The Effect of Aging on the Skeletal Response to Intermittent Treatment with Parathyroid Hormone
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内分泌学杂志 2005年第4期
Departments of Medicine (E.K., B.-h.S., K.L., J.D., K.I.) and Orthopaedics (N.T., C.G.), Yale School of Medicine, New Haven, Connecticut 06520-8020; and Department of Orthopaedics (M.B.), Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
Address all correspondence and requests for reprints to: Eleanor Knopp, TAC S141, 300 Cedar Street, New Haven, Connecticut 06520-8020. E-mail: eleanor.knopp@yale.edu.
Abstract
Little is known about the modifying effects of age on the skeletal response to intermittent treatment with PTH. We therefore compared the response of 63 aged (18 month old) and 61 young-adult (3 month old) C57BL/6 mice to 4 wk of daily sc injections of either vehicle or h(1–34)PTH at a dose of 95 ng/g body weight. The increase in total body bone mineral density (BMD), compared with vehicle-treated animals, was similar in aged and young-adult mice (+5.6 vs. +6.3%). Aged animals demonstrated a greater increase in spinal BMD than their younger counterparts (+12.0 vs. +5.1%, P = 0.01; absolute increment: 57 x 10–4 vs. 28 x 10–4 g/cm2). Microcomputed tomography analyses in a subset of the vertebrae showed a trend toward higher L5 trabecular bone volume fraction in the PTH-treated aged animals (+40.2 vs. +19.6%). Vertebral histomorphometry demonstrated a greater PTH-induced increase in osteoblast number in aged vs. young-adult animals (694 vs. 396 cells/mm2). In contrast, in the femur the PTH-induced increase in BMD tended to be greater in the young-adult than the aged animals, although this did not reach statistical significance (8.1 vs. 4.2%). The numbers of osteoblast progenitors and mineralizing colonies in cultured marrow were unaffected by PTH treatment in either group. We conclude that aging differentially impacts the regional skeletal response to PTH such that the increase in BMD in the spine is augmented, whereas that in the femur is unaffected. Effects on osteoblast progenitor recruitment do not seem to be the basis for these changes.
Introduction
THE INCIDENCE OF osteoporotic fracture rises exponentially with aging such that 50–60% of women in their mid- to late 70s will have had an osteoporotic spine fracture (1, 2). In women, first-fracture incidence rates increase from 12/100,000 person-years in the less than 50-yr-old age group to 1200/100,000 person-years for women 80–85 yr old (3). An increased incidence of hip fracture also occurs in older men (4), and the death rate after hip fracture is even greater in men than in women (5).
Until recently, therapies for osteoporosis were limited to antiresorptive agents such as estrogens, the related selective estrogen receptor modulators, and bisphosphonates. There is a great need for anabolic therapies because, particularly in elderly patients with severe osteoporosis, antiresorptive therapy cannot return bone mass to normal.
PTH is the first effective anabolic agent for the treatment of osteoporosis. Animal studies have demonstrated convincingly that intermittent administration of PTH leads to an increase in bone mass (6, 7). The clinical efficacy of PTH in the treatment of osteoporosis was established in a large, prospective, double-blind, and randomized clinical trial by Neer et al. (8). These investigators showed that in women with osteoporosis, 18 months of once-daily administration of sc PTH dramatically increased bone mass in the spine and hip and reduced spinal fracture rates. Despite nearly 3 decades of experimentation, however, the cellular mechanisms by which PTH exerts this anabolic effect remain obscure.
In addition to uncertainty about the cellular mechanisms underlying the anabolic effect of PTH, the influence of aging on the skeletal response to this treatment is largely unstudied. Because PTH will primarily be used in older patients, determining how aging influences the response to this new therapeutic is of considerable clinical relevance. We therefore compared the anabolic response to intermittent PTH treatment in young-adult and aged mice to determine whether the anabolic effect of PTH is influenced by the aging process.
Materials and Methods
Materials
Human (1–34) PTH was from Bachem Bioscience Inc. (King of Prussia, PA). Metofane was from Medical Developments Australia (Victoria, Australia), ketamine and xylazine were from Henry Schein Inc. (Melville, NY). Phenol red-free MEM, ascorbic acid, ?-glycerophosphate, penicillin-streptomycin, L-glutamine, and tetracycline were from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was from Summit Biotechnology (Fort Collins, CO). Dulbecco’s phosphate-buffered saline was from Gibco BRL (Rockville, MD). Millonig’s fixative was from Surgipath Inc. (Richmond, IL). Toluidine blue powder was from Fisher Scientific Inc. (Pittsburgh, PA). Uvinert was from Biomedical Specialties, Inc. (Santa Monica, CA). The Vector Red alkaline phosphatase substrate kit I was from Vector Laboratories, Inc. (Burlingame, CA).
Experimental Animals
Three- and 18-month-old male C57BL/6 mice (n = 61 and n = 63, respectively) were obtained from the National Institute on Aging. Mice were fed standard chow ad libitum and were maintained and used according to National Institutes of Health guidelines. This study was approved by the Yale Animal Care and Use Committee.
Treatment protocol
Animals from each age group were randomly assigned to receive either single daily sc doses of vehicle (10 mM acetic acid containing 2% heat-inactivated mouse serum) or h(1–34) PTH at 95 ng/g body weight reconstituted in vehicle.
The 4-wk treatment schedule was as follows: 1) on d 1–29, the animals received single daily doses of either vehicle or PTH; animals were weighed every 7 d, and the dose of PTH was adjusted for any change in weight; 2) on d 1, 8, 15, 22, and 29, total body and regional bone mineral density (BMD) was determined by PIXImus (see below); 3) on d 22 and 27, mice were injected ip with tetracycline at 15 μg/g body weight dissolved in sterile 0.9% NaCl to label the mineralizing front in bone; and 4) on d 30, the day of killing, animals were anesthetized and serum samples collected from each mouse for determination of osteocalcin and intact PTH. Urine was also collected for determination of C-terminal telopeptides of type I collagen.
The left femur and lumbar spine were harvested, fixed in Millonig’s fixative, embedded in methylmethacrylate, and processed for static and dynamic histomorphometric analyses as previously reported (9, 10, 11). Eight left femurs and the corresponding L5 vertebra were reserved from each group, fixed in 70% EtOH, and subsequently scanned by microcomputed tomography (microCT).
The marrow was flushed from the right femur and tibia of half the mice and cultured to determine the numbers of osteoblast-progenitors.
Killing
Animals were deeply anesthetized with Metofane and killed by cervical dislocation after blood and urine were collected on d 30.
Bone densitometry
PIXImus.
Serial BMD determinations of total body, spine, and femur were obtained weekly (beginning on d 1) using a PIXImus densitometer (GE-Lunar Corp., Madison, WI) running software version 1.45. The coefficient of variation for total BMD as measured in our laboratory is 1.0 ± 0.2%. Scan acquisition time is 5 min/animal and is performed on anesthetized animals in the prone position. The total body window is defined as the whole-body image minus the calvarium, mandible, and teeth. The spine window is a rectangle spanning a length of the spine from L1 to the beginning of the sacrum. The femur window encompasses the entire right femur of each mouse. BMD values are expressed in grams per square centimeter x 10–4. Anesthesia for densitometric measurements was accomplished using a mixture of ketamine and xylazine administered ip at a dose of 50 and 5 μg/kg, respectively, in the 3-month-old animals and 30 μg/kg ketamine, 3 μg/kg xylazine in the 18-month-old animals. Mice were warmed under a heating lamp until they regained consciousness and were water restricted for 1 h post anesthesia.
MicroCT.
Femurs and vertebrae were evaluated using desktop microtomographic imaging systems equipped with a 10- and 5-μm focal spot microfocus x-ray tube, respectively (μCT20 and μCT40, Scanco Medical AG, Bassersdorf, Switzerland) (12). The entire femur was scanned using a 34-μm slice increment, requiring approximately 100–150 microCT slices per specimen. Images were reconstructed, filtered, and thresholded as previously described (13). The images were stored in three-dimensional (3D) arrays with an isotropic voxel size of 34 μm. Morphometric parameters were computed using a direct 3D approach that does not rely on assumptions about whether the underlying structure is either plate or rod like (14). Cancellous bone volume was measured in the secondary spongiosa of the distal metaphysis. Specifically, the region began 0.25 mm proximal to the distal femur growth plate and extended proximally for 2 mm. Cortical bone morphology was evaluated in a 1-mm-thick section at the femoral midshaft. For assessment of cancellous bone in the vertebral body, the entire vertebra was scanned using a 12-μm slice increment, requiring approximately 300 microCT slices per vertebra. Images were stored in 3D arrays with an isotropic voxel size of 12 μm. For this report, the trabecular bone in the vertebral body was analyzed, excluding the endplate regions (approximately 200–250 microCT slices). Precision data from our laboratory for repeat measurements for microCT-based morphometric measurements range from 1.3 to 5.7.
Bone histomorphometry
Undecalcified lumbar vertebrae and femurs embedded in methylmethacrylate were cut into 4-μm longitudinal sections, mounted on chrome-alum gelatin-coated slides, and stained with 2% toluidine blue (pH 3.7) in citric acid buffer for light microscopy. Similarly, 8-μm longitudinal sections were placed on chrome-alum gelatin-coated slides, left unstained, and mounted with Uvinert for fluorescent analysis. Sections were examined in a blinded fashion using the Osteomeasure software program (Osteometrics, Atlanta, GA) to measure light and fluorescent histomorphometric parameters. These parameters are as described by Parfitt et al. (15). All measurements were taken at a final magnification of x250 beginning at a constant distance from the growth plate of the femur. Histomorphometric analyses of lumbar vertebrae included trabecular bone only and excluded endosteal and cortical bone surfaces. Two full lumbar vertebral body sections and an entire femoral section per mouse were read.
Assays for markers of bone turnover
Serum osteocalcin was measured by a species-specific RIA as previously described (16). Urine samples were assayed for C-terminal telopeptides of type I collagen using the RatLaps ELISA kit (Osteometer BioTech A/S, Herlev, Denmark). Results were normalized to urine creatinine, which was determined using the end point colorimetric creatinine kit (Sigma) adapted to a 96-well microplate.
Assay for intact PTH
Serum samples from the vehicle-treated animals (n = 20/group) were assayed for intact PTH using a commercially available Rat PTH IRMA kit following the manufacturer’s recommended protocol (Immutopics Inc., San Clemente, CA).
Detection and quantification of osteoblast progenitors
Triplicate primary bone marrow cultures were made from the right tibia and femur of half the mice. The skin was removed from the right leg of each mouse; the leg was then placed in a dish containing cold 1x Dulbecco’s PBS. The femur and tibia were separated, the muscle removed, and the bones placed in a fresh 1x PBS with 10% FBS. The ends of each bone were snipped off using scissors, and again the bones were placed in fresh 1x PBS with 10% FBS. Marrow was flushed out with 1x PBS containing 10% FBS using a 20-gauge needle for the femurs and a 22-gauge needle for the tibias. Cells were collected by centrifugation at 250 x g for 5 min, and nucleated cells were counted using a hemocytometer. Cells were plated in 10-cm2 dishes, in triplicate, at a density of 2.5 x 106 nucleated cells/dish in a final volume of 10 ml of phenol red-free MEM containing 10% FBS, 50 μM ascorbic acid, 10 mM ?-glycerophosphate, 1% penicillin-streptomycin, and 1% L-glutamine. Cells were maintained for 21 or 28 d at 37 C in a humidified incubator supplied with a 5%CO2-95% air mixture. Half of the media was replaced every 5 d. Two of the three plates from each mouse were stained at 21 d for alkaline phosphatase and subsequently counted macroscopically for positive staining colonies by two independent readers. The remaining group of plates was stained at 28 d by the Von Kossa method to identify colonies containing calcium phosphate. For both the alkaline phosphatase-stained plates and the Von Kossa-stained plates, the between-observer correlation coefficients were quite high: 0.97 and 0.95, respectively.
Statistics
An intention-to-treat analysis was used in the assessment of change in BMD. Accordingly, the last bone density value for mice that died during the treatment period was carried forward. The PIXImus data were modeled using a random-effects regression (17). In this analysis, fixed effects are modeled for treatment, time, the treatment-by-time interaction, and the age-by-time-by-treatment interaction representing the average difference in treatment outcome means, the average rate of change in the outcome, the average difference between treatments in the rate of change in the outcome, and the average difference in treatment effect between the young and old, respectively. In addition, random effects were also included to allow the slope and intercept of the trajectory of outcome change over time to vary randomly among animals and cause a separate regression line to be fitted for each animal. Two-way ANOVA was used with post hoc Bonferroni-corrected tests to assess the effect of age and treatment on nonrepeated assessments, i.e. the osteocalcin and C-terminal telopeptides of type I collagen data. Data presented are the mean ± SEM, unless otherwise noted.
Results
Baseline characteristics of animals and tolerance of treatment
The 124 animals generally tolerated the study protocol well. The 3-month-old animals (young-adults, n = 61) gained an average of 0.5 g (28.7 ± 0.4 g 29.2 ± 0.4 g; mean initial final weight) during the course of the study, whereas the 18-month-old (aged, n = 63) animals lost an average of 1.2 g (32.0 ± 0.4 g 30.8 ± 0.4 g). Although the aged mice were heavier at the start of the study, total body BMD as determined by PIXImus was comparable in the two groups at the start of the study, whereas L-spine and femur BMD were lower in the aged group (see Table 1).
TABLE 1. BMD values as measured by PIXImus at baseline and d 29 in PTH- and vehicle-treated animals
Six young-adult mice and nine aged mice died during the study. Of the 3-month-old mice that died, three were in the vehicle group and three were in the PTH group. Of the 18-month-old mice that died, one was from the vehicle group and eight were from the PTH group. The 18-month-old mice became increasingly sensitive to the administration of anesthesia during the course of the study, which may, in addition to their age, explain the somewhat higher death rate in this group. As noted, an intention-to-treat analysis was used so that the final BMD value taken before the death of a mouse was carried forward throughout the remainder of the study. At the end of the study, there were the following numbers of animals in each of the four groups: 27 young-adult vehicle, 28 young-adult PTH, 30 aged vehicle, and 24 aged PTH. Results did not significantly differ when nonsurvivors were excluded so only the intention-to-treat analysis is presented.
Changes in bone density with treatment
As assessed by PIXImus, BMD increased significantly with PTH treatment in both young-adult and aged mice in each of the measured parameters: total body, spine, and femur. Vehicle-treated animals showed minor fluctuations in BMD over the 4-wk treatment period with no consistent changes noted. Absolute BMD values at baseline and after 4 wk of vehicle or PTH treatment are summarized in Table 1.
Changes in total body, femur, and spine BMD in response to PTH or vehicle treatment in the young-adult and aged animals are summarized in Fig. 1, A–C. Average trajectories of BMD change for the four treatment groups are shown. As can be seen in Fig. 1A, the increase in total body BMD was comparable in the young-adult and aged animals in response to PTH treatment. The total body bone density increased by 6.3 ± 0.9% in the young-adult animals and 5.6 ± 0.6% in the aged animals over the 4 wk of treatment; these increments were not statistically different from one another. There was no effect of vehicle treatment in the two groups.
FIG. 1. Rate of increase in total body (A), spine (B), and femur (C) BMD as measured by PIXImus in each of the four age/treatment groups over the course of the 4-wk experiment. *, The rate of increase in spine BMD in the aged animals in response to PTH was significantly greater than the rate of increase in the younger animals (P for age-treatment-time interaction = 0.01). The increase in total body and femur BMD with PTH treatment was similar between the young-adult and aged animals. There was no effect of vehicle (Veh) treatment in any of the skeletal envelopes or either age group.
Surprisingly, the response to PTH in the spine differed significantly between the young-adult and aged groups. As seen in Fig. 1B, the rate of increase in spinal bone density in the aged animals in response to PTH was significantly greater than the rate of increase in the younger animals (P for age-treatment-time interaction = 0.01). It should be emphasized that this analysis is independent of the starting bone density in each group. What is plotted is the change in bone mass over time with treatment. As the data clearly demonstrate by the divergent slopes of the two PTH-treated groups, the response to PTH was enhanced in the aged spine, compared with the spine in the young-adult animals. Specifically, the aged animals evidenced a 12.0 ± 1.4% increase over baseline spine BMD values vs. a 5.1 ± 1.4% increase in the young-adult animals. The absolute increment in the aged spine was 57 ± 5.8 g/cm2 x 10–4, whereas the absolute increment in the young-adult spine was 28 ± 7.4 g/cm2 x 10–4, (P < 0.01).
In contrast to the findings in the spine, the increment in femoral BMD in the young-adult animals was nearly twice as great as that observed in the aged animals (8.1 ± 1.5 vs. 4.2 ± 1.1%). This corresponds to an absolute increase of 61 ± 10.7 g/cm2 x 10–4 in the young-adult animals vs. 30 ± 7.5 g/cm2 x 10–4 in the aged group. However, there was an effect of age insofar as the bone density was higher at all treatment points in the young-adult animals, compared with the aged animals.
Because the spine is rich in trabecular bone, we sought to further analyze the response in this skeletal compartment by using microCT, which permits a quantitative analysis restricted to the trabecular envelope. Figure 2, A and B, show the results of microCT analyses of the trabecular compartment of the L5 vertebra and left trabecular femur, respectively. Eight animals were analyzed per group. Although not statistically significant by two-way ANOVA, there was a trend toward a greater increase in L5 trabecular bone volume (BV/TV) with PTH treatment in the aged animals than was observed in the young-adult animals. This is true whether the data are considered as an absolute increment with respect to vehicle controls 0.068 (aged) vs. 0.046 (young-adult) [0.237 ± 0.0113 minus 0.169 ± 0.009 (aged PTH minus aged vehicle-treated), compared with 0.281 ± 0.019 minus 0.235 ± 0.015 (young-adult PTH minus young-adult vehicle-treated)] or as a percent increase of PTH treatment groups over controls 40.2 vs. 19.6% (aged vs. young-adult).
FIG. 2. BV/TV of L5 vertebra (A) and trabecular femur (B) as measured by microCT. Trabecular bone in the L5 vertebral body was analyzed, excluding the endplate regions. In the femur, cancellous bone volume was measured in the distal metaphysis (n = 8 in all experimental groups). Data are shown as mean ± SEM. *, Mean value is significantly different from the mean value in the vehicle-treated group (P < 0.001 by two-way ANVOA).
MicroCT analysis of the femoral trabecular bone (Fig. 2B) mirrored the trend seen in the PIXImus data. Specifically, the younger animals responded more robustly to PTH treatment in this compartment. Young-adult animals demonstrated a 152.2% rise in femoral trabecular bone density (absolute BV/TV increase over vehicle controls of 0.032), whereas aged animals demonstrated an 83.1% increase (absolute increment of 0.014). Although significantly different from vehicle controls, the difference in the degree of response between aged and young-adult PTH-treated animals did not reach statistical significance.
Histomorphometric analyses
As might be anticipated and consistent with the known effect of aging on parameters of bone formation, histomorphometric analysis of L5 vertebrae demonstrated a trend toward fewer numbers of osteoblasts in the aged vehicle-treated animals than the vehicle-treated young-adult animals (Table 2A). However, after PTH-treatment the opposite was true. The number of osteoblasts per tissue area (NOb/TAR) increased 655% with PTH treatment (over vehicle controls) in the aged animals, compared with a 240% increase in the younger mice; absolute values at the end of treatment were higher in the aged animals: 800 ± 71 aged PTH vs. 561 ± 83 young-adult PTH (P < 0.02). The number of osteoclasts increased to a greater degree in the aged animals with PTH treatment than in the younger mice, consistent with a greater overall increase in bone turnover induced by PTH in this group (221 vs. 129%, respectively), although this did not reach statistical significance.
TABLE 2. NOb/TAR in lumbar vertebrae of vehicle- and PTH-treated animals
Table 2B shows the data from static histomorphometric analyses of the femur (n = 5/group). Again, aged animals seem to have fewer osteoblasts than do the young-adult animals with vehicle treatment alone. After PTH treatment the average number of osteoblasts increased in both age groups with a trend toward a greater increase in NOb/TAR in the femurs of the young-adult, compared with the aged animals (653 ± 77 vs. 290 ± 50). The number of osteoclasts also increased to a greater extent in the young-adult, compared with the aged animals (220 vs. 42%, respectively, P = NS).
TABLE 3. NOb/TAR in femurs of vehicle- and PTH-treated animals
Dynamic histomorphometry performed on five lumbar specimens from each experimental group confirmed that bone formation rates tended to be lower in the vehicle-treated aged animals than the vehicle-treated young-adult animals (Table 2C). Despite this there was a trend toward a greater increase in the bone formation rate (BFR/BV) with PTH treatment in the aged animals than seen in the young-adult animals. Aged mice show an absolute increment of 294 in the BFR to BV ratio, a 250% increase over vehicle treatment vs. an absolute increment of 194, a 95% increase over vehicle treatment in the younger mice.
TABLE 4. BFR/BV in lumbar vertebrae of vehicle- and PTH-treated animals
Bone turnover markers
Table 3 summarizes the data from assays for bone turnover. As expected, treatment with PTH increased osteocalcin levels in both age groups (n = 25–30 per experimental group). Young-adult PTH-treated animals had osteocalcin levels that were 131% higher than vehicle controls (P < 0.001); the corresponding increase was 156% in the aged PTH-treated animals (P < 0.001). C-terminal telopeptides of type I collagen (at n = 6 in each experimental group) also increased significantly in both age groups after PTH treatment; the increase was greater in the aged group. Levels of these bone turnover markers were similar in the vehicle-treated animals from both age groups.
TABLE 5. Markers of bone turnover: osteocalcin and C-terminal telopeptides of type I collagen (CTX) in vehicle-treated and PTH-treated animals
Intact PTH
Intact PTH levels did not differ significantly between the young and aged vehicle-treated animals. The mean serum PTH values were 25.6 ± 3.9 pg/ml in the young-adult animals and 26.7 ± 4.6 pg/ml in the aged animals.
Effect of PTH treatment on osteoblast progenitors
There were no significant differences in the number of marrow-derived alkaline phosphatase-positive colonies between vehicle- and PTH-treated animals in either age group. Alkaline phosphatase-positive colony numbers were significantly higher in cultures obtained from 18-month-old animals, compared with those from the 3-month-old animals. The mean number of colonies in each group was as follows: 91 ± 11 (young-adult vehicle) vs. 89 ± 12 (young-adult PTH), P = NS; and 124 ± 12 (aged vehicle) vs. 144 ± 19 (aged PTH), P = NS. Given that there was no effect of PTH treatment, pooling the data for the respective age groups yields the following average colony counts: 90 ± 8 (young adult) vs. 134 ± 11 (aged), P < 0.01. The numbers of mineralizing colonies as assessed by Von Kossa staining also did not vary significantly between marrow samples cultured from PTH-treated and vehicle-treated animals. Again in this assay, the mean colony counts in cultures of cells isolated from aged animals were more numerous than in those isolated from the young-adult mice: 28 ± 5 (aged) vs. 8 ± 1 (young-adult); P < 0.001. The mean number of colonies in each subgroup was as follows: 6 ± 2 (young-adult vehicle) vs. 9 ± 2 (young-adult PTH), P = NS; and 30 ± 7 (aged vehicle) vs. 26 ± 6 (aged PTH), P = NS.
Discussion
This study was designed to determine the effect of aging on the anabolic response to PTH. Using both PIXImus and microCT methodologies, we found that intermittent treatment with PTH increases bone mass in mice, as it does in rats and humans. However, there were surprising differences in the regional skeletal responses in young-adult and aged mice.
Whereas the magnitude of the increase in total body BMD was very similar in young and aged mice, the increment in spinal BMD was twice as great in the aged animals as in the young-adult animals when measured by PIXImus. The greater response in the aged spine, which is rich in trabecular bone, is surprising because trabecular bone tends to disappear with aging. The microCT data, although limited by sample size, are consistent with the conclusion that this response is indeed seen in the trabecular envelope: the spinal BV/TV ratio was 1.5-fold greater in the aged animals, compared with the young-adult animals.
The spinal BMD findings are consistent with the dramatic increase in spinal BMD seen in older patients treated with PTH: Neer et al. (8) reported a 14% increase in spine BMD in 70-yr-old postmenopausal women treated daily with 40 μg PTH for 21 months. To the extent that this model applies to patients with osteoporosis, our results suggest that the anabolic response to PTH in the spine is maintained or even enhanced with aging. In agreement with this, Marcus et al. (18) recently reported that women over 65 yr of age had a greater increase in vertebral BMD after treatment with PTH than did women under 65 yr. Of course, the direct extension of our findings in mice to the anabolic response to PTH in humans is limited by the fact that the murine skeleton differs from the human one in a number of ways, including that the epiphyses of long bones do not close in mice and that gravitational force is experienced differently because mice are quadripeds and humans bipedal.
In contrast to the findings in the spine, the trabecular response to PTH in the femur was nearly twice as great in the young-adult mice as it was in the aged animals. This divergent response in the long bones may reflect regional differences in PTH responsiveness at a cellular level. Consistent with this notion, there was a trend toward a greater increase in the number of osteoblasts per tissue area after PTH treatment in the femurs of the young-adult animals, compared with the aged animals.
Static and dynamic histomorphometric analyses in the lumbar spine suggest that without anabolic therapy the number of osteoblasts per tissue area and bone formation rates are depressed in the aged state. This is consistent with the diminution in basal osteoblast activity that occurs with advanced age. However, the capacity of the aged animals to respond to PTH at a cellular level is well preserved, especially in the spine. After PTH therapy, the aged mice show higher osteoblast counts and levels of bone formation in the spine than do their younger counterparts; the magnitude of the increase in the aged animals clearly outstrips that of the young-adult mice and provides the cellular basis for the greater increases in BMD measured by dual-energy x-ray absorptiometry and microCT.
The mechanism for this increased responsiveness of osteoblasts in the spine of the aged animals is not clear but may reflect a difference in the number of osteoblast progenitors. The number of osteoblast progenitor cells appears to be greater in the marrow cultures from aged animals than those from young-adult mice. It is highly unlikely that these results are due to methodological variation because the same tendency was noted in two essentially separate experiments. That is, the whole experimental protocol, including the cell culture, was staggered so that marrow from half of the mice from each of the four experimental groups were cultured on 1 d, and 3 d later marrow was isolated from the remaining half. Two independent observers counted each plate, making counting bias unlikely.
Support for the notion that osteoblast progenitor number increases with age can be found in two recent studies. Stenderup et al. (19) cultured mesenchymal stem cells from the marrow of young (aged 22–44 yr), old (66–74 yr), and osteoporotic individuals (58–83 yr). In this study there was a clear trend toward increasing colony number with increasing age with mean colony counts of 87 ± 12, 99 ± 19, and 129 ± 13 in the three groups, respectively. Pfeilschifter et al. (20) examined the mitogenic responsiveness to growth factors and hormones (including PTH) of osteoblast-like cells isolated from women ranging in age from 50 to 90 yr. Alkaline phosphatase levels measured in cells cultured from femoral head trabecular bone from patients age 61–70 yr were 14.8 ± 1.4 nmol/min·mg cell protein; these levels rose progressively with increasing age to 23.0 ± 6.4 in women age 71–80 yr and 35 ± 8.6 in women age 81–90 yr. A similar trend is seen in osteocalcin production by these cultures, 3.3 ± 0.7 3.4 ± 0.8 6.0 ± 2.1 ng/mg cell protein. These studies are consistent with our data showing that osteoblast progenitor number actually increases with age and that another process must account for the decrease in osteoblast activity.
Two theories have been proposed to account for the increased BMD seen with intermittent administration of PTH. It is widely held that PTH increases osteoblast progenitor recruitment and differentiation. In this scenario, PTH would increase the number of colony-forming unit osteoblast cells derived from stromal cells and therefore, the number of cells committed to becoming differentiated osteoblasts. Alternatively, Jilka et al. (21) suggested that the principal mechanism by which PTH exerts its anabolic effect in bone is by a prosurvival effect on osteoblasts. Normally mouse osteoblasts have a relatively short life span of approximately 200 h (21). Jilka et al. found that the programmed cell death of osteoblasts can be delayed by the action of PTH, thereby extending the life span and productivity of osteoblasts. The net result is increased bone deposition with time.
We found that the number of osteoblast progenitor cells, as assessed by both alkaline phosphatase-positive and mineralizing marrow-derived colonies was not increased with PTH administration. Because PTH had no stimulatory effect on osteoblast progenitors, these data suggest that PTH does not act by influencing osteoblast progenitor differentiation in our model. Assuming then that PTH acts primarily by inhibiting apoptosis and if apoptosis increases with aging, one might anticipate a more robust response to PTH in aged bone. Thus, the in vivo decline in baseline osteoblast activity with aging would not be due to a defect in differentiation but rather a shortened osteoblast survival time, compared with the young-adult animals. The increase in progenitor number observed in vitro may reflect an attempt to compensate for the increased apoptotic rate in vivo.
In the face of an expanded progenitor pool, one would anticipate a more robust response in the aged animals as a result of the prosurvival effect of PTH. The direct test of this formulation would be to measure apoptotic rates in bone tissue taken from the four groups of animals studied in this experiment. Unfortunately, whereas we were able to stain apoptotic cells in bone, we were not able to accurately identify and quantify those cells as osteoblasts despite numerous attempts using several methodologies.
Serum levels of intact PTH were similar in the young-adult and aged vehicle-treated mice. To the extent that PTH serves as an integrated estimate of baseline mineral homeostasis, it would seem that gross disorders of mineral metabolism such as vitamin D deficiency were not present in either group of animals. Consistent with this, baseline markers of bone turnover were normal in both groups. As occurs in humans, PTH treatment increased markers of bone formation and resorption in both groups of animals. However, in contrast to humans, it has been shown that creatinine is not a reliable marker of glomerular filtration rate in rodents (22), limiting the interpretation of urine markers in this study.
In conclusion, our data suggest that aged bone is fully capable of responding to the anabolic stimulus of intermittent PTH exposure. Aged bone responds as well as if not better than young bone in each parameter measured in the spine, whereas in the femoral trabecular compartment, the response was preserved. Whether osteoblast apoptotic rates change with aging and whether the prosurvival effect of PTH is influenced by the aging process remain to be determined. Exploring whether these phenomena contribute to the differences in regional response to the hormone will be of considerable interest.
Acknowledgments
We gratefully acknowledge the support of the Yale Core Center for Musculoskeletal Disorders (YCCMD), in particular Kimberly Wilson in the densitometry subunit of the YCCMD Physiology Core for her assistance, as well as the expert help of Christiane Coady in the Department of Orthopaedics. We also thank Julie Crawford and the laboratories of Robert Weinstein and Robert Jilka for providing the FBS as well as advice on cell culture.
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Address all correspondence and requests for reprints to: Eleanor Knopp, TAC S141, 300 Cedar Street, New Haven, Connecticut 06520-8020. E-mail: eleanor.knopp@yale.edu.
Abstract
Little is known about the modifying effects of age on the skeletal response to intermittent treatment with PTH. We therefore compared the response of 63 aged (18 month old) and 61 young-adult (3 month old) C57BL/6 mice to 4 wk of daily sc injections of either vehicle or h(1–34)PTH at a dose of 95 ng/g body weight. The increase in total body bone mineral density (BMD), compared with vehicle-treated animals, was similar in aged and young-adult mice (+5.6 vs. +6.3%). Aged animals demonstrated a greater increase in spinal BMD than their younger counterparts (+12.0 vs. +5.1%, P = 0.01; absolute increment: 57 x 10–4 vs. 28 x 10–4 g/cm2). Microcomputed tomography analyses in a subset of the vertebrae showed a trend toward higher L5 trabecular bone volume fraction in the PTH-treated aged animals (+40.2 vs. +19.6%). Vertebral histomorphometry demonstrated a greater PTH-induced increase in osteoblast number in aged vs. young-adult animals (694 vs. 396 cells/mm2). In contrast, in the femur the PTH-induced increase in BMD tended to be greater in the young-adult than the aged animals, although this did not reach statistical significance (8.1 vs. 4.2%). The numbers of osteoblast progenitors and mineralizing colonies in cultured marrow were unaffected by PTH treatment in either group. We conclude that aging differentially impacts the regional skeletal response to PTH such that the increase in BMD in the spine is augmented, whereas that in the femur is unaffected. Effects on osteoblast progenitor recruitment do not seem to be the basis for these changes.
Introduction
THE INCIDENCE OF osteoporotic fracture rises exponentially with aging such that 50–60% of women in their mid- to late 70s will have had an osteoporotic spine fracture (1, 2). In women, first-fracture incidence rates increase from 12/100,000 person-years in the less than 50-yr-old age group to 1200/100,000 person-years for women 80–85 yr old (3). An increased incidence of hip fracture also occurs in older men (4), and the death rate after hip fracture is even greater in men than in women (5).
Until recently, therapies for osteoporosis were limited to antiresorptive agents such as estrogens, the related selective estrogen receptor modulators, and bisphosphonates. There is a great need for anabolic therapies because, particularly in elderly patients with severe osteoporosis, antiresorptive therapy cannot return bone mass to normal.
PTH is the first effective anabolic agent for the treatment of osteoporosis. Animal studies have demonstrated convincingly that intermittent administration of PTH leads to an increase in bone mass (6, 7). The clinical efficacy of PTH in the treatment of osteoporosis was established in a large, prospective, double-blind, and randomized clinical trial by Neer et al. (8). These investigators showed that in women with osteoporosis, 18 months of once-daily administration of sc PTH dramatically increased bone mass in the spine and hip and reduced spinal fracture rates. Despite nearly 3 decades of experimentation, however, the cellular mechanisms by which PTH exerts this anabolic effect remain obscure.
In addition to uncertainty about the cellular mechanisms underlying the anabolic effect of PTH, the influence of aging on the skeletal response to this treatment is largely unstudied. Because PTH will primarily be used in older patients, determining how aging influences the response to this new therapeutic is of considerable clinical relevance. We therefore compared the anabolic response to intermittent PTH treatment in young-adult and aged mice to determine whether the anabolic effect of PTH is influenced by the aging process.
Materials and Methods
Materials
Human (1–34) PTH was from Bachem Bioscience Inc. (King of Prussia, PA). Metofane was from Medical Developments Australia (Victoria, Australia), ketamine and xylazine were from Henry Schein Inc. (Melville, NY). Phenol red-free MEM, ascorbic acid, ?-glycerophosphate, penicillin-streptomycin, L-glutamine, and tetracycline were from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was from Summit Biotechnology (Fort Collins, CO). Dulbecco’s phosphate-buffered saline was from Gibco BRL (Rockville, MD). Millonig’s fixative was from Surgipath Inc. (Richmond, IL). Toluidine blue powder was from Fisher Scientific Inc. (Pittsburgh, PA). Uvinert was from Biomedical Specialties, Inc. (Santa Monica, CA). The Vector Red alkaline phosphatase substrate kit I was from Vector Laboratories, Inc. (Burlingame, CA).
Experimental Animals
Three- and 18-month-old male C57BL/6 mice (n = 61 and n = 63, respectively) were obtained from the National Institute on Aging. Mice were fed standard chow ad libitum and were maintained and used according to National Institutes of Health guidelines. This study was approved by the Yale Animal Care and Use Committee.
Treatment protocol
Animals from each age group were randomly assigned to receive either single daily sc doses of vehicle (10 mM acetic acid containing 2% heat-inactivated mouse serum) or h(1–34) PTH at 95 ng/g body weight reconstituted in vehicle.
The 4-wk treatment schedule was as follows: 1) on d 1–29, the animals received single daily doses of either vehicle or PTH; animals were weighed every 7 d, and the dose of PTH was adjusted for any change in weight; 2) on d 1, 8, 15, 22, and 29, total body and regional bone mineral density (BMD) was determined by PIXImus (see below); 3) on d 22 and 27, mice were injected ip with tetracycline at 15 μg/g body weight dissolved in sterile 0.9% NaCl to label the mineralizing front in bone; and 4) on d 30, the day of killing, animals were anesthetized and serum samples collected from each mouse for determination of osteocalcin and intact PTH. Urine was also collected for determination of C-terminal telopeptides of type I collagen.
The left femur and lumbar spine were harvested, fixed in Millonig’s fixative, embedded in methylmethacrylate, and processed for static and dynamic histomorphometric analyses as previously reported (9, 10, 11). Eight left femurs and the corresponding L5 vertebra were reserved from each group, fixed in 70% EtOH, and subsequently scanned by microcomputed tomography (microCT).
The marrow was flushed from the right femur and tibia of half the mice and cultured to determine the numbers of osteoblast-progenitors.
Killing
Animals were deeply anesthetized with Metofane and killed by cervical dislocation after blood and urine were collected on d 30.
Bone densitometry
PIXImus.
Serial BMD determinations of total body, spine, and femur were obtained weekly (beginning on d 1) using a PIXImus densitometer (GE-Lunar Corp., Madison, WI) running software version 1.45. The coefficient of variation for total BMD as measured in our laboratory is 1.0 ± 0.2%. Scan acquisition time is 5 min/animal and is performed on anesthetized animals in the prone position. The total body window is defined as the whole-body image minus the calvarium, mandible, and teeth. The spine window is a rectangle spanning a length of the spine from L1 to the beginning of the sacrum. The femur window encompasses the entire right femur of each mouse. BMD values are expressed in grams per square centimeter x 10–4. Anesthesia for densitometric measurements was accomplished using a mixture of ketamine and xylazine administered ip at a dose of 50 and 5 μg/kg, respectively, in the 3-month-old animals and 30 μg/kg ketamine, 3 μg/kg xylazine in the 18-month-old animals. Mice were warmed under a heating lamp until they regained consciousness and were water restricted for 1 h post anesthesia.
MicroCT.
Femurs and vertebrae were evaluated using desktop microtomographic imaging systems equipped with a 10- and 5-μm focal spot microfocus x-ray tube, respectively (μCT20 and μCT40, Scanco Medical AG, Bassersdorf, Switzerland) (12). The entire femur was scanned using a 34-μm slice increment, requiring approximately 100–150 microCT slices per specimen. Images were reconstructed, filtered, and thresholded as previously described (13). The images were stored in three-dimensional (3D) arrays with an isotropic voxel size of 34 μm. Morphometric parameters were computed using a direct 3D approach that does not rely on assumptions about whether the underlying structure is either plate or rod like (14). Cancellous bone volume was measured in the secondary spongiosa of the distal metaphysis. Specifically, the region began 0.25 mm proximal to the distal femur growth plate and extended proximally for 2 mm. Cortical bone morphology was evaluated in a 1-mm-thick section at the femoral midshaft. For assessment of cancellous bone in the vertebral body, the entire vertebra was scanned using a 12-μm slice increment, requiring approximately 300 microCT slices per vertebra. Images were stored in 3D arrays with an isotropic voxel size of 12 μm. For this report, the trabecular bone in the vertebral body was analyzed, excluding the endplate regions (approximately 200–250 microCT slices). Precision data from our laboratory for repeat measurements for microCT-based morphometric measurements range from 1.3 to 5.7.
Bone histomorphometry
Undecalcified lumbar vertebrae and femurs embedded in methylmethacrylate were cut into 4-μm longitudinal sections, mounted on chrome-alum gelatin-coated slides, and stained with 2% toluidine blue (pH 3.7) in citric acid buffer for light microscopy. Similarly, 8-μm longitudinal sections were placed on chrome-alum gelatin-coated slides, left unstained, and mounted with Uvinert for fluorescent analysis. Sections were examined in a blinded fashion using the Osteomeasure software program (Osteometrics, Atlanta, GA) to measure light and fluorescent histomorphometric parameters. These parameters are as described by Parfitt et al. (15). All measurements were taken at a final magnification of x250 beginning at a constant distance from the growth plate of the femur. Histomorphometric analyses of lumbar vertebrae included trabecular bone only and excluded endosteal and cortical bone surfaces. Two full lumbar vertebral body sections and an entire femoral section per mouse were read.
Assays for markers of bone turnover
Serum osteocalcin was measured by a species-specific RIA as previously described (16). Urine samples were assayed for C-terminal telopeptides of type I collagen using the RatLaps ELISA kit (Osteometer BioTech A/S, Herlev, Denmark). Results were normalized to urine creatinine, which was determined using the end point colorimetric creatinine kit (Sigma) adapted to a 96-well microplate.
Assay for intact PTH
Serum samples from the vehicle-treated animals (n = 20/group) were assayed for intact PTH using a commercially available Rat PTH IRMA kit following the manufacturer’s recommended protocol (Immutopics Inc., San Clemente, CA).
Detection and quantification of osteoblast progenitors
Triplicate primary bone marrow cultures were made from the right tibia and femur of half the mice. The skin was removed from the right leg of each mouse; the leg was then placed in a dish containing cold 1x Dulbecco’s PBS. The femur and tibia were separated, the muscle removed, and the bones placed in a fresh 1x PBS with 10% FBS. The ends of each bone were snipped off using scissors, and again the bones were placed in fresh 1x PBS with 10% FBS. Marrow was flushed out with 1x PBS containing 10% FBS using a 20-gauge needle for the femurs and a 22-gauge needle for the tibias. Cells were collected by centrifugation at 250 x g for 5 min, and nucleated cells were counted using a hemocytometer. Cells were plated in 10-cm2 dishes, in triplicate, at a density of 2.5 x 106 nucleated cells/dish in a final volume of 10 ml of phenol red-free MEM containing 10% FBS, 50 μM ascorbic acid, 10 mM ?-glycerophosphate, 1% penicillin-streptomycin, and 1% L-glutamine. Cells were maintained for 21 or 28 d at 37 C in a humidified incubator supplied with a 5%CO2-95% air mixture. Half of the media was replaced every 5 d. Two of the three plates from each mouse were stained at 21 d for alkaline phosphatase and subsequently counted macroscopically for positive staining colonies by two independent readers. The remaining group of plates was stained at 28 d by the Von Kossa method to identify colonies containing calcium phosphate. For both the alkaline phosphatase-stained plates and the Von Kossa-stained plates, the between-observer correlation coefficients were quite high: 0.97 and 0.95, respectively.
Statistics
An intention-to-treat analysis was used in the assessment of change in BMD. Accordingly, the last bone density value for mice that died during the treatment period was carried forward. The PIXImus data were modeled using a random-effects regression (17). In this analysis, fixed effects are modeled for treatment, time, the treatment-by-time interaction, and the age-by-time-by-treatment interaction representing the average difference in treatment outcome means, the average rate of change in the outcome, the average difference between treatments in the rate of change in the outcome, and the average difference in treatment effect between the young and old, respectively. In addition, random effects were also included to allow the slope and intercept of the trajectory of outcome change over time to vary randomly among animals and cause a separate regression line to be fitted for each animal. Two-way ANOVA was used with post hoc Bonferroni-corrected tests to assess the effect of age and treatment on nonrepeated assessments, i.e. the osteocalcin and C-terminal telopeptides of type I collagen data. Data presented are the mean ± SEM, unless otherwise noted.
Results
Baseline characteristics of animals and tolerance of treatment
The 124 animals generally tolerated the study protocol well. The 3-month-old animals (young-adults, n = 61) gained an average of 0.5 g (28.7 ± 0.4 g 29.2 ± 0.4 g; mean initial final weight) during the course of the study, whereas the 18-month-old (aged, n = 63) animals lost an average of 1.2 g (32.0 ± 0.4 g 30.8 ± 0.4 g). Although the aged mice were heavier at the start of the study, total body BMD as determined by PIXImus was comparable in the two groups at the start of the study, whereas L-spine and femur BMD were lower in the aged group (see Table 1).
TABLE 1. BMD values as measured by PIXImus at baseline and d 29 in PTH- and vehicle-treated animals
Six young-adult mice and nine aged mice died during the study. Of the 3-month-old mice that died, three were in the vehicle group and three were in the PTH group. Of the 18-month-old mice that died, one was from the vehicle group and eight were from the PTH group. The 18-month-old mice became increasingly sensitive to the administration of anesthesia during the course of the study, which may, in addition to their age, explain the somewhat higher death rate in this group. As noted, an intention-to-treat analysis was used so that the final BMD value taken before the death of a mouse was carried forward throughout the remainder of the study. At the end of the study, there were the following numbers of animals in each of the four groups: 27 young-adult vehicle, 28 young-adult PTH, 30 aged vehicle, and 24 aged PTH. Results did not significantly differ when nonsurvivors were excluded so only the intention-to-treat analysis is presented.
Changes in bone density with treatment
As assessed by PIXImus, BMD increased significantly with PTH treatment in both young-adult and aged mice in each of the measured parameters: total body, spine, and femur. Vehicle-treated animals showed minor fluctuations in BMD over the 4-wk treatment period with no consistent changes noted. Absolute BMD values at baseline and after 4 wk of vehicle or PTH treatment are summarized in Table 1.
Changes in total body, femur, and spine BMD in response to PTH or vehicle treatment in the young-adult and aged animals are summarized in Fig. 1, A–C. Average trajectories of BMD change for the four treatment groups are shown. As can be seen in Fig. 1A, the increase in total body BMD was comparable in the young-adult and aged animals in response to PTH treatment. The total body bone density increased by 6.3 ± 0.9% in the young-adult animals and 5.6 ± 0.6% in the aged animals over the 4 wk of treatment; these increments were not statistically different from one another. There was no effect of vehicle treatment in the two groups.
FIG. 1. Rate of increase in total body (A), spine (B), and femur (C) BMD as measured by PIXImus in each of the four age/treatment groups over the course of the 4-wk experiment. *, The rate of increase in spine BMD in the aged animals in response to PTH was significantly greater than the rate of increase in the younger animals (P for age-treatment-time interaction = 0.01). The increase in total body and femur BMD with PTH treatment was similar between the young-adult and aged animals. There was no effect of vehicle (Veh) treatment in any of the skeletal envelopes or either age group.
Surprisingly, the response to PTH in the spine differed significantly between the young-adult and aged groups. As seen in Fig. 1B, the rate of increase in spinal bone density in the aged animals in response to PTH was significantly greater than the rate of increase in the younger animals (P for age-treatment-time interaction = 0.01). It should be emphasized that this analysis is independent of the starting bone density in each group. What is plotted is the change in bone mass over time with treatment. As the data clearly demonstrate by the divergent slopes of the two PTH-treated groups, the response to PTH was enhanced in the aged spine, compared with the spine in the young-adult animals. Specifically, the aged animals evidenced a 12.0 ± 1.4% increase over baseline spine BMD values vs. a 5.1 ± 1.4% increase in the young-adult animals. The absolute increment in the aged spine was 57 ± 5.8 g/cm2 x 10–4, whereas the absolute increment in the young-adult spine was 28 ± 7.4 g/cm2 x 10–4, (P < 0.01).
In contrast to the findings in the spine, the increment in femoral BMD in the young-adult animals was nearly twice as great as that observed in the aged animals (8.1 ± 1.5 vs. 4.2 ± 1.1%). This corresponds to an absolute increase of 61 ± 10.7 g/cm2 x 10–4 in the young-adult animals vs. 30 ± 7.5 g/cm2 x 10–4 in the aged group. However, there was an effect of age insofar as the bone density was higher at all treatment points in the young-adult animals, compared with the aged animals.
Because the spine is rich in trabecular bone, we sought to further analyze the response in this skeletal compartment by using microCT, which permits a quantitative analysis restricted to the trabecular envelope. Figure 2, A and B, show the results of microCT analyses of the trabecular compartment of the L5 vertebra and left trabecular femur, respectively. Eight animals were analyzed per group. Although not statistically significant by two-way ANOVA, there was a trend toward a greater increase in L5 trabecular bone volume (BV/TV) with PTH treatment in the aged animals than was observed in the young-adult animals. This is true whether the data are considered as an absolute increment with respect to vehicle controls 0.068 (aged) vs. 0.046 (young-adult) [0.237 ± 0.0113 minus 0.169 ± 0.009 (aged PTH minus aged vehicle-treated), compared with 0.281 ± 0.019 minus 0.235 ± 0.015 (young-adult PTH minus young-adult vehicle-treated)] or as a percent increase of PTH treatment groups over controls 40.2 vs. 19.6% (aged vs. young-adult).
FIG. 2. BV/TV of L5 vertebra (A) and trabecular femur (B) as measured by microCT. Trabecular bone in the L5 vertebral body was analyzed, excluding the endplate regions. In the femur, cancellous bone volume was measured in the distal metaphysis (n = 8 in all experimental groups). Data are shown as mean ± SEM. *, Mean value is significantly different from the mean value in the vehicle-treated group (P < 0.001 by two-way ANVOA).
MicroCT analysis of the femoral trabecular bone (Fig. 2B) mirrored the trend seen in the PIXImus data. Specifically, the younger animals responded more robustly to PTH treatment in this compartment. Young-adult animals demonstrated a 152.2% rise in femoral trabecular bone density (absolute BV/TV increase over vehicle controls of 0.032), whereas aged animals demonstrated an 83.1% increase (absolute increment of 0.014). Although significantly different from vehicle controls, the difference in the degree of response between aged and young-adult PTH-treated animals did not reach statistical significance.
Histomorphometric analyses
As might be anticipated and consistent with the known effect of aging on parameters of bone formation, histomorphometric analysis of L5 vertebrae demonstrated a trend toward fewer numbers of osteoblasts in the aged vehicle-treated animals than the vehicle-treated young-adult animals (Table 2A). However, after PTH-treatment the opposite was true. The number of osteoblasts per tissue area (NOb/TAR) increased 655% with PTH treatment (over vehicle controls) in the aged animals, compared with a 240% increase in the younger mice; absolute values at the end of treatment were higher in the aged animals: 800 ± 71 aged PTH vs. 561 ± 83 young-adult PTH (P < 0.02). The number of osteoclasts increased to a greater degree in the aged animals with PTH treatment than in the younger mice, consistent with a greater overall increase in bone turnover induced by PTH in this group (221 vs. 129%, respectively), although this did not reach statistical significance.
TABLE 2. NOb/TAR in lumbar vertebrae of vehicle- and PTH-treated animals
Table 2B shows the data from static histomorphometric analyses of the femur (n = 5/group). Again, aged animals seem to have fewer osteoblasts than do the young-adult animals with vehicle treatment alone. After PTH treatment the average number of osteoblasts increased in both age groups with a trend toward a greater increase in NOb/TAR in the femurs of the young-adult, compared with the aged animals (653 ± 77 vs. 290 ± 50). The number of osteoclasts also increased to a greater extent in the young-adult, compared with the aged animals (220 vs. 42%, respectively, P = NS).
TABLE 3. NOb/TAR in femurs of vehicle- and PTH-treated animals
Dynamic histomorphometry performed on five lumbar specimens from each experimental group confirmed that bone formation rates tended to be lower in the vehicle-treated aged animals than the vehicle-treated young-adult animals (Table 2C). Despite this there was a trend toward a greater increase in the bone formation rate (BFR/BV) with PTH treatment in the aged animals than seen in the young-adult animals. Aged mice show an absolute increment of 294 in the BFR to BV ratio, a 250% increase over vehicle treatment vs. an absolute increment of 194, a 95% increase over vehicle treatment in the younger mice.
TABLE 4. BFR/BV in lumbar vertebrae of vehicle- and PTH-treated animals
Bone turnover markers
Table 3 summarizes the data from assays for bone turnover. As expected, treatment with PTH increased osteocalcin levels in both age groups (n = 25–30 per experimental group). Young-adult PTH-treated animals had osteocalcin levels that were 131% higher than vehicle controls (P < 0.001); the corresponding increase was 156% in the aged PTH-treated animals (P < 0.001). C-terminal telopeptides of type I collagen (at n = 6 in each experimental group) also increased significantly in both age groups after PTH treatment; the increase was greater in the aged group. Levels of these bone turnover markers were similar in the vehicle-treated animals from both age groups.
TABLE 5. Markers of bone turnover: osteocalcin and C-terminal telopeptides of type I collagen (CTX) in vehicle-treated and PTH-treated animals
Intact PTH
Intact PTH levels did not differ significantly between the young and aged vehicle-treated animals. The mean serum PTH values were 25.6 ± 3.9 pg/ml in the young-adult animals and 26.7 ± 4.6 pg/ml in the aged animals.
Effect of PTH treatment on osteoblast progenitors
There were no significant differences in the number of marrow-derived alkaline phosphatase-positive colonies between vehicle- and PTH-treated animals in either age group. Alkaline phosphatase-positive colony numbers were significantly higher in cultures obtained from 18-month-old animals, compared with those from the 3-month-old animals. The mean number of colonies in each group was as follows: 91 ± 11 (young-adult vehicle) vs. 89 ± 12 (young-adult PTH), P = NS; and 124 ± 12 (aged vehicle) vs. 144 ± 19 (aged PTH), P = NS. Given that there was no effect of PTH treatment, pooling the data for the respective age groups yields the following average colony counts: 90 ± 8 (young adult) vs. 134 ± 11 (aged), P < 0.01. The numbers of mineralizing colonies as assessed by Von Kossa staining also did not vary significantly between marrow samples cultured from PTH-treated and vehicle-treated animals. Again in this assay, the mean colony counts in cultures of cells isolated from aged animals were more numerous than in those isolated from the young-adult mice: 28 ± 5 (aged) vs. 8 ± 1 (young-adult); P < 0.001. The mean number of colonies in each subgroup was as follows: 6 ± 2 (young-adult vehicle) vs. 9 ± 2 (young-adult PTH), P = NS; and 30 ± 7 (aged vehicle) vs. 26 ± 6 (aged PTH), P = NS.
Discussion
This study was designed to determine the effect of aging on the anabolic response to PTH. Using both PIXImus and microCT methodologies, we found that intermittent treatment with PTH increases bone mass in mice, as it does in rats and humans. However, there were surprising differences in the regional skeletal responses in young-adult and aged mice.
Whereas the magnitude of the increase in total body BMD was very similar in young and aged mice, the increment in spinal BMD was twice as great in the aged animals as in the young-adult animals when measured by PIXImus. The greater response in the aged spine, which is rich in trabecular bone, is surprising because trabecular bone tends to disappear with aging. The microCT data, although limited by sample size, are consistent with the conclusion that this response is indeed seen in the trabecular envelope: the spinal BV/TV ratio was 1.5-fold greater in the aged animals, compared with the young-adult animals.
The spinal BMD findings are consistent with the dramatic increase in spinal BMD seen in older patients treated with PTH: Neer et al. (8) reported a 14% increase in spine BMD in 70-yr-old postmenopausal women treated daily with 40 μg PTH for 21 months. To the extent that this model applies to patients with osteoporosis, our results suggest that the anabolic response to PTH in the spine is maintained or even enhanced with aging. In agreement with this, Marcus et al. (18) recently reported that women over 65 yr of age had a greater increase in vertebral BMD after treatment with PTH than did women under 65 yr. Of course, the direct extension of our findings in mice to the anabolic response to PTH in humans is limited by the fact that the murine skeleton differs from the human one in a number of ways, including that the epiphyses of long bones do not close in mice and that gravitational force is experienced differently because mice are quadripeds and humans bipedal.
In contrast to the findings in the spine, the trabecular response to PTH in the femur was nearly twice as great in the young-adult mice as it was in the aged animals. This divergent response in the long bones may reflect regional differences in PTH responsiveness at a cellular level. Consistent with this notion, there was a trend toward a greater increase in the number of osteoblasts per tissue area after PTH treatment in the femurs of the young-adult animals, compared with the aged animals.
Static and dynamic histomorphometric analyses in the lumbar spine suggest that without anabolic therapy the number of osteoblasts per tissue area and bone formation rates are depressed in the aged state. This is consistent with the diminution in basal osteoblast activity that occurs with advanced age. However, the capacity of the aged animals to respond to PTH at a cellular level is well preserved, especially in the spine. After PTH therapy, the aged mice show higher osteoblast counts and levels of bone formation in the spine than do their younger counterparts; the magnitude of the increase in the aged animals clearly outstrips that of the young-adult mice and provides the cellular basis for the greater increases in BMD measured by dual-energy x-ray absorptiometry and microCT.
The mechanism for this increased responsiveness of osteoblasts in the spine of the aged animals is not clear but may reflect a difference in the number of osteoblast progenitors. The number of osteoblast progenitor cells appears to be greater in the marrow cultures from aged animals than those from young-adult mice. It is highly unlikely that these results are due to methodological variation because the same tendency was noted in two essentially separate experiments. That is, the whole experimental protocol, including the cell culture, was staggered so that marrow from half of the mice from each of the four experimental groups were cultured on 1 d, and 3 d later marrow was isolated from the remaining half. Two independent observers counted each plate, making counting bias unlikely.
Support for the notion that osteoblast progenitor number increases with age can be found in two recent studies. Stenderup et al. (19) cultured mesenchymal stem cells from the marrow of young (aged 22–44 yr), old (66–74 yr), and osteoporotic individuals (58–83 yr). In this study there was a clear trend toward increasing colony number with increasing age with mean colony counts of 87 ± 12, 99 ± 19, and 129 ± 13 in the three groups, respectively. Pfeilschifter et al. (20) examined the mitogenic responsiveness to growth factors and hormones (including PTH) of osteoblast-like cells isolated from women ranging in age from 50 to 90 yr. Alkaline phosphatase levels measured in cells cultured from femoral head trabecular bone from patients age 61–70 yr were 14.8 ± 1.4 nmol/min·mg cell protein; these levels rose progressively with increasing age to 23.0 ± 6.4 in women age 71–80 yr and 35 ± 8.6 in women age 81–90 yr. A similar trend is seen in osteocalcin production by these cultures, 3.3 ± 0.7 3.4 ± 0.8 6.0 ± 2.1 ng/mg cell protein. These studies are consistent with our data showing that osteoblast progenitor number actually increases with age and that another process must account for the decrease in osteoblast activity.
Two theories have been proposed to account for the increased BMD seen with intermittent administration of PTH. It is widely held that PTH increases osteoblast progenitor recruitment and differentiation. In this scenario, PTH would increase the number of colony-forming unit osteoblast cells derived from stromal cells and therefore, the number of cells committed to becoming differentiated osteoblasts. Alternatively, Jilka et al. (21) suggested that the principal mechanism by which PTH exerts its anabolic effect in bone is by a prosurvival effect on osteoblasts. Normally mouse osteoblasts have a relatively short life span of approximately 200 h (21). Jilka et al. found that the programmed cell death of osteoblasts can be delayed by the action of PTH, thereby extending the life span and productivity of osteoblasts. The net result is increased bone deposition with time.
We found that the number of osteoblast progenitor cells, as assessed by both alkaline phosphatase-positive and mineralizing marrow-derived colonies was not increased with PTH administration. Because PTH had no stimulatory effect on osteoblast progenitors, these data suggest that PTH does not act by influencing osteoblast progenitor differentiation in our model. Assuming then that PTH acts primarily by inhibiting apoptosis and if apoptosis increases with aging, one might anticipate a more robust response to PTH in aged bone. Thus, the in vivo decline in baseline osteoblast activity with aging would not be due to a defect in differentiation but rather a shortened osteoblast survival time, compared with the young-adult animals. The increase in progenitor number observed in vitro may reflect an attempt to compensate for the increased apoptotic rate in vivo.
In the face of an expanded progenitor pool, one would anticipate a more robust response in the aged animals as a result of the prosurvival effect of PTH. The direct test of this formulation would be to measure apoptotic rates in bone tissue taken from the four groups of animals studied in this experiment. Unfortunately, whereas we were able to stain apoptotic cells in bone, we were not able to accurately identify and quantify those cells as osteoblasts despite numerous attempts using several methodologies.
Serum levels of intact PTH were similar in the young-adult and aged vehicle-treated mice. To the extent that PTH serves as an integrated estimate of baseline mineral homeostasis, it would seem that gross disorders of mineral metabolism such as vitamin D deficiency were not present in either group of animals. Consistent with this, baseline markers of bone turnover were normal in both groups. As occurs in humans, PTH treatment increased markers of bone formation and resorption in both groups of animals. However, in contrast to humans, it has been shown that creatinine is not a reliable marker of glomerular filtration rate in rodents (22), limiting the interpretation of urine markers in this study.
In conclusion, our data suggest that aged bone is fully capable of responding to the anabolic stimulus of intermittent PTH exposure. Aged bone responds as well as if not better than young bone in each parameter measured in the spine, whereas in the femoral trabecular compartment, the response was preserved. Whether osteoblast apoptotic rates change with aging and whether the prosurvival effect of PTH is influenced by the aging process remain to be determined. Exploring whether these phenomena contribute to the differences in regional response to the hormone will be of considerable interest.
Acknowledgments
We gratefully acknowledge the support of the Yale Core Center for Musculoskeletal Disorders (YCCMD), in particular Kimberly Wilson in the densitometry subunit of the YCCMD Physiology Core for her assistance, as well as the expert help of Christiane Coady in the Department of Orthopaedics. We also thank Julie Crawford and the laboratories of Robert Weinstein and Robert Jilka for providing the FBS as well as advice on cell culture.
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