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Keywords:

  • teriparatide;
  • transiliac bone biopsy;
  • bone formation;
  • modeling;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Transiliac bone biopsies were obtained from 55 women treated with teriparatide or placebo for 12–24 months. We report direct evidence that modeling bone formation at quiescent surfaces was present only in teriparatide-treated patients and bone formation at remodeling sites was higher with teriparatide than placebo.

Introduction: Recombinant teriparatide [human PTH(1-34)], a bone formation agent for the treatment of osteoporosis when given once daily subcutaneously, increases biochemical markers of bone turnover and activation frequency in histomorphometry studies.

Materials and Methods: We studied the mechanisms underlying this bone-forming action of teriparatide at the basic multicellular unit by the appearance of cement lines, a method used to directly classify surfaces as modeling or remodeling osteons, and by the immunolocalization of IGF-I and IGF-II. Transiliac bone biopsies were obtained from 55 postmenopausal women treated with teriparatide 20 or 40 μg or placebo for 12–24 months (median, 19.8 months) in the Fracture Prevention Trial.

Results: A dose-dependent relationship was observed in modeling and mixed remodeling/modeling trabecular hemiosteons. Trabecular and endosteal hemiosteon mean wall thicknesses were significantly higher in both teriparatide groups than in placebo. There was a dose-dependent relationship in IGF-II immunoreactive staining at all bone envelopes studied. The greater local IGF-II presence after treatment with teriparatide may play a key role in stimulating bone formation.

Conclusions: Direct evidence is presented that 12–24 months of teriparatide treatment induced modeling bone formation at quiescent surfaces and resulted in greater bone formation at remodeling sites, relative to placebo.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Osteoporotic fractures are a biomechanical consequence of a decrease in bone quality; that is, the components of bone strength, such as structural integrity or microarchitectural deterioration that occurs with increasing age that are not measured by bone densitometry.(1–3) Therapeutic drugs for the treatment of osteoporosis increase bone strength, as shown by a decrease in the incidence of fragility fractures, through their effects on bone turnover.(4) Biochemical markers of bone formation have more rapid and larger increases than resorption markers within the first 3 months of teriparatide therapy, suggesting an early imbalance of bone turnover in favor of formation that remain positive over the first 6–12 months of treatment.(5–7) Markers of bone turnover may reflect underlying changes in bone histology and provide indirect evidence of a dissociation of bone formation and resorption.(8–10)

The predominant mechanism of bone turnover in adults is bone remodeling, the cellular basis for age-related bone loss when the processes of bone resorption and formation become unbalanced. Frost hypothesized that modeling may occur throughout life, although modeling activity is less frequent after skeletal maturity.(11,12) Antiresorptive agents reduce bone turnover, decreasing the size of the remodeling space and the number of basic multicellular units (BMUs), resulting in a preservation of bone microarchitecture.(13,14)

Recombinant teriparatide {human PTH(1-34) [hPTH (1-34)]}, currently the only bone-forming osteoporosis drug available for clinical use,(15,16) increases bone turnover with a greater stimulation of formation than resorption. Teriparatide increases the size of the remodeling space and the number of BMUs, resulting in a positive bone balance and improved macro- and microarchitecture.(10,17–19) Changes in bone microarchitecture with teriparatide treatment, associated with an increase in bone strength, include increased trabecular bone volume, bone width, connectivity density, improved morphology with a shift toward more plate-like structure, and increased cortical thickness.(17,18,20–23)

Mechanisms for improvements in microarchitecture by bone-forming agents may be through modeling or remodeling.(24) In short-duration treatment (1–6 months) studies with bone-forming agents in animals(24–26) and humans,(20,22,23,27) bone modeling plays an important role. Reeve et al.(20) suggested that 6 months treatment with teriparatide caused dissociation between formation and resorption resulting in a positive bone balance in patients with osteoporosis. Hodsman and Steer(22) hypothesized that new bone formation occurred on previously quiescent surfaces after 28 days of teriparatide treatment in women with osteoporosis. This hypothesis was supported by indirect evidence from increases in activation frequency and double tetracycline-labeled surfaces and is consistent with the well-known effects of teriparatide in increasing markers of bone formation.(15,28)

The appearance of cement lines and collagen fiber orientation are used to directly classify surfaces as modeling (i.e., new bone formation occurring on a quiescent surface without preceding osteoclast bone resorption).(29) At modeling sites, cement lines are smooth, with parallel collagen fiber orientation, absent of prior osteoclastic activity.(29) In the remodeling process, the cement line is scalloped, indicative of the osteoclastic activity that precedes bone formation in a BMU.(29)

Dempster et al.(23) reported that 28 days of teriparatide treatment directly stimulated bone formation without prior resorption on both trabecular and endocortical bone surfaces in postmenopausal women with osteoporosis, based on the presence of scalloped or smooth cement lines. They found modeling-based formation on trabecular and endocortical surfaces in teriparatide-treated patients, but not in biopsy samples from controls.(23) Using the technique of quadruple labeling to obtain longitudinal results from a single biopsy after 7 weeks of teriparatide treatment, Lindsay et al.,(27) in their abstract, suggested that bone formation occurred either on previously quiescent surfaces or by osteoblast spillover from remodeling sites onto previously quiescent surfaces.

IGFs play an important role in the regulation of skeletal remodeling by upregulating osteoblast and osteoclast activities associated with acquisition of BMD.(30–32) Growth factors may also mediate the effects of PTH in stimulating new bone formation.(33) Although the age-related decline in BMD and bone strength is well known, less recognized as a risk factor for osteoporotic fracture is the decline in serum IGF concentrations with age.(34,35)

The aim of this study was to investigate the working mechanism of the bone-forming action of teriparatide by analyzing the occurrence and dimensions of modeling and remodeling osteons and IGF-I or IGF-II expression in transiliac bone biopsies obtained from patients after either 12 months of treatment or at the end of treatment (range, 19–24 months) in the Fracture Prevention Trial.(15)

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Subjects

Transiliac bone biopsies were obtained at study baseline from 102 women who volunteered for the biopsy substudy at selected study sites. The volunteers were randomly assigned to provide a second biopsy from the contralateral side after either 12 months of treatment or at the end of the study (median, 22.0 months; range, 19–24 months). Approximately one third of the women were randomized to have the second biopsy after 12 months of treatment and two thirds after 24 months of treatment. More patients were assigned to the longer-duration time to improve the likelihood of acquiring the desired number of paired biopsies adequate for evaluation. The volunteers were a subset of the 1637 postmenopausal women with osteoporosis enrolled in the randomized, multicenter, double-blind, placebo-controlled Fracture Prevention Trial.(15) To enroll in the Fracture Prevention Study, a patient's laboratory value for 25-hydroxyvitamin D had to be between the lower limit of normal and three times the upper limit of normal at baseline. Normal was defined by the central laboratory reference range. All of the patients in this substudy had 25-hydroxy and 1,25 dihydroxyvitamin D levels above the lower limit of normal at baseline.

Participants were treated with once-daily self-injections of either placebo or teriparatide 20 or 40 μg. Details of this study have been published.(15) Baseline BMD measurements of the lumbar spine and femoral neck were obtained by DXA as previously described.(15) Biochemical markers of bone formation (serum bone-specific alkaline phosphatase [BALP], serum procollagen type I C-terminal propeptide [PICP]) and resorption (urinary N-terminal telopeptide cross-linked collagen type I [NTX]) were measured at baseline on fasting samples.

No evidence of modeling was found in the baseline sections obtained before treatment; therefore, the analysis and comparisons were restricted to samples obtained after treatment. Of the 61 follow-up biopsies obtained, 52 samples were of sufficient quality for histomorphometrical analyses for modeling/remodeling activity. Nine biopsy specimens were excluded from histomorphometry because they were crushed or broken (six of nine) or did not have pair-matched sections for both cement line and polarized light quantification (three of nine). Immunohistochemistry was performed on a total of 39 samples from among 55 unbroken biopsy specimens; 13 samples randomly selected from the intact specimens for each experimental group (placebo and teriparatide 20 and 40 groups). Thirty-six biopsies were used for both histomorphometric and immunohistochemistry analyses. The technician who performed the analysis was blinded to the treatment group assignment.

Biopsies used for the histomorphometry modeling and remodeling analyses were distributed as follows—placebo: 12 months (n = 7), study endpoint (n = 13); teriparatide 20 μg: 12 month (n = 6), endpoint (n = 13); teriparatide 40 μg: 12 month (n = 5), endpoint (n = 8). Immunocytochemistry analyses were performed on samples obtained at 12 months (placebo, n = 5; teriparatide, 20 μg, n = 4; teriparatide 40 μg, n = 6) and study endpoint (placebo, n = 8; teriparatide 20 μg, n = 9; teriparatide 40 μg, n = 7).

The Institutional Review Board for Research Involving Human Subjects at each participating center approved the protocols for the Fracture Prevention Trial and the biopsy substudy. Volunteers gave written informed consent before participation in the treatment and biopsy studies.

Treatment and biopsy schedule

Participants self-administered a once-daily injection of placebo for 2 weeks and were randomly assigned to receive placebo or 20 μg or 40 μg of teriparatide daily for 3 years. The Fracture Prevention Trial(15) was terminated early because of a finding of osteosarcoma in rats(36,37); therefore, the treatment duration was shorter than originally planned. Participants received daily supplements of 1000 mg of elemental calcium and 400–1200 IU of vitamin D3 for the duration of the trial.

The transiliac bone biopsies were carried out using a modified Bordier needle or similar large bore (6–8 mm) trephine system after in vivo double tetracycline labeling (tetracycline HCL 250 mg, four times a day) given orally in a 3:12:3-day sequence, as described by Jiang et al.(17) The methods for biopsy specimen evaluations have been published previously.(38,39) All measured and derived variables were expressed according to standard nomenclature recommended by the American Society of Bone and Mineral Research nomenclature committee.(39)

Histomorphometry

Biopsies were dehydrated in graded ethanol and embedded in methylmethacrylate at −20°C. From each block, several 8-μm-thick undecalcified sections were cut with 100-μm spacing and prepared for different staining methods or left unstained. Of each section pair, the unstained section was used for tetracycline fluorochrome and polarized light analyses. The toluidine blue–stained sections were used to view cement lines, osteoblasts, and wall thickness, whereas Goldner's trichrome–stained sections were used to measure tissue area, bone area, and bone surface under transmitted light. All biopsy measurements were made by laboratory personnel blinded to individual treatment assignments.

Active bone-forming, tetracycline-labeled osteons on trabecular and endocortical surfaces were studied using polarized light for collagen orientation and transmitted light for cement line stains (Fig. 1). Modeling and remodeling analyses were performed on the trabecular structure hemiosteons of the entire trabecular tissue area. Only those trabecular structural units containing either tetracycline label or osteoblast surfaces as evidence for active bone formation at the time of biopsy were analyzed. The osteons were classified according to the presence of smooth (modeling) or scalloped (remodeling) cement lines. A remodeling site was defined by the presence of a scalloped cement line with interrupted collagen fibers indicating that formation followed previous bone resorption or osteoclastic activity.(29) In bone-forming modeling sites, the cement lines are smooth, showing evidence that no osteoclastic bone resorption preceded bone formation. The modeling unit was further assessed under polarized light to ensure that collagen fibers in the whole unit followed the same orientation as those of the adjacent bone tissue. Bone-forming units with the presence of very short scalloped (less than one sixth of the total cement line length) and long smooth cement lines were classified as prolonged remodeling sites or mixed remodeling-modeling hemiosteons (Fig. 1). To avoid the cutting orientation effect, all the modeling and mixed remodeling-modeling units were identified in longitudinal cutting orientation and further confirmed by polarized light used to examine the collagen fiber patterns in the whole unit. The percentage of each type of the hemiosteons from the total numbers of formation osteons was calculated.

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Figure Figure 1. Remodeling hemiosteons. (A) Unstained fluorescence light image and (B) transmitted light for toluidine blue staining image specific for cement lines. Newly formed lamellar bone is indicated by the red arrows. The presence of double label is evident here. Reversal or scalloped cement line in the adjacent bone or interrupted collagen fiber orientation from adjacent bone tissue underlying the forming site outlined by black arrows, indicating previous bone resorption surface, evidenced by scalloped curved fine lines (62-year-old woman treated with teriparatide 40 μg). Mixed remodeling–modeling hemiosteons. (C) Unstained fluorescence light image and (D) transmitted light for toluidine blue staining image specific for cement lines. Newly formed lamellar bone is indicated by the red arrows. Very short scalloped cement line and a long smooth cement line in the adjacent bone underlying the forming site outlined by black arrows, indicating previous bone resorption surface, evidenced by scalloped curved fine lines. Arrows indicate that formation was initiated on the resorption site and then extended beyond the previous resorption surface. Smooth cement lines are indicated by the yellow arrows. The inset in C shows the presence of lamellar bone (73-year-old woman treated with teriparatide 40 μg). Modeling hemiosteons. (E) Unstained fluorescence light image and (F) polarized light. Newly formed lamellar bone is indicated by the red arrows. Smooth cement lines and parallel collagen fiber orientation as the adjacent bone tissue, without interruption, indicating absence of previous bone resorption (yellow arrows; 73-year-old woman treated with 40 μg teriparatide).

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Bone area, bone surface, tissue area, and trabecular and endocortical mean wall thickness were analyzed semiautomatically by a direct tracing method using a Digitizing Image Analysis System (DIAS) consisting of an epifluorescent microscope and a digitizing pad (Summagraphic, Fairfield, CT, USA) coupled to an Apple Macintosh SE computer, using histomorphometry software (KSS Scientific Consultants, Magna, UT, USA).(40) Wall thickness (μm) was measured on toluidine blue–stained sections as the average distance between cement lines and quiescent trabecular surfaces, corrected for obliquity. Wall thickness was measured only on tetracycline-labeled osteons covered with lining cells, indicating finished (quiescent) bone remodeling units formed during the treatment period. All measurements were performed at ×100 magnification on the entire trabecular and endocortical envelopes. The parameters were normalized to bone, surface, and tissue reference. On average, 16–30 formation osteons were measured per section (range, 6–68). Six to 16 such finished remodeling osteons were used for identification of wall thickness in each biopsy sample.

Three types of hemiosteons were observed (Fig. 1): remodeling hemiosteons (Figs. 1A and 1B) identified by scalloped reversal lines that interrupt surrounding collagen fibers, indicating prior resorption; mixed remodeling-modeling hemiosteons (Figs. 1C and 1D) identified by a mixture of very short scalloped cement lines and long smooth cement lines; and modeling hemiosteons (Figs. 1E and 1F) identified by smooth cement lines with parallel collagen fibers without interruption of adjacent bone tissue.

Immunocytochemistry

To maximize immunoreactivity, bone biopsies were embedded in methylmethacrylate at −20°C.(41) Unstained 7-μm sections were deplasticized in two successive 20-minute washes of xylene, followed by two washes in ethanol and a brief wash in PBS containing 0.1% Tween 20 (PBS-T), pH 7.4. Sections were treated with 1% acetic acid for 10 minutes to expose antigenic sites. Before the addition of the primary antibody, nonspecific tissue binding was blocked by incubating the tissue section in 10% BSA for 30 minutes at room temperature. The collagen antibodies used in this study were supplied by Dr Juha Risteli, from the University of Oulu, Finland, and tested by him to have <1% cross-reactivity with other collagen species. Subsequently, sections were incubated overnight with primary antibody against human IGF-I, II, bone morphometric protein (BMP)-2, TGFβ1, and type I collagen. The IGF-I and IGF-II monoclonal antibodies were obtained from BIOSOURCE (Camarillo, CA, USA); TGFβ1, rabbit polyclonal, and BMP-2, goat, polyclonal, affinity-purified antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After rinsing, the sections were incubated with peroxidase labeled secondary biotin-avidin antibodies for 1 h and subsequently developed in ethylcarbazole. After washing in buffer, the chromagen, diaminobenzidine was applied for 5 minutes followed by a counterstain with Mayer's hematoxylin.

Negative controls included substituting the primary antisera with preimmune sera from the same species, and omitting the primary antibody. All controls revealed the expected negative results. The total length of IGF-II–positive stained cement lines was first measured in trabecular and cortical bones using the same image system described above. The length was normalized to bone area and surface perimeter for comparison. The entire trabecular bone in the sample for IGF staining was quantified.

Statistical analysis

Demographics and relevant biopsy and markers of bone turnover variables at baseline were summarized by group mean and SD. To compare the baseline characteristics among the three treatment groups, each pairwise comparison was performed. Two-sample t-tests were used for age, years postmenopausal, body mass index, lumbar spine, and femoral neck T score. The Wilcoxon rank sum test was used for the bone markers: NTX, BALP, and PICP. Separate Fisher's exact tests were performed for the number of patients with zero, one, and at least two prevalent fractures.

The postbaseline data were similar between the two time-points (12 months and study endpoint) for each of the treatment groups; therefore, the postbaseline biopsies were pooled for analyses. For each parameter, separate two-sample tests were performed to compare teriparatide 20 μg versus placebo and to compare teriparatide 40 μg versus placebo. The Wilcoxon rank sum test was used for each comparison; p values for each comparison were obtained using Monte Carlo simulation because of the small sample size. α level of p < 0.05 was the criterion for statistical significance with no adjustment for multiplicity. All analyses were performed using SAS version 8 statistical software (SAS Institute, Cary, NC, USA). Data are presented as mean ± SD in the text and tables and as mean ± SE in the figures.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Demographics

There were no statistically significant differences in baseline characteristics among groups (Table 1). Baseline demographics for the biopsy substudy did not differ from those of the Fracture Prevention Trial population.(15)

Table Table 1.. Baseline Characteristics of Patients*
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Histomorphometry

There was no woven bone, osteomalacia, or other histological abnormality observed in the postbaseline biopsies for any of the patients in this substudy. Three types of hemiosteons were observed in biopsy samples after teriparatide treatment: remodeling hemiosteons (Figs. 1A and 1B); mixed remodeling-modeling hemiosteons (Figs. 1C and 1D); and modeling hemiosteons (Figs. 1E and 1F).

No modeling hemiosteons or mixed remodeling-modeling hemiosteons were found on trabecular surfaces in placebo-treated patients. Compared with placebo, a significantly greater percentage of active, tetracycline labeled modeling hemiosteons on trabecular surfaces was seen in women treated with teriparatide 40 μg (3.8%, p < 0.001), but not in women treated with 20 μg (0.4%, Fig. 2). There were also significantly greater percentages of mixed remodeling/modeling osteons on trabecular surfaces for both teriparatide groups (teriparatide 20 μg, 2.4%; teriparatide 40 μg, 3.9%, p < 0.05 versus placebo for both teriparatide groups; Fig. 2). There were 6–68 such formation osteons in each biopsy samples: 0–6 modeling; 6–65 remodeling; 0–5 mixed remodeling-modeling.

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Figure Figure 2. Percent of modeling, mixed remodeling-modeling, and remodeling hemiosteons in trabeculae during teriparatide treatment (mean ± SE). No modeling or mixed remodeling-modeling hemiosteons were observed in the placebo group. Bars to the right of the dotted line (remodeling) use the right axis. ap < 0.001 vs. placebo.

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Mean wall thickness of trabecular hemiosteons was significantly greater than placebo (p < 0.05) in teriparatide 20 and 40 μg (Fig. 3). The difference was more pronounced on the endocortical surfaces in both teriparatide groups compared with placebo (Fig. 3).

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Figure Figure 3. Mean wall thickness in cancellous and endocortical hemiosteons (mean ± SE). ap < 0.05 vs. placebo.

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Immunocytochemistry

Immunoreactivity for IGF-I, IGF-II, BMP-2, TGFβ1, and collagen I was detectable in bone matrix with preferential localization in cement lines, reversal lines of bone, and the outer lining cell layer of bone remodeling compartments. Among them, collagen I showed a high signal and qualitative analysis, suggesting that teriparatide treatment markedly upregulated the expression of collagen I on bone matrix, but the staining was too poorly delineated to enable quantification (Fig. 4). Only IGF-II showed sufficiently high levels and had clear borders to warrant quantification. IGF-II staining was evaluated in periosteal, cortical, endocortical, and trabecular bone (Table 2). The highest expression of IGF-II was found in cement lines (Fig. 5). When enumerating the length of cement lines with positive IGF-II immunoreactivity, we found that both teriparatide treatment groups upregulated IGF-II expression compared with placebo treatment (Table 2). The in situ expression of IGF-II, per unit cortical area, cortical osteon number, trabecular surface, and trabecular area, was dose-dependently greater with teriparatide treatment than with placebo treatment (Table 2). IGF-II expression was also significantly greater on periosteal surfaces for both teriparatide groups compared with placebo (Table 2).

Table Table 2.. Effect of Teriparatide Treatment on In Situ Expression of IGF-II in Trabecular and Cortical Bone and on the Periosteal and Endocortical Surfaces
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Figure Figure 4. Effect of teriparatide treatment on in situ staining of type I collagen in trabeculae detected by immunohistochemistry. Transiliac bone biopsy sections were counterstained with Mayer's hematoxylin after incubated with primary antibody against human collagen I. Greater intensity of staining in teriparatide-treated women than in placebo is apparent. (Women treated with placebo [62 years old] or teriparatide 20 μg [66 years old] or 40 μg [58 years old], respectively.)

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Figure Figure 5. Microimages showing the effect of teriparatide treatment on immunocytochemical staining of IGF-II in trabeculae and periosteum. Transiliac bone biopsy sections were counterstained with Mayer's hematoxylin after incubated with primary antibody against human IGF-II. Teriparatide treatment resulted in a dose-dependent increase in immunocytochemical staining of IGF-II on bone matrix. Black arrows indicate staining on major cement lines. (Women treated with placebo [69 years old] or teriparatide 20 μg [70 years old] or 40 μg [73 years old], respectively.)

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Our findings of modeling and mixed remodeling-modeling hemiosteons in postmenopausal women with osteoporosis provide direct evidence that 12–24 months of recombinant teriparatide treatment induced new bone formation at quiescent surfaces. That findings of modeling were evident after 12–24 months of teriparatide treatment highlights a distinction of this research from the work of Hodsman and Steer,(22) Dempster et al,(23) and Lindsay et al.,(27) who reported that an early effect determined after just a month of teriparatide treatment was increased bone modeling. Hodsman and Steer(22) hypothesized that the increases in double tetracycline-labeled surfaces were compatible with new bone formation occurring on previously quiescent surfaces after 28 days of teriparatide therapy in postmenopausal women. Dempster et al.(23) reported that modeling-based formation activity on trabecular surfaces was found after 28 days of teriparatide treatment. Lindsay et al.(27)observed that 70% of new bone formation was remodeling based, and 30% was modeling based. The greater proportion of remodeling-based formation is likely a function of the longer duration of teriparatide treatment in our study.

Bone turnover can be measured by activation frequency, which is the probability that a new cycle of remodeling will be initiated,(39) that is, an index to show how many new BMUs (remodeling packet) are initiated in a given time. Activation frequency is increased during the early months of treatment, but by 18 months, activation frequency in teriparatide-treated patients was comparable with that of untreated patients.(10) With continued treatment, although activation frequency may have returned to untreated levels, a positive bone balance is maintained, as indicated by higher mean wall thickness.(42) BMD continues to increase throughout the teriparatide treatment,(15) and we suggest that this gain in BMD occurs through a combination of modeling and remodeling.

Biochemical markers of bone turnover provide supporting evidence that, during the first month of teriparatide therapy, new bone formation may be modeling-based because only formation markers show responses during this early phase.(9,15,28,43) After 1 month, resorption markers steadily increase, although they remain proportionately lower than formation markers, suggesting that bone formation gradually becomes more remodeling-based.

The classification of cement lines as smooth or scalloped was used to determine whether modeling- or remodeling-based new bone formation occurred, as described in other studies.(12,27,29) As discussed recently by Lindsay et al,(27) although smooth cement lines are associated with modeling-based new bone formation, this assumption is unlikely to be confirmed by research findings. Lindsay et al. postulated that a lack of scalloped cement lines may result from the resorption of lamellar bone by osteoclasts. Another explanation is that smooth cement lines occur when osteoclasts resorb along collagen fibers. Nevertheless, we and others found that new bone formation osteons with smooth cement lines are uncommon under normal circumstances.

We also report the presence of new structures in recombinant teriparatide-treated bone, mixed remodeling-modeling osteons, consisting of very short scalloped and long smooth cement lines. We did not observe such a modeling-dominant structure in placebo-treated participants. We interpret this structure as an indication that formation is initiated at active remodeling sites on the resorption surface and then extended beyond the previous resorption surface during the bone formation period with teriparatide treatment. The appearance of this structure with modeling areas surrounding the resorption lacunae suggests that teriparatide causes extension or “overfilling” of trabecular hemiosteons beyond the boundaries of the original remodeling site onto a quiescent surface, thus greatly enhancing the positive remodeling balance at each remodeling unit.(18,44)

In agreement with Dempster et al.(23) and Lindsay et al.,(27) who found an absence of modeling in their control patients, we likewise found no typical modeling hemiosteons in our placebo-treated patients. In contrast, Kobayashi et al.(12) reported modeling activity in the iliac crest samples collected during the total hip arthroplasty, although the extent of minimodeling was very low (1% of bone volume and 2% of bone surface). The finding of significantly more modeling and mixed remodeling-modeling osteons in teriparatide-treated than in placebo-treated patients supports the hypothesis that teriparatide increases and induces bone formation at Frost's intermediary organizational level (modeling and remodeling).(45)

Wall thickness is a measure of the final bone balance after a remodeling cycle has finished. Increased mean wall thickness, indicative of a positive bone balance,(46) was higher in the teriparatide-treated groups than in placebo, as shown previously.(18,27,42,44) We confirmed greater mean wall thickness of endosteal osteons relative to placebo as previously reported,(23,42) and also observed a greater mean wall thickness of cancellous hemiosteons relative to placebo that may indicate stimulation of osteoblastic synthesis of new bone matrix, especially type I collagen. The apparent increase in collagen I levels associated with teriparatide treatment in our study supports this hypothesis.

Although IGF-I has been primarily established in rodents as the dominant mediator, our results suggest that IGF-II is more abundant than IGF-I in human bone, in agreement with Mohan and Baylink,(47) who reported that the growth factor showing the highest concentrations in human bone matrix is IGF-II. Recently, Pepene et al.(48) reported that some antiresorptive treatments (estrogen therapy and calcitonin) had no direct influence on serum or bone matrix growth factor levels. Boonen et al.(35) found that IGF-II concentrations were 40% lower in elderly women with hip fractures compared with healthy, age-matched controls. Although periosteal bone formation occurs at a negligible level during adulthood, particularly in women, our results suggest that teriparatide may reactivate periosteal bone formation. From a biomechanical view, an increase in periosteal apposition contributes to improved bone strength and improved resistance to fracture.(49,50)

IGF-II is a known stimulator of osteoblastic activity and has been positively associated with osteoblast surface.(51,52) Our study showed for the first time that IGF-II immunostaining was twice as high on periosteal surfaces in teriparatide-treated patients than in placebo-treated patients, which may play a key role in the creation of a positive bone balance and improved trabecular and cortical bone architecture previously shown in these biopsies. Our inability to show significant changes in other growth factors such as BMP-2, and TGFβ1 may signify either an earlier response or lesser involvement by these factors in the bone-forming response.

There is a limitation in characterizing bone structure units by microscopy in histological sections. Although we call the hemiosteons with smooth cement lines “modeling” units, they might conceivably be part of a remodeling site, where we have sectioned through the more peripheral parts. Thus, in this study, all the modeling and mixed remodeling-modeling units were identified in longitudinal cutting orientation and further confirmed by polarized light to examine the collagen fiber patterns in the whole unit. 3D construction of these hemiosteons would help to confirm whether they are true modeling or remodeling-modeling units.

In summary, these results provide the first direct evidence that teriparatide treatment of 12- to 24-month duration induces modeling bone formation at quiescent surfaces and that bone formation at remodeling sites in teriparatide-treated groups is greater than in the placebo group. These mechanisms may contribute to the improvement of trabecular and cortical architecture shown after teriparatide treatment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was funded by Eli Lilly and Company. The authors thank Juha Risteli, MD, PhD, at the University of Oulu, Finland, who supplied the collagen antibodies and checked their specificity, Webster Jee, PhD, at the University of Utah for critical review of the manuscript, and Melinda Rance for preparation of the graphs.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
  • 1
    Parfitt AM, Mathews CH, Villanueva AR, Kleerekoper M, Frame B, Rao DS 1983 Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss. J Clin Invest 72: 13961409.
  • 2
    NIH Consensus Development Panel 2001 Osteoporosis prevention, diagnosis, and therapy. JAMA 285: 785795.
  • 3
    Recker RR, Barger-Lux MJ 2004 The elusive concept of bone quality. Curr Osteoporos Rep 2: 97100.
  • 4
    Riggs BL, Parfitt AM 2005 Drugs used to treat osteoporosis: The critical need for a uniform nomenclature based on their action on bone remodeling. J Bone Miner Res 20: 177184.
  • 5
    Reeve J, Bradbeer JN, Arlot M, Davies UM, Green JR, Hampton L, Edouard C, Hesp R, Hulme P, Ashby JP 1991 hPTH 1-34 treatment of osteoporosis with added hormone replacement therapy: Biochemical, kinetic and histological responses. Osteoporos Int 1: 162170.
  • 6
    Hodsman AB, Fraher LJ, Ostbye T, Adachi JD, Steer BM 1993 An evaluation of several biochemical markers for bone formation and resorption in a protocol utilizing cyclical parathyroid hormone and calcitonin therapy for osteoporosis. J Clin Invest 91: 11381148.
  • 7
    Hodsman AB, Fraher LJ, Watson PH, Ostbye T, Stitt LW, Adachi JD, Taves DH, Drost D 1997 A randomized controlled trial to compare the efficacy of cyclical parathyroid hormone versus cyclical parathyroid hormone and sequential calcitonin to improve bone mass in postmenopausal women with osteoporosis. J Clin Endocrinol Metab 82: 620628.
  • 8
    Eastell R, Delmas PD, Hodgson SF, Eriksen EF, Mann KG, Riggs BL 1988 Bone formation rate in older normal women: Concurrent assessment with bone histomorphometry, calcium kinetics, and biochemical markers. J Clin Endocrinol Metab 67: 741748.
  • 9
    Dobnig H, Sipos A, Jiang Y, Fahrleitner-Pammer A, Ste-Marie LG, Gallagher JC, Pavo I, Wang J, Eriksen EF 2005 Early changes in biochemical markers of bone formation correlate with improvements in bone structure during teriparatide therapy. J Clin Endocrinol Metab 90: 39703977.
  • 10
    Arlot M, Meunier PJ, Boivin G, Haddock L, Tamayo J, Correa-Rotter R, Jasqui S, Donley DW, Dalsky GP, Martin JS, Eriksen EF 2005 Differential effects of teriparatide and alendronate on bone remodeling in postmenopausal women assessed by histomorphometric parameters. J Bone Miner Res 20: 12441253.
  • 11
    Frost HM 1990 Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolff's law: The bone modeling problem. Anat Rec 226: 403413.
  • 12
    Kobayashi S, Takahashi HE, Ito A, Saito N, Nawata M, Horiuchi H, Ohta H, Ito A, Iorio R, Yamamoto N, Takaoka K 2003 Trabecular minimodeling in human iliac bone. Bone 32: 163169.
  • 13
    Chavassieux PM, Arlot ME, Reda C, Wei L, Yates AJ, Meunier PJ 1997 Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis. J Clin Invest 100: 14751480.
  • 14
    Eriksen EF, Melsen F, Sod E, Barton I, Chines A 2002 Effects of long-term risedronate on bone quality and bone turnover in women with postmenopausal osteoporosis. Bone 31: 620625.
  • 15
    Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB, Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH 2001 Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 344: 14341441.
  • 16
    Orwoll E, Scheele WH, Paul S, Adami S, Syversen U, Diez-Perez A, Kaufman JM, Clancy AD, Gaich G 2003 The effect of teriparatide [human parathyroid hormone (1-34)] therapy on bone mineral density in men with osteoporosis. J Bone Miner Res 18: 917.
  • 17
    Jiang Y, Zhao JJ, Mitlak BH, Wang O, Genant HK, Eriksen EF 2003 Teriparatide [recombinant human parathyroid hormone (1-34)] improves both cortical and cancellous bone structure. J Bone Miner Res 18: 19321941.
  • 18
    Dempster DW, Cosman F, Kurland ES, Zhou H, Nieves J, Woelfert L, Shane E, Plavetic K, Muller R, Bilezikian J, Lindsay R 2001 Effects of daily treatment with parathyroid hormone on bone microarchitecture and turnover in patients with osteoporosis: A paired biopsy study. J Bone Miner Res 16: 18461853.
  • 19
    Seeman E, Delmas PD 2001 Reconstructing the skeleton with intermittent parathyroid hormone. Trends Endocrinol Metab 12: 281283.
  • 20
    Reeve J, Meunier PJ, Parsons JA, Bernat M, Bijvoet OL, Courpron P, Edouard C, Klenerman L, Neer RM, Renier JC, Slovik D, Vismans FJ, Potts JT Jr 1980 Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: A multicentre trial. BMJ 280: 13401344.
  • 21
    Bradbeer JN, Arlot ME, Meunier PJ, Reeve J 1992 Treatment of osteoporosis with parathyroid peptide (hPTH 1-34) and oestrogen: Increase in volumetric density of iliac cancellous bone may depend on reduced trabecular spacing as well as increased thickness of packets of newly formed bone. Clin Endocrinol (Oxf) 37: 282289.
  • 22
    Hodsman AB, Steer BM 1993 Early histomorphometric changes in response to parathyroid hormone therapy in osteoporosis: Evidence for de novo bone formation on quiescent cancellous surfaces. Bone 14: 523527.
  • 23
    Dempster DW, Zhou H, Cosman F, Nieves J, Adachi JD, Fraher LJ, Watson PH, Lindsay R, Hodsman AB 2001 PTH treatment directly stimulates bone formation in cancellous and cortical bone in humans. J Bone Miner Res 16: S1; S179.
  • 24
    Yao W, Jee WS, Zhou H, Lu J, Cui L, Setterberg R, Liang T, Ma Y 1999 Anabolic effect of prostaglandin E2 on cortical bone of aged male rats comes mainly from modeling-dependent bone gain. Bone 25: 697702.
  • 25
    Dobnig H, Turner RT 1995 Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 136: 36323638.
  • 26
    Hock JM, Hummert JR, Boyce R, Fonseca J, Raisz LG 1989 Resorption is not essential for the stimulation of bone growth by hPTH-(1-34) in rats in vivo. J Bone Miner Res 4: 449458.
  • 27
    Lindsay R, Cosman F, Zhou H, Bostrom MP, Shen VW, Cruz JD, Nieves JW, Dempster DW 2006 A novel tetracycline labeling scheduling for longitudinal evaluation of the short-term effects of anabolic therapy with a single iliac crest bone biopsy: Early actions of teriparatide. J Bone Miner Res 21: 366373.
  • 28
    Lindsay R, Nieves J, Formica C, Henneman E, Woelfert L, Shen V, Dempster D, Cosman F 1997 Randomised controlled study of effect of parathyroid hormone on vertebral-bone mass and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 350: 550555.
  • 29
    Erben RG 1996 Trabecular and endocortical bone surfaces in the rat: Modeling or remodeling? Anat Rec 246: 3946.
  • 30
    Canalis E 1995 Growth hormone, skeletal growth factors and osteoporosis. Endocr Pract 1: 3943.
  • 31
    Yakar S, Rosen CJ, Beamer WG, Ackert-Bicknell CL, Wu Y, Liu JL, Ooi GT, Setser J, Frystyk J, Boisclair YR, LeRoith D 2002 Circulating levels of IGF-1 directly regulate bone growth and density. J Clin Invest 110: 771781.
  • 32
    Yakar S, Rosen CJ 2003 From mouse to man: Redefining the role of insulin-like growth factor-I in the acquisition of bone mass. Exp Biol Med (Maywood) 228: 245252.
  • 33
    Canalis E, McCarthy TL, Centrella M 1990 Differential effects of continuous and transient treatment with parathyroid hormone related peptide (PTHrp) on bone collagen synthesis. Endocrinology 126: 18061812.
  • 34
    Kasukawa Y, Miyakoshi N, Mohan S 2004 The anabolic effects of GH/IGF system on bone. Curr Pharm Des 10: 25772592.
  • 35
    Boonen S, Mohan S, Dequeker J, Aerssens J, Vanderschueren D, Verbeke G, Broos P, Bouillon R, Baylink DJ 1999 Down-regulation of the serum stimulatory components of the insulin-like growth factor (IGF) system (IGF-I, IGF-II, IGF binding protein [BP]-3, and IGFBP-5) in age-related (type II) femoral neck osteoporosis. J Bone Miner Res 14: 21502158.
  • 36
    Vahle JL, Sato M, Long GG, Young JK, Francis PC, Engelhardt JA, Westmore M, Ma L, Nold JB 2002 Skeletal changes in rats given daily subcutaneous injections of recombinant human parathyroid hormone(1-34) for 2 years and relevance to human safety. Toxicol Pathol 30: 312321.
  • 37
    Vahle JL, Long GG, Sandusky G, Westmore M, Ma YL, Sato M 2004 Bone neoplasms in F344 rats given teriparatide [rhPTH(1-34)] are dependent on duration of treatment and dose. Toxicol Pathol 32: 426438.
  • 38
    Eriksen EF 1986 Normal and pathological remodeling of human trabecular bone: Three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev 7: 379408.
  • 39
    Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2: 595610.
  • 40
    Ma Y, Jee WS, Chen Y, Gasser J, Ke HZ, Li XJ, Kimmel DB 1995 Partial maintenance of extra cancellous bone mass by antiresorptive agents after discontinuation of human parathyroid hormone (1-38) in right hindlimb immobilized rats. J Bone Miner Res 10: 17261734.
  • 41
    Reinhold G Erben 1997 Embedding of bone samples in methymethacrylate: An improved method suitable for bone histomorphometry, histochemistry, and immunohistochemistry. J Histochem Cytochem 45: 307313.
  • 42
    Hodsman AB, Kisiel M, Adachi JD, Fraher LJ, Watson PH 2000 Histomorphometric evidence for increased bone turnover without change in cortical thickness or porosity after 2 years of cyclical hPTH(1-34) therapy in women with severe osteoporosis. Bone 27: 311318.
  • 43
    McClung MR, San Martin J, Miller PD, Civitelli R, Bandeira F, Omizo M, Donley DW, Dalsky GP, Eriksen EF 2005 Teriparatide and alendronate increase bone mass by opposite effects on bone remodeling. Arch Intern Med 165: 17621768.
  • 44
    Misof BM, Roschger P, Cosman F, Kurland ES, Tesch W, Messmer P, Dempster DW, Nieves J, Shane E, Fratzl P, Klaushofer K, Bilezikian J, Lindsay R 2003 Effects of intermittent parathyroid hormone administration on bone mineralization density in iliac crest biopsies from patients with osteoporosis: A paired study before and after treatment. J Clin Endocrinol Metab 88: 11501156.
  • 45
    Frost HM 1986 Intermediary Organization of the Skeleton. CRC Press, Boca Raton, FL, USA.
  • 46
    Eriksen EF, Mosekilde L, Melsen F 1986 Kinetics of trabecular bone resorption and formation in hypothyroidism: Evidence for a positive balance per remodeling cycle. Bone 7: 101108.
  • 47
    Mohan S, Baylink DJ 1991 Evidence that the inhibition of TE85 human bone cell proliferation by agents which stimulate cAMP production may in part be mediated by changes in the IGF-II regulatory system. Growth Regul 1: 110118.
  • 48
    Pepene CE, Seck T, Diel I, Minne HW, Ziegler R, Pfeilschifter J 2004 Influence of fluor salts, hormone replacement therapy and calcitonin on the concentration of insulin-like growth factor (IGF)-I, IGF-II and transforming growth factor-beta 1 in human iliac crest bone matrix from patients with primary osteoporosis. Eur J Endocrinol 150: 8191.
  • 49
    Turner CH 2002 Biomechanics of bone: Determinants of skeletal fragility and bone quality. Osteoporos Int 13: 97104.
  • 50
    Martin RB 2002 Size, structure and gender: Lessons about fracture risk. J Musculoskelet Neuronal Interact 2: 209211.
  • 51
    Seck T, Scheppach B, Scharla S, Diel I, Blum WF, Bismar H, Schmid G, Krempien B, Ziegler R, Pfeilschifter J 1998 Concentration of insulin-like growth factor (IGF)-I and -II in iliac crest bone matrix from pre- and postmenopausal women: Relationship to age, menopause, bone turnover, bone volume, and circulating IGFs. J Clin Endocrinol Metab 83: 23312337.
  • 52
    Ishibe M, Ishibashi T, Kaneda K, Koda T, Rosier RN, Puzas JE 1998 Stimulation of bone formation in vivo by insulin-like growth factor-II in rats. Calcif Tissue Int 63: 3638.