal sites were analyzed by computed tomography (QCT), histomorphometry, and biomechanical testing. The large sample size (n = 26–35) of this study was useful in confirming the anabolic, dose-dependent effects of PTH at trabecular and cortical bone sites. Longitudinal analysis of tibias by QCT confirmed a small age-dependent reduction in bone density, with further reductions observed for ovariectomized controls (OVX). Subtle but deleterious effects of ovariectomy on bone quality are described. Additionally, the strength of the femoral neck was shown not to differ between baseline, sham-operated controls, or OVX in this model, suggesting limited utility of this measurement in aged rats. Both doses of PTH induced substantial gains above OVX, in bone mass and connectivity for the proximal tibia, distal femur, proximal femur, and spine. Ovariectomy significantly decreased the toughness of vertebral bone. However, PTH at 8 µg/kg reversed this deleterious effect on bone quality, while 40 µg/kg PTH significantly improved both toughness and brittleness beyond baseline controls. Cortical bone analyses at the femoral or tibial diaphysis confirmed the PTH stimulation of endosteal and periosteal bone formation with resulting increase in cortical thickness, moment of inertia, strength, and stiffness of the femur. PTH treatment significantly improved the intrinsic properties, Young’s modulus and toughness, of the femur compared with OVX. At 40 µg/kg PTH, bone mass and strength were typically greater than sham or baseline controls, confirming that PTH is functionally anabolic at trabecular and cortical bone sites. Interestingly, PTH dose-dependently increased the femora length, in the absence of differences between baseline, sham, and OVX controls. PTH slightly increased body weight above OVX. In addition, PTH did not interfere with the beneficial effects of ovariectomy on rat longevity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERMITTENT sc administration of human PTH (1–34) was shown previously to significantly increase bone mass in ovariectomized rats (see review in 1 . Specifically, x-ray densitometry, histomorphometry, and other techniques were used to show that PTH (1–34) stimulates trabecular bone formation in the distal femur (2, 3, 4, 5), proximal tibia (5, 6, 7, 8, 9), femoral neck (10, 11), and vertebra (4, 12, 13, 14, 15). That is, even in aged rats with established osteopenia, intermittent sc dosing of PTH was shown to be capable of restoring lost trabecular bone to control levels (5, 15, 16, 17, 18) provided that remnants of trabecular bone spicules remain (19).

Substantial gains in the cortical bone of rats have also been reported with PTH (1–34) (20, 21, 22, 23, 24, 25, 26, 27, 28). The increase in cortical bone mass has been attributed to an increase in cortical wall thickness through stimulation of bone formation on both endocortical and periosteal surfaces. Consequently, these PTH (1–34) effects have translated to substantial gains in the mechanical integrity of the rat diaphysis (28).

Many of these animal studies have been found to parallel clinical data generated in postmenopausal women (29, 30, 31, 32, 33). That is, intermittent sc administration of low doses of PTH (1–34) enhanced bone formation at several sites, including the spine and femoral neck of osteoporotic women and men (29, 30, 31, 32, 33, 34). Early clinical data suggested the possibility that trabecular bone increased at the expense of cortical bone (30); but this has not been observed in detailed animal studies (2, 10, 12, 20, 21, 25).

However, earlier studies tended to use younger animals (5, 35, 36) and were relatively short in duration, or had insufficient statistical power, especially for the statistical analyses of biomechanical effects. Therefore, a well powered experiment was initiated to quantitate the functional effects of PTH (1–34) on the axial and appendicular skeleton of ovariectomized, aged rats. To more closely model postmenopausal women, our study used nongrowing, 9-month-old rats (5, 35, 36, 37) that were scanned at baseline, ovariectomized, and administered PTH at 8 or 40 µg/kg for 6 months. Novel findings are presented for ovariectomy and PTH effects on bone quality in trabecular and cortical bone compartments and on body weight and longevity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovariectomized rat model
Nine-month old, virus antibody-free, virgin Sprague-Dawley female rats (Charles River, Portage, MI) were maintained on a 12-h light, 12-h dark cycle at 22 C with ad libitum access to food (TD 89222 with 0.5% Ca and 0.4% P, Teklad, Madison, WI) and water. One hundred sixty six rats were selected from a pool of 172 rats based on the baseline bone mineral density (BMD), measured at the proximal tibia metaphysis by computed tomography (QCT) (960A, Norland, Ft. Atkinson, WI) (38, 39). Rats were then randomized into baseline and four treatment groups. Baseline animals (n = 26) were euthanized, and then bilateral ovariectomies were performed on the remaining rats, except for sham-ovariectomy controls. Treatment groups included: 1) sham-operated controls (sham, n = 35), 2) ovariectomized controls (OVX, n = 35), 3) OVX treated with 8 µg/kg biosynthetic human PTH(1–34) (LY333334, Lilly) (n = 35) administered daily by sc injection (s.c.), or 4) OVX treated with 40 µg/kg/day sc PTH(1–34) (n = 35). Sham and OVX rats were administered 100 µl/100 g body weight of the injection vehicle, consisting of 20 mM NaH2PO4, 0.9% NaCl, and mannitol (2.13 g/liter). Proximal tibias of rats were scanned longitudinally at monthly intervals by QCT using voxel dimensions of 0.195 x 0.195 x 1.2 mm. After 6 months of treatment, rats were injected ip with calcein (10 mg/kg, Sigma, St. Louis, MO) buffered with bicarbonate (pH 7.3) at 18 days and 4 days before death. All animal procedures were reviewed before implementation by an internal animal welfare committee, to ensure compliance with NIH guidelines.

Tissue collection, processing, and histomorphometry
After the final dose of treatment, rats were euthanized by CO2 inhalation. The left femur and L-6 vertebra were removed from each rat, cleaned of soft tissue, and frozen at -20 C, until biomechanical testing. Right tibias, femurs, and L-3 were removed, cleaned, and fixed in 50% ethanol/saline. Tibias and femurs were analyzed by QCT, as described previously (38, 39).

For histomorphometry, right tibias and L-3 were trimmed, using a low-speed diamond saw (Buehler Ltd., Lake Bluff, IL) and fixed in 70% ethanol. Specimens were stained for 4 days in Villanueva osteochrome bone stain (Polysciences Inc., Warrington, PA), destained, dehydrated in a graded series of alcohols, and defatted in acetone. Proximal tibia, tibia midshaft, and L-3 vertebrae were then infiltrated with methyl methacrylate, as described by Schenk et al. (40). Specimens were then embedded in a mixture of methyl methacrylate (75 ml)-dibutyl phthalate (19 ml)-benzoyl peroxide (2.5 g) (Kodak, Rochester, NY) and polymerized at room temperature. Longitudinal sections of the proximal tibia (4 and 8 µm) were cut on a Reichert-Jung 2065 microtome (Magee Scientific Inc., Dexter, MI). The 4-µm sections were stained with 6% silver nitrate (Von Kossa stain) before coverslipping; the 8-µm sections were mounted unstained for dynamic measurements. Cross-sections of the midshaft proximal to the tibia-fibular junction were sawed to 50-µm thick specimens (Diamond wire saw, DE Diamond Knives Inc., Wilmington, DE). Sections were glued onto slides dipped in 0.5% gelatin, dried overnight, and coverslipped with Eukitt (Calibrated Instruments Inc., Hawthorne, NY).

Histomorphometric measurements were made using an Optiphot-2 fluorescence microscope (Nikon, Melville, NY) and a semiautomatic digitizing system (SummaSketch III, Summagraphics Co., Seymour, CT; 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). For proximal tibial metaphysis, trabecular bone morphometry measurements were performed on the area beginning 0.3 to 3.3 mm distal to the epiphyseal growth plate. The sampling site excluded cortical edges. For L-3, the entire marrow region within the cortical shell was measured to derive trabecular bone parameters. Specifically, measurements were made of cancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th, µm), and number (Tb.N, No./mm), according to the nomenclature of Parfitt et al. (41). In addition, indices of trabecular bone connectivity were measured as described by Garrahan and co-workers (42, 43, 44). Specifically, trabecular bone images were skeletonized using NIH Image 1.59 to measure the number of trabecular nodes, node to node (NTN), free end to free end, node to free end, cortical to free end, cortical to node, and cortical to cortical. These measures were normalized to total tissue area for density calculations, as described previously (45).

For the tibia midshaft, sections were examined using an Olympus stereo zoom microscope (Olympus, Lake Success, NY). Cortical bone measurements included total cortical area, marrow cavity area, cortical area, periosteal perimeter, endocortical perimeter, single-labeled perimeter, double-labeled perimeter, and interlabeled width. Mineral apposition rate was derived from the interlabel width divided by the interval. Bone formation rate was the labeled perimeter multiplied by mineral apposition rate divided by the cortical perimeter.

Biomechanical analyses
Femurs were thawed before testing, and bone strength was measured on intact femurs using a three-point bending test. Load was applied midway between two supports that were 15 mm apart. The femurs were positioned so the loading point was 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 3 min before testing to allow equilibration of temperature. Load-displacement curves were recorded at a crosshead speed of 1 mm/sec using a servo-hydraulic materials testing machine (MTS Corp., Minneapolis, MN) and an x-y recorder (Hewlett Packard 7090A, Palo Alto, CA). Ultimate load (Fu) was calculated as the maximum force sustained by the bone. Ultimate stress was calculated using the following equation:

(1)
where {varsigma}u is ultimate stress, L is the distance between the loading supports (15 mm), b is the width of the femur in the anterior-posterior direction, and I is the moment of inertia (46). The value for moment of inertia used in stress analysis was calculated under the assumption that the femoral cross-sections were elliptically shaped (47) using the following equation:

(2)
where a is the width of the cross-section in the medial-lateral direction, b is the width of the bone in the anterior-posterior direction, and t is the average cortical thickness. Average cortical thickness was calculated from thickness measurements made in each of four quadrants of the femoral cross-section with a pair of digital calipers. Widths a and b were measured at the location of the femur where the top loader contacted the bone.

The bending stiffness (S) of the bone was calculated as the maximum slope of the force-displacement curve. The Young’s modulus (E) was calculated as

(3)
and modulus of toughness, or toughness (u) was calculated as

(4)
where area is the area under the load-displacement curve. The ultimate strain ({epsilon}u) was calculated as

(5)
where du is the ultimate displacement.

Femoral neck strength was measured by mounting the proximal half of the femur vertically in a chuck and applying downward force at a rate of 1 mm/sec on the femoral head until the neck failed. The ultimate load was calculated as the maximum force sustained by the femoral neck. All tests were done at room temperature using the MTS system.

Bone strength of the L-6 vertebrae was measured after the posterior processes were removed and the ends of the centrum made parallel using a diamond wafering saw (Buehler Isomet, Evanston, IL). Ultimate load (Fu), stiffness (S), ultimate stress ({varsigma}u), Young’s modulus (E), toughness (u), and ultimate strain ({epsilon}u) for each vertebra was measured in compression at a load rate of 50 N/sec using the MTS machine (Fig. 1Go). The compressive load was applied through a pivoting platen to correct for nonparallel alignment of the faces of the vertebral body (46). Specimens were tested in saline solution at 37 C. Ultimate load was calculated as the maximum load sustained by the specimen. Ultimate stress was calculated 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 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, and ultimate strain was calculated as the ultimate displacement divided by T.



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  		Figure 1. Stress strain diagram for a rat vertebra. Ultimate stress ({varsigma}u), Young’s modulus (E), toughness (u), and ultimate strain ({epsilon}u) were measured as shown. These parameters describe the bone quality: {varsigma}u reflects vertebral strength, E reflects stiffness, u reflects toughness, and {epsilon}u is the inverse of brittleness.

 
Statistics
Data are presented as means and SDs. 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. Longitudinal data were analyzed cross-sectionally at respective time points.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Longitudinal analyses of ovariectomized rats
Aging effects were observed as a small but significant decline in volumetric BMD of 12% for Sham tibias, by 5 months postsurgery (Fig. 2Go). By comparison, a 23% loss in BMD was observed for OVX compared with baseline, by 2 months postsurgery. Both PTH treatment groups steadily increased BMD at 5 months with the high-dose BMD 58% higher and the low-dose BMD 28% higher than baseline. These data confirm the anabolic effect of PTH (1–34) on the proximal tibias metaphysis of aged, ovariectomized rats (5, 7).



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  		Figure 2. Longitudinal analysis of tibias by QCT. The proximal tibias of rats were scanned longitudinally in vivo at monthly intervals, using voxel dimensions of 0.195 x 0.195 x 1.2 mm. Compared with baseline, a small but significant loss in volumetric BMD (mg/cc) of 12% was observed for the sham proximal tibias by 5 months postsurgery. A 23% loss in OVX BMD was observed by 2 months postsurgery for the ovariectomized rats. Both PTH treatment groups steadily increased BMD over the period examined. Each group were significantly different from the others by 2 months post-OVX (Fisher’s PLSD). Data are mean ± SD.

 
Longitudinal examination of body weight showed no gain in the Sham group (312 ± 36 g) compared with baseline (293 ± 23 g); however, a significant 24% increase was observed for the OVX group (357 ± 43 g) by 2 months postovariectomy. After 6 months, increases in body weights of the low (373 ± 47 g) and high groups (380 ± 36 g) were observed compared with OVX; however, only the latter difference was significant. Ovariectomy had beneficial effects on the survival of aged rats, as seven of 35 rats were lost from the Sham group but only one of 35 from the OVX (Table 1Go). PTH (1–34) did not interfere with the improved survival induced by ovariectomy, as only one rat was lost from either low (1/35) or high (0/35) groups.


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  		Table 1. Cortical bone analysis of femoral or tibial diaphysis

 
Analysis of femurs and tibias at 6 months postovariectomy
Examination of femoral length showed no differences between baseline, sham, or OVX groups at termination (Table 1Go). However, PTH induced dose-dependent increases in femur length as compared with controls. Sham and OVX groups did not differ in femoral width, but low and high-dose PTH groups had significantly wider femurs along both the M-L and A-P axes compared with baseline, sham, and OVX. Cortical thickness of the femur declined by 15% from baseline due to ovariectomy. PTH increased cortical thickness dose-dependently to above OVX, sham, and baseline. The femoral moments of inertia were not different between baseline, sham, or OVX; however, PTH increased moment of inertia in a dose-dependent manner.

Histological examination of the tibial diaphysis showed no significant differences in the periosteal cross-sectional area between baseline, sham, and OVX (Table 1Go). PTH significantly increased the periosteal area. The marrow cavity increased with age, as evidenced by a 22% increase in Sham compared with baseline. OVX further increased the marrow cavity by 43% compared with baseline. PTH substantially decreased the marrow cavity by 40% and 60% for low and high, respectively, compared with OVX. Significant, dose-dependent PTH effects were also observed on the endocortical and periosteal bone formation rates that caused increased cortical bone area above baseline, sham, and OVX.

The distal metaphysis and middiaphysis of femurs were examined by QCT (Fig. 3Go). In the metaphysis, ovariectomy reduced BMD by 26% compared with Sham, which was primarily due to a 22% decrease in bone mineral content (BMC) (Fig. 3Go, A and C). No differences in metaphyseal area were observed between OVX and sham (data not shown). Low-dose PTH induced a 86% increase in BMD and a 89% increase in BMC, while high-dose PTH induced a 241% increase in BMD and a 257% increase in BMC compared with OVX. Both doses also increased the metaphyseal area (data not shown). In the middiaphysis, ovariectomy induced an 11% decrease in BMD compared with Sham, which was due to an 11% decrease in the BMC (Fig. 3Go, B and D). Low-dose PTH induced a 30% increase in BMD and a 36% increase in BMC, while high-dose PTH induced a 47% increase in BMD and a 51% increase in BMC compared with OVX. Additionally, an increase in cross-sectional area due to low and high doses was observed. These data confirm the significant, dose-dependent PTH effects on femoral bone mass at the metaphysis and diaphysis, which more than compensate for the ovariectomy effects.



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  		Figure 3. Analysis of femurs by QCT. The distal metaphysis and middiaphysis of femurs were examined at termination ex vivo by QCT, using voxel dimensions of 0.147 x 0.147 x 1.2 mm. Analysis of the metaphysis showed that ovariectomy induces an 26% decrease in BMD compared with sham that was primarily due to a 22% decrease in BMC. No differences in cross-sectional area were observed between OVX and sham (data not shown). PTH induced dose-dependent, significant increases in BMD, BMC, and cross-sectional area at both sites. Data are mean ± SD. Significant differences from sham, OVX, low, and high are designated "s," "o," "l," and "h," respectively (Fisher PLSD, P < 0.001).

 
Tibia histomorphometry
PTH effects on the proximal tibias were analyzed by histomorphometry (Table 2Go). Ovariectomy reduced BV/TV and Tb.N by greater than 73% compared with baseline or sham. However, a small but significantly larger Tb.Th was observed in OVX, as the few remaining trabeculas were thicker on average than spicules found in baseline or sham. PTH significantly increased BV/TV, Tb.Th, and Tb.N compared with OVX, sham, and baseline.


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  		Table 2. Histomorphometry of the proximal tibial metaphysis

 
Additionally, ovariectomy and PTH effects were evaluated in the epiphysis (data not shown). Ovariectomy induced a 16% and 27% reduction in BV/TV compared with baseline and sham, respectively, that was attributable to significant reductions in Tb.N. PTH significantly increased BV/TV, Tb.Th, and Tb.N compared with OVX and sham, but to a lesser degree than observed in the metaphysis. The magnitude of change was less because less bone was lost due to ovariectomy. These data show that the epiphysis loses bone due to ovariectomy, although to a lesser extent than the metaphysis. Interestingly, the trabecular bone attained for low and high doses was similar to levels observed, respectively, for the metaphysis.

Trabecular bone architecture of the metaphysis was analyzed by quantitating connectivity, as described in Materials and Methods. Connectivity was found to be largely described in terms of the number density of nodes, free-ends, and by ratios of nodes to free ends (Node/F) (Table 2Go). OVX reduced the number of nodes and NTN by 87–92%, as compared with sham or baseline. PTH dose-dependently increased connectivity to above OVX, sham, and baseline levels.

Vertebrae histomorphometry
PTH effects on L-3 lumbar vertebrae were examined by histomorphometry (Table 3Go). Ovariectomy reduced BV/TV by 40% and Tb.N by 29% compared with sham, while Tb.Th was not different between sham or OVX at this site. PTH increased BV/TV, Tb.Th, and Tb.N compared with OVX; and increased BV/TV and Tb.Th by 38–104% compared with Sham. High-dose PTH had a significantly greater effect on BV/TV and Tb.Th than low-dose PTH. Trabecular bone architecture of vertebra was analyzed in a manner similar to the metaphysis. OVX reduced the connectivity of trabeculas, number of nodes, and NTN by 20–24%, compared with sham. PTH increased connectivity compared with OVX and sham.


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  		Table 3. Vertebrae histomorphometry

 
Biomechanical analysis of femurs
The functional consequences of ovariectomy and PTH treatment on the integrity of femurs were evaluated by biomechanical analyses (Table 4Go). Ovariectomy decreased the ultimate force (Fu), stiffness (S), ultimate stress ({varsigma}u), and toughness (u) compared with baseline and sham. Ovariectomy in rats was shown to reduce the Young’s modulus (E) by 19% compared with sham. PTH improved structural measures (Fu, S) to above OVX, sham, and baseline levels. PTH improved material properties ({varsigma}u, E, u) to above OVX levels. Neither OVX nor PTH affected failure strain ({epsilon}u), indicating that the brittleness of the femoral bone was unaffected.


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  		Table 4. Biomechanical analyses of the femoral diaphysis and femoral neck

 
There was no difference in femoral neck Fu between the baseline, sham, and OVX groups suggesting that the femoral neck bone strength was not affected by age or OVX. However, low- and high-dose PTH significantly increased Fu by 31% and 41%, respectively, over Sham.

Biomechanical properties of vertebrae
Compression analysis of vertebrae showed that OVX reduced ultimate force, Fu, by 23% compared with sham, while low and high PTH doses increased Fu by 90% and 132% over Sham, respectively (Table 5Go). Ovariectomy had no effect on stiffness, while low and high PTH doses increased stiffness by 54% and 61%, respectively, over Sham. OVX reduced the ultimate stress, {varsigma}u, by 23% compared with Sham. PTH raised {varsigma}u by 70% and 103% above sham level.


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  		Table 5. Biomechanical analyses of L-6 vertebra

 
OVX had insignificant effects on the Young’s modulus, E, but PTH increased E by 37% and 47% above sham. OVX reduced the toughness, u, by 44% compared with sham, while PTH treatment increased u by 89% and 157% over sham. In addition, PTH significantly increased the failure strain, {epsilon}u, over OVX in a dose-dependent manner. These data show that PTH reduces the brittleness of vertebrae.

Finally, correlation analyses were conducted to examine the relationship between bone strength and mass at appendicular and axial sites for these animals (Fig. 4Go). Strength of the femoral midshaft (Fu) was strongly correlated with the BMC of the middiaphysis, measured by QCT. Specifically, regression analysis showed a significant correlation with r = 0.92 (P < 0.0001). A similar analysis (data not shown) showed that the ultimate strength of L-6 vertebra ({varsigma}u) correlated with the trabecular bone volume (BV/TV) of L-3 vertebra with r = 0.78 (P < 0.0001). These data clearly show that the PTH effects on bone mass and architecture are functionally beneficial.



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  		Figure 4. Correlation analysis of strength of the femoral diaphysis to midshaft BMC. Strength of the femoral midshaft, represented by ultimate force (Fu), was strongly correlated with the BMC of the middiaphysis, determined ex vivo by QCT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH (1–34) was evaluated in vivo with group sizes capable of discerning subtle effects on aged, ovariectomized rats. Longitudinal analyses confirmed the significant effect of ovariectomy on body weight in aged rats. PTH (1–34) induced a small but significant increase in body weight beyond OVX controls. Additionally, ovariectomy was observed to have beneficial effects on the survival of aged rats, as fewer OVX animals (1/35) were lost during the experiment compared with sham (7/35). PTH (1–34) did not alter the beneficial effect of ovariectomy on survival.

In distal femurs and proximal tibias, 8 and 40 µg/kg PTH (1–34) dose-dependently increased the volumetric BMD, BMC, X-Area, bone volume, trabecular thickness, trabecular number, and connectivity of trabecular bone in the metaphysis. In addition, PTH (1–34) increased trabecular bone parameters in the tibial epiphysis to comparable levels observed in the metaphysis. These data show that PTH is anabolic to trabecular bone sites, including the epiphysis, which is minimally responsive to estrogen levels (48).

Ovariectomy was observed to diminish trabecular bone mass and connectivity in the epiphysis, contrary to a previous report (48). Differences from the previous report are the shorter duration of the current study (6 months vs. 11.5 months), the use of older rats (9 months vs. 3 months), and sample size (26–35 vs. 8 per group). The latter is likely to be the most important difference. However, both studies agree in showing that the magnitude of the difference between OVX and Sham was considerably less in the epiphysis than metaphysis.

In addition, PTH was shown to improve connectivity in the proximal tibias and vertebrae in agreement with some previous efforts (7). By contrast, Lane et al. (9) showed that PTH does not affect connectivity. The different conclusions may be explained because our measurments are two dimensional instead of three dimensional; unfortunately, our QCT lacked sufficient spatial resolution for spicule mapping in rat bones. Nevertheless, our connectivity analysis was useful in confirming the substantial architectural effects of PTH on trabecular bone in aged, ovariectomized rats. We would suggest that conventional parameters do not adequately describe the architectural effects or functional consequences of treatments.

Dramatic anabolic effects of PTH on cortical bone that were observed previously (20, 21, 22, 23, 24, 25, 26, 27, 28) were confirmed in our study of aged, ovariectomized rats. QCT and histomorphometry confirmed significant dose-dependent effects of PTH on BMD, BMC, cross-sectional area, cortical thickness, and moment of inertia, at the expense of the marrow cavity. The data clearly show the PTH stimulation of endocortical and periosteal bone formation and detail the substantial increases of the mass, density, and geometrical properties of cortical bone to clearly beyond that of baseline controls.

Previous clinical studies suggested that the significant enhancement of trabecular bone mass occurs at the expense of cortical bone (30, 34). No evidence of this was observed, and these data confirm and extend previous studies in rats clearly showing that trabecular and cortical bone compartments increase mass along all three dimensions in response to PTH (2, 7, 10, 11, 12, 20, 21, 25, 26, 27).

Increased bone in trabecular and cortical bone compartments was coupled with impressive gains in the structural integrity of the bone tissue, as well as the material quality of vertebra. We take bone quality to represent the resistance of a bone to fracture. This resistance is best defined as the toughness, which represents the amount of energy the bone can absorb before it breaks. Increased toughness can result from either increased strength or decreased brittleness, i.e. increased ultimate strain. The high dose PTH treatment both increased vertebral strength and decreased brittleness, resulting in more than a 4-fold increase in toughness of the vertebrae compared with OVX and a 2-fold improvement over baseline controls. For the first time, ovariectomy was shown to significantly reduce the strength and Young’s modulus of the femoral diaphysis, indicating an effect on the structural quality of the cortical bone. High-dose PTH improved the strength and Young’s modulus of the femoral diaphysis to above that of baseline controls. The toughness of cortical bone was also reduced by OVX and returned to baseline levels by PTH treatment. However, the effects of PTH on cortical bone toughness were not as dramatic as seen in vertebral bone; and cortical bone brittleness was not affected by either OVX or PTH treatment. Therefore, the cortical bone results suggest that the PTH treatment produced a subtle improvement of bone quality. The more dramatic effects of PTH on toughness in vertebral bone probably resulted from a combination of improved structural integrity of the trabecular lattice and, possibly, improved bone tissue quality.

These results show clearly that the PTH-stimulated bone formation results in substantially improved bone mass, architecture, and quality, as a function of dose. This suggests a significant advantage to the therapeutic usage of PTH compared with agents that primarily inhibit bone resorption (49, 50). Additional studies will be required to substantiate this possibility.


   Acknowledgments
 
The authors gratefully acknowledge the excellent technical assistance of Tongyu Wang, Tina Fuson, Ellen Rowley, Shawn Smith, M. Dee Adrian, Pam Shetler, and Harlan Cole.

Received February 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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