Part of these results was presented in abstract form as a plenary poster at the 24th Annual Meeting of the American Society for Bone and Mineral Research, San Antonio, Texas, September 20-24, 2002.
The authors have no conflict of interest
The concept of lose, restore, maintain (LRM) for reversing existing osteoporosis was tested in rats. The withdrawal of PTH results in the loss of the acquired bone mass, but sequential therapy with zoledronate quite effectively maintained the PTH(1-84)-acquired bone quantity and quality.
Introduction: Because antiresorptive agents against osteoporosis are presently quite limited, strong anabolic agents such as human parathyroid hormone (hPTH) are quite helpful. However, because hPTH(1-34) is available only through injection and has a critical side effect of causing bone tumors during life-long administration in the rat, it would be practical to use PTH for the shortest possible duration to obtain the maximal effect. To determine the effectiveness of the osteoporosis-reversing concept of lose, restore, and maintain (LRM), recombinant hPTH(1-84) [rhPTH(1-84)] and the respective antiresorptive agents were sequentially studied.
Materials and Methods: Thirty-six, 20-week-old Sprague-Dawley rats were used. Treatment started at the 25th week after ovariectomy, which was performed at 20 weeks of age, with 5 weeks of rhPTH(1-84) 100 μg/kg/day, 5 days/week, followed by the respective sequential therapies for 5 weeks as follows: (1) ovariectomized rats (OVX; n = 6), (2) sham-operated rats (SHAM; n = 6), (3) OVX rats with PTH maintenance (PTH-M; n = 6), (4) OVX rats treated with PTH and then PTH was withdrawn (PTH-W; n = 6), (5) PTH-treated OVX rats treated with 17β-estradiol (PTH-E; 10 μg/day SC, 5 days/week; n = 6), and (6) PTH-treated OVX rats treated with zoledronate (PTH-Z; 12.5 μg/kg SC weekly; n = 6). BMD of the right femora was measured by DXA. μCT was used to measure the structural parameters of the second lumbar vertebrae. Three-point bending test of the femora and compressive tests of vertebrae were also performed.
Results: Bone quantity data showed that the BMD and most of the microstructural parameters were significantly higher in the PTH-M and PTH-Z groups than in the OVX and PTH-W groups (p < 0.05). Measurement of the cortical thickness revealed that only the PTH-M group showed a significant increase (p = 0.001). The ultimate force (Fu) at the midshaft of the femora was similar in the treated groups and stronger than in the OVX group (p < 0.05). However, in the vertebrae, the Fu of the PTH-M and PTH-Z groups was significantly higher, by ∼44-47%, than in the OVX and PTH-E groups and showed a higher tendency than in the PTH-W group.
Conclusion: PTH withdrawal resulted in the loss of acquired BMD, and sequential therapy with antiresorptives prevented further loss (17β-estradiol versus zoledronate). The zoledronate after rhPTH(1-84) as a sequential regimen was quite consistently effective.
OSTEOPOROSIS IS A DISEASE defined by a decreased bone mass and altered microarchitecture, resulting in increased bone fragility and an accompanying increased risk of fracture.(1) Fracture is the major complication of osteoporosis and is attributed to lowered bone strength, which is a recently emphasized concept, and is now used in the newly revised definition of osteoporosis.(2) Thus, any effective treatment of osteoporosis should imply an improvement in overall bone strength. Of the various potential anabolic agents, human parathyroid hormone (hPTH) has currently emerged as the most promising treatment.
PTH is the main regulator of calcium homeostasis. Aside from this primary effect, PTH's paradoxical anabolic effect has been well known since 1932 and recently received Food and Drug Administration (FDA) approval.(3) Many reports detail PTH's effects on both animal and human bones. These effects include increased bone mass in both trabecular and cortical bones, with accompanying enhanced bone strength, and has been proven in rats, rabbits, monkeys, and most recently, mice.(4–8)
PTH's potential in increasing bone mass and strength is tempered with concerns about its use as an anabolic agent in osteoporosis. Practically, because PTH is a peptide and easily degraded in the gastrointestinal tract, it should be administered only through a parenteral route. Long-term studies (18-24 months) using high-dose hPTH(1-34) administered to 6-week-old Fisher 344 rats showed an increased risk of osteogenic sarcoma,(9) and the recent report on the possible relationship of osteogenic sarcoma with underlying primary hyperparathyroidism leaves some concerns on the use of recombinant hPTH (rhPTH) for long term.(10) Along with an increase in cancellous bone mass, there is a fear of cortical bone loss, or a cortical steal phenomenon.(11) If cortical bone is lost, the enriched cortical bone sites could be placed in jeopardy of fracturing. Therefore, it would be prudent and most appropriate to administer PTH for a limited time period to achieve the maximal anabolic effect on bone. However, the increased bone mass caused by hPTH(1-34) was lost after hPTH withdrawal.(7,12,13) Therefore, using antiresorptive agents sequentially after PTH would be considered a good alternative for several reasons: (1) to minimize the dosage and the duration of rhPTH and (2) to fill the increased cortical bone porosity during PTH treatment by turning the weak points previously discussed into strong ones.(4,7)
In this study, we attempted to prove the efficacy of the lose, restore, and maintain (LRM) concept as a practical approach in treating osteoporosis.(14) Therefore, rhPTH(1-84) was chosen for the restoration, and the 17β-estradiol and zoledronate were selected as sequential regimens. Furthermore, we endeavored to prove the effect of the sequential therapy in both aspects bone quantity and bone quality using conventional methods, such as DXA and biomechanical tests, as well as μCT.
MATERIALS AND METHODS
Thirty-six, 5-month-old virgin female Sprague-Dawley rats were purchased from the Department of Laboratory Animal Medicine at Yonsei Medical Research Center (Seoul, Korea). They were housed in a room maintained at 22.2°C with 12-h light and12-h dark cycles. The animals were fed freely with Purina laboratory rodent chow (Hagribrand Purina Korea Co., Kunsan, Korea), which contained 1.17% calcium, 0.77% phosphorous, and 2.6 IU vitamin D/g. All animals were treated in accordance with the guidelines and regulations for the use and care of animals of Yonsei University, Seoul, Korea.
The animals were randomized into the following six groups at the age of 5 months, with six rats per group, and two rats were housed per cage. The sham-operated group (SHAM) and the ovariectomized group (OVX) were treated with a vehicle for 10 consecutive weeks starting 5 weeks after the operation. All the rats in the remaining four groups were ovariectomized at 5 months old, left untreated for 5 weeks, and then treated with rhPTH(1-84) for 5 weeks. They were divided into different treatment regimen modes. One group was maintained on rhPTH(1-84) for another 5 weeks (PTH-M), and another group was given the vehicle for a further 5 weeks to observe the effects of PTH withdrawal (PTH-W). The other two groups were administered antiresorptive agents, either 17β-estradiol (PTH-E) or zoledronate (PTH-Z). The rhPTH(1-84), kindly donated by the Mogam Biotechnology Research Institute (Kyungi, Korea), was dissolved in 0.9% normal saline. The rhPTH(1-84) groups were treated daily for 5 days/week with intermittent subcutaneous injections of 100 μg/kg/day. The control group was injected subcutaneously with an equivalent volume of 0.9% normal saline at the same frequency. The 17β-estradiol (Sigma Aldrich, St Louis, MO, USA), was given subcutaneously at 10 μg/kg/day for 5 days/week, which is the dose proven to be effective to prevent osteopenia in OVX rats.(15,16) Zoledronate, kindly donated by Novartis Pharmaceutical (Basel, Switzerland), was subcutaneously injected 12.5 μg/kg, once a week.(17) The detailed protocol used is shown in Table 1. At the end of the 10 weeks of treatment, the rats were killed, and the femur and vertebrae were harvested and stored in saline-soaked gauze at −20°C until required for analysis.
Table Table 1.. Experimental Protocols
The BMD of the excised right femora were measured by DXA (QDR-4500A; Hologic, Waltham, MA, USA). The femora were scanned at a resolution of 0.5 mm with a scanning speed of 2 mm/s. The region was analyzed for BMC, projected bone area, and BMD. The triplicate determinations of the five different femora, with repositioning, showed a CV of 0.59%.
The μCT and software used for this experiment were from Skyscan (Antwerpen, Belgium). The computer system was adapted to allow a simultaneous acquisition step on one sample and an analysis step on another sample as previously described.(18) Using this technique, the static parameters were measured and included the trabecular bone volume (BV), volume fraction (Vol.F), trabecular thickness (Tb.Th.), trabecular separation (Tb. Sp.), surface area per bone volume, trabecular number (Tb.N.), degree of anisotropy (DOA), and structural model index (SMI).
Measurement of the cortical thickness and cross-sectional area at the mid-diaphysis of the femur
The cortical thicknesses at the femur's mid-diaphysis were measured using a Mitutoyo digital caliper (Cole Parmer, Vernon Hills, IL, USA). The average cortical thickness was calculated using the thickness measurements made in each of four quadrants, that is, the anterior-posterior (AP) and medial-lateral (ML) sides of the femoral cross-section, using a pair of digital calipers. The diameter at the AP and ML positions were measured, and the cross-sectional area was calculated.
Cross-sectional images of the middle of the vertebral body from the μCT were used to measure the thicknesses of the cortical shell and the cross-sectional area of the vertebral body (Fig. 1). Image-analyzing software (Image-Proplus; Media Cybernetics, Silver Spring, MD, USA) was used to calculate those parameters.
The femurs were thawed before testing, and the bone strength of the intact femurs was measured using a three-point bending test. A 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. Load-displacement curves were recorded at a crosshead speed of 1 mm/s using a servo-hydraulic materials testing machine (Instron, Buckinghamshire, UK) and an X-ray recorder (7090A; Hewlett Packard, Palo Alto, CA, USA).
Before the measurement of the L2 vertebrae's bone strength, its posterior processes were removed, and the ends of the centrum were made parallel with a diamond wafering saw (Buehler Isomet, Evanston, IL, USA). The biomechanical parameters of each vertebra were measured by compression, at a load rate of 50 N/s, using the Instron machine.
All data were expressed as means and SD. SPSS 10.0 software (Chicago, IL, USA) was used for the statistical analysis. The differences in the BMD, cortical thickness, various microstructural parameters, and biomechanical data between the groups were compared using ANOVA with a Bonferroni's multiple group comparison procedure. A p value of <0.05 was accepted as statistically significant.
The BMC and BMD of the right femora were 10.9% and 9.9% lower in the OVX group, respectively, than in the SHAM group (p < 0.05; Figs. 2 and 3). The discontinuation of rhPTH(1-84; PTH-W) resulted in a lower BMD than in the SHAM group, but it was still higher than in the OVX group. Treatment of the OVX rats with rhPTH(1-84) for 10 weeks (PTH-M) resulted in a greater BMD than in the original OVX group (15.3%, p < 0.001) or the PTH-W or PTH-E groups (average, 8.3%; p < 0.05). The OVX rats and rhPTH(1-84) rats, which were pretreated with sequential 17β-estradiol (PTH-E), maintained BMD, but at a level similar to the rhPTH(1-84) withdrawal group (PTH-W). In contrast, the sequential administration of zoledronate after rhPTH(1-84) (PTH-Z) resulted in higher BMD values compared with the OVX group (13.7%, p < 0.001) and the PTH-E or PTH-W group (average, 6.6% higher; p = 0.095).
Figure 4 shows the typical features of the 3D trabecular microstructure of the L2 vertebrae in the rats from each group. The microstructure showed deterioration in the OVX group compared with the rhPTH(1-84) pretreated groups. However, microstructural improvements were only seen in the bones with rhPTH(1-84) for 10 weeks (PTH-M) or bones that had the sequential use of zoledronate after rhPTH(1-84) (PTH-Z).
Table 2 shows the microstructural parameters of the OVX and other groups with different modalities. An ovariectomy significantly reduced the BV and Vol.F. by 47.1% and 42.0%, respectively (p < 0.001), compared with the sham controls. The BV and Vol.F. of the PTH-M group were significantly higher than found in the OVX group (50.0% and 46.0%, respectively, p < 0.001).
Table Table 2.. Effects of Sequential Therapy After rhPTH(1-84) Treatment on the Microstructural Parameters of L2 Vertebrae
However, these parameters in the PTH-W and PTH-E groups showed no differences compared with the OVX group. In the PTH-Z group, these same parameters were as high as in the PTH-M group (50.0% and 48.9%, respectively, p < 0.001), and compared with the OVX group, they were also higher than in the PTH-W group (38.9% and 37.4%, p < 0.01).
Trabecular thicknesses were also thicker in the PTH-M and PTH-Z groups by an average of 35.7% (p < 0.001) compared with the OVX, PTH-W, and PTH-E groups, as well as the SHAM controls. The total of the trabecular surfaces, per given volume, was smaller by ∼20.0% in the PTH-M and PTH-Z groups than in the OVX group (p < 0.001). However, trabecular separation in the PTH-Z group was less than in the OVX group (p < 0.05).
The DOA showed higher values in the PTH-M and PTH-Z groups than in the OVX, PTH-W, and PTH-E groups by an average of 14% (p < 0.05). The SMI revealed lower values in the SHAM, PTH-M, and PTH-Z groups by 16.9%, 16.4%, and 14.4%, respectively, than in the OVX group (p < 0.05).
Cortical thickness and cross-sectional area
The average cortical thicknesses at the mid-diaphyses of the femora were greater in the PTH-M group only (Fig. 5; p < 0.001). However, there were no significant differences in CSA between the groups (data not shown).
At the lumbar vertebrae, no group showed significant differences in the following parameters: thickness of the cortical shell, CSA of the vertebral body, or canal size (data not shown).
The femora diaphyses were evaluated with the three-point bending test at mid-shaft (Table 3). All groups treated with rhPTH(1-84) for 5 or 10 weeks showed significantly higher values for ultimate force (Fu), stiffness (S; p < 0.0001), ultimate stress (σu), and Young's modulus (E; p < 0.05). In addition, the femora of the PTH-Z group showed stiffer bone than the SHAM group (p < 0.05).
Table Table 3.. Biomechanical Parameters of the Femora
The mechanical properties of the lumbar vertebrae were evaluated by the compression test (Table 4). The 10-week treatment of rhPTH(1-84) or sequential therapy with zoledronate improved the Fu by 47.7% and 65.6%, respectively, compared with the OVX group (p < 0.05). The σu was also improved in both previously described groups by 44.1% and 64.0%, respectively, compared with the OVX group (p < 0.05).
Table Table 4.. Biomechanical Parameters of the Lumbar Vertebrae
Recently, the concept of osteoporosis rapidly evolved from a definition focused on low bone mass toward a definition that emphasizes the status of deteriorated bone strength.(2) Bone strength is mostly determined by the bone quantity, but the qualitative aspect of the bone contributes an∼30% effect. The qualitative element is best exemplified by a much-reduced risk of fracture relative to a limited increase in the BMD with the present use of antiresorptive agents. However, limitations of the antiresorptive agents probably stem from dramatically reduced bone remodeling and significantly increased microdamages, which would gradually stiffen the bone and make it prone to fracture.(19,20)
On the contrary, rhPTH(1-34), the only anabolic agent approved by the FDA (approved in 2002), stimulates bone formation and improves bone strength quite effectively. However, rhPTH has several shortcomings. First, because of its characteristic as a peptide hormone, it rapidly degrades in the gastrointestinal tract, making subcutaneous injection inevitable for patients, which causes discomfort and apprehension. Second, it is currently very expensive. Third, there are some concerns on the development of the osteosarcoma, because preclinical animal carcinogenesis data revealed that life-long treatment of rhPTH(1-34) in a rat model resulted in the occurrence of osteosarcoma,(9) and a case report of a highly possible relation of osteosarcoma to primary hyperparathyroidism has recently been reported.(10) Therefore, bone mass should ideally be acquired within a short period of hPTH treatment and maintained using other antiresorptive agents sequentially.
This experiment was performed to confirm the usefulness of the LRM concept of Tang et al.(14) To make the bone “lose,” 5-month-old rats were ovariectomized, and osteopenia was expected to happen after 5 weeks of estrogen depletion.(21) To “restore” the osteopenic bone, the rats were treated with rhPTH(1-84) for 5 weeks. The rhPTH(1-84) was chosen because its C terminus was pro-apoptotic when bound to the receptors that recognize the C-PTH fragments (CPTHRs) in osteocytic cells,(22) suggesting that there is less chance of tumorigenesis than with rhPTH(1-34), which strongly inhibits apoptosis of osteoblasts and osteocytes.(23) To “maintain” the bone pretreated with rhPTH(1-84) a well-known antiresorptive agent, 17β-estradiol and zoledronate, a third-generation bisphosphonate, were compared in this study. Both quantitative and qualitative methods such as DXA, μCT, and biomechanical tests were used to evaluate bone and to prove the effects of the LRM concept.
The administration of an initial 5-week course of intermittent high-dose hPTH(1-84) (100 μg/kg/day, five times per week) completely restored the femoral BMD to the same level of the age-matched, intact control rats (SHAM). As expected, the PTH-induced bone mass gains were lost in the PTH-W group after only 5 weeks after treatment termination. However, only zoledronate, among all the maintenance agents, was effective at maintaining the PTH-induced bone mass gains. The estrogen therapy, at 10 μg/kg/day, five times per week, was less effective in protecting against bone loss, especially in the vertebrae, and showed similar levels as in the PTH-withdrawn group (PTH-W). In contrast, zoledronate maintained the PTH-induced bone gain, not only in the BMD, but also in the parameters assessed by μCT. The trabecular bone volume, trabecular thickness, surface volume, and trabecular separation were comparable with those in the PTH-M group and were significantly better than in the OVX, PTH-W, and even the PTH-E groups. Therefore, zoledronate seemed to be more effective than estrogen in inhibiting bone resorption and maintaining the new bone formed with rhPTH(1-84).
Interestingly, the data on the trabecular and cortico-trabecular mixed sites were somewhat different, especially in the biomechanical analyses results. In the predominantly cortical site, measurement of the ultimate force, ultimate stress, stiffness, and Young's modulus of the mid-diaphysis at the femur was higher in all the treated groups compared with the OVX group. However, those parameters showed no significant differences between the treated groups. However, in the trabecular-rich vertebrae, the ultimate force and ultimate stress were significantly higher in the PTH-M and PTH-Z groups than in the OVX and the PTH-E groups, which was consistent with the positive changes in the microstructural analyses. This site-specific result might be explained by the different weight-bearing status and the rate of bone turnover between these two sites. However, the biomechanical parameters in the PTH-W group showed intermediate values, indicating that, even if the bone mass was decreased with the termination of the rhPTH(1-84) treatment, residual effects from the improved bone quality existed in this group. This suggests that the effects of these agents on bone, at the prescribed doses of rhPTH(1-84) of 100 μg/kg/day, over 5 weeks, were mainly restricted to changes in the trabecular bone, and that the anabolic effects of the PTH on the trabecular bone in rats was not achieved at the expense of the cortical bone, as previously suspected.
Other investigators have shown a reversal in acquired bone mass with the use of hPTH after 12-24 weeks.(16,24,25) This report confirms that the anabolic effects on the bone mass may be rapidly reversible once the anabolic stimulus is removed. In addition, it was quite surprising to see that, as early as 5 weeks after hPTH(1-84) withdrawal, the bone mass and microstructural parameters were similar to those in the OVX rats (OVX). To overcome the loss of bone mass and maintain the anabolic effects of hPTH, a few studies have reported trying sequential therapy, including hPTH(1-34), followed by antiresorptive agents, such as selective estrogen receptor modulator (SERM), bisphosphonate, or estrogen, and found that only the SERM and bisphosphonates, such as risedronate, showed effective maintenance.(16,26) In this study, the effects of the sequential therapy with zoledronate, have been proven for the first time. Although the dosage of zoledronate was relatively high, significant improvements in the bone quality were revealed. Conversely, the quantitative parameters in the PTH-E group, obtained by DXA or μCT and the measurement of Fu of the L2 vertebrae, were not as good as those found in the PTH-Z group, with the exception of the Fu of the right femur, which showed similar levels. The differences between zoledronate and estradiol might be attributed to their different potencies against bone resorption and their abilities to reduce the cortical porosity.(17)
In conclusion, the effective maintenance of bone strength at the predominantly trabecular (lumbar spine) and cortical-trabecular sites (femur), in the OVX-osteopenic rat model, was accomplished by the sequential treatment with zoledronate after rhPTH(1-84). Estrogen was insufficient in preserving the bone mass achieved with rhPTH(1-84) as much as the maintenance of the bone mass with sequential zoledronate. These data suggest that bisphosphonates, used properly, may be suitable agents for maintaining the bone mass acquired from using rhPTH(1-84) for short periods.
This work was supported by Korea Health 21 R & D Project Grant 01-PJ1-PG1-01CH08-0001, Ministry of Health and Welfare, Republic of Korea, and the Brain Korea 21 project for medical sciences.