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Keywords:

  • parathyroid hormone;
  • osteoporosis;
  • bone microarchitecture;
  • human

Abstract

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

We examined paired iliac crest bone biopsy specimens from patients with osteoporosis before and after treatment with daily injections of 400 U of recombinant, human parathyroid hormone 1–34 [PTH(1–34)]. Two groups of patients were studied. The first group was comprised of 8 men with an average age 49 years. They were treated with PTH for 18 months. The second group was comprised of 8 postmenopausal women with an average age 54 years. They were treated with PTH for 36 months. The women had been and were maintained on hormone replacement therapy for the duration of PTH treatment. Patients were supplemented to obtain an average daily intake of 1500 mg of elemental calcium and 100 IU of vitamin D. The biopsy specimens were subjected to routine histomorphometric analysis and microcomputed tomography (CT). Cancellous bone area was maintained in both groups. Cortical width was maintained in men and significantly increased in women. There was no increase in cortical porosity. There was a significant increase in the width of bone packets on the inner aspect of the cortex in both men and women. This was accompanied by a significant decrease in eroded perimeter on this surface in both groups. Micro-CT confirmed the foregoing changes and, in addition, revealed an increase in connectivity density, a three dimensional (3D) measure of trabecular connectivity in the majority of patients. These findings indicate that daily PTH treatment exerts anabolic action on cortical bone in patients with osteoporosis and also can improve cancellous bone microarchitecture. The results provide a structural basis for the recent demonstration that PTH treatment reduces the incidence of osteoporosis-related fractures.


INTRODUCTION

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

THE ANABOLIC action of parathyroid hormone (PTH) on the mammalian skeleton was first appreciated in the late 1920s and early 1930s.(1, 2) However, it took until the mid-70s before the first clinical trials were conducted in both men and women with osteoporosis.3-5) The 1990s saw further clinical trials of PTH alone and in combination with various antiresorptive treatments such as estrogen and calcitonin.6-9) Combined PTH and estrogen also was used to treat glucocorticoid-induced osteoporosis.(10) Recently, a small clinical trial(11) and a large multicenter trial(12) have confirmed the antifracture efficacy originally indicated in 1997.(7)

Few clinical studies have used bone biopsy as an endpoint; indeed, there have only been three studies in which paired biopsy specimens before and after PTH treatment have been analyzed.(4, 13, 14) These investigations yielded variable results in terms of the cancellous bone response. The first two studies(4, 13) showed a significant increase in cancellous bone volume but the latest study(14) did not. Furthermore, cortical bone variables were assessed only in one of the studies(14) and there have been no investigations in which the three-dimensional (3D) structure of bone has been assessed.

Therefore, the aim of this study was to perform both 2D and 3D morphometric analysis of cancellous and cortical bone structure in paired biopsy specimens from patients with osteoporosis treated with PTH.

MATERIALS AND METHODS

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

This study was approved by the Institutional Review Boards of Helen Hayes Hospital and Columbia-Presbyterian Medical Center. All subjects gave informed consent. Paired biopsy specimens before and after treatment were obtained from two groups of patients. The first was a group of 8 postmenopausal women with osteoporosis (average age, 54 years) who were treated with 400 U/day of PTH(1-34) for 36 months. These women had been and were maintained on hormone replacement therapy for the duration of the study. The characteristics of these patients have been provided elsewhere.(7) The second group consisted of 8 eugonadal men with idiopathic osteoporosis (average age, 49 years) who were treated with the same regimen of PTH for 18 months. The characteristics of these patients have been described previously in full.(15, 16) All patients were supplemented, if necessary, to achieve a daily intake of 1500 mg of elemental calcium and 400 IU of vitamin D. Bone mineral density (BMD) of the lumbar spine was measured by dual-energy X-ray absorptiometry as previously described.(7, 15)

Before biopsy all patients were double-labeled with tetracycline in a 3:12:3 day sequence. For the first biopsy, tetracycline hydrochloride was used to produce the first label, and demethylchlortetracycline was used to produce the second label. The labels produced by each tetracycline differ in color under UV light. For the second biopsy, the order of tetracyclines was reversed to aid distinction between the two sets of labels. The biopsy specimens were obtained, processed, and subjected to histomorphometric analysis as previously described in detail from our laboratory.17-21) All variables were expressed according to the recommendations of the ASBMR nomenclature committee.(22) The observer was blinded as to whether the biopsy specimens were pre- or posttreatment, except for dynamic variables in which blinding was not possible. Indices of bone structure were evaluated using Goldner-stained, 7-μm-thick sections. Before the measurements, the cancellous space was demarcated by well-established criteria.(23) Cancellous bone area, as a percentage of total cancellous tissue area, trabecular number, trabecular width, and trabecular separation, were derived from automated measurement (Optomax V AMS system; Optomax, Inc., Hollis, NH, USA) of bone area and perimeter at 31× magnification and defined as previously described.(22)

All static indices of remodeling activity were measured on 7-μm-thick sections stained with Goldner's trichrome. Unstained sections (20 μm thick) were used to measure dynamic indices of bone formation. Perimeter indices were expressed as percentages of total trabecular perimeter and were measured by the point counting method at 125× magnification. Mineral apposition rate was calculated from the semiautomated measurement of interlabel distance (Optomax VIDS V) and the interval in days between the two labels. Bone formation rate was calculated according to the standard formula(22) using the double- plus one-half of the single-labeled surface as the mineralizing perimeter and was expressed in cubic micrometers per square micrometer per day. Wall width of all completed trabecular and endocortical bone packets in three to four sections per biopsy specimen was measured semiautomatically (VIDS IV; Optomax) at four equidistant points along the length of each packet at 160× magnification.

After histomorphometric analysis, the embedded samples were analyzed by high-resolution microcomputed tomography (micro-CT) using a μCT20 system (Scanco Medical, Bassersdorf, Switzerland) with a spatial resolution of 28 μm. Eight pairs of biopsy specimens from the men were available for micro-CT analysis; six pairs were available for the women. The variables and method of analysis are described in full by Hildebrand et al.(24) The analytical method used has advantages over previous techniques in that it does not make the assumptions of an underlying fixed plate or rod model of trabecular structure. Bone surface (BS) area was calculated using the marching cubes method to triangulate the surface of the mineralized bone phase.(25) Bone volume was calculated using tetrahedrons corresponding to the enclosed volume of the triangulated surface.(26) Total volume (TV) was the volume of the sample that was examined. To compare samples of varying size, normalized indices, BV/TV and BS/TV, were used. Trabecular thickness was determined by filling maximal spheres into the structure with distance transformation(27) and then calculating the average thickness of all bone voxels. Trabecular separation was calculated by the same procedure except the voxels representing nonbone tissue were filled with maximal spheres. Trabecular number was calculated as the inverse of the mean distance between the midaxes of the trabeculae. Connectivity density expresses the number of connections per cubic millimeter and was derived from the Euler number(28) as follows: ConnD = (1 − Euler number)/TV. The Euler number (χ) is fundamental to all determinations of connectivity. In cancellous bone, it is defined as follows(29): χ=β0 − β1 + β2, where β0 is the number of bone particles (traditionally assumed to be 1); β1 is the connectivity, that is, the maximum number of connections that must be broken to split the structure into two parts; and β2 is the number of marrow cavities fully surrounded by bone. Cortical thickness was expressed as the average thickness of the 3D cortex using the same method as that for trabecular thickness. The cortical margins were defined by a semiautomatic contouring algorithm that locates the most probable edge of the inner and outer cortical surfaces.

Statistical analysis

Data are expressed as mean ± SEM for men and women separately. The significance of differences between mean values for variables before and after PTH treatment was assessed using a paired t-test. Regression analysis was performed using the method of least squares. All data analysis was performed using PsiPlot (Polysoftware International, Pearl River, NY, USA) and Microsoft Excel Data Analysis software (Microsoft Corp., Redmond, WA, USA).

RESULTS

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

BMD and bone histomorphometry

Values for lumbar spine BMD and histomorphometric variables before and after PTH treatment are given in Table 1. The patients displayed an anabolic response to PTH treatment as evidenced by a significant increase in lumbar spine BMD in both men and women. After PTH treatment, there was a trend toward an increase in eroded perimeter in both men and women and in bone formation rate in women (Table 1). Cancellous bone area was maintained in women and there was almost a significant increase in men (p = 0.07). Consistent with the changes in cancellous bone area, the wall width of trabecular packets was maintained in women and was significantly increased in men. Cortical width was slightly increased in men and was significantly (p < 0.02) increased in women. Cortical porosity was unchanged.

Table Table 1.. BMD and Bone Histomorphometry Variables in Patients Before and After PTH Treatment
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Micro-CT

Analysis of cancellous bone structure in 3D by micro-CT revealed maintenance of cancellous bone volume in women and a trend toward an increase in men (Table 2). The individual responses are shown in Fig. 1. There also was a strong trend toward an increase in connectivity density, a 3D index of trabecular connectivity, in both men and women (Table 2). Although there was some heterogeneity in individual responses, 75% of the men and 66% of the women showed maintenance or improvement, by 12-253% in connectivity density (Fig. 1).

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Figure FIG. 1. Percentage changes in three variables of iliac bone structure after PTH treatment. All variables were measured by micro-CT.

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Table Table 2.. Structural Variables Obtained by Micro-CT in Patients Before and After PTH Treatment
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Consistent with the results from the 2D analysis, cortical thickness was slightly increased in men and was significantly (p < 0.01) increased in women (Table 2). Although cortical thickness was maintained or improved (>15%) in 6 of the 8 men, 2 men showed a decrease; however, all of the women showed an increase (Fig. 1). Figure 2 shows images of bone structure from 1 woman and 1 man before and after PTH treatment. Note the improvement in trabecular architecture and increase in cortical thickness after PTH treatment.

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Figure FIG. 2. (A) Paired biopsy specimens from a 64-year-old woman and (B) a 47-year-old man before and after treatment with PTH. All biopsy specimens are shown at the same magnification. In the woman, cortical thickness increased from 320 to 420 μm after treatment and connectivity density increased from 2.9 to 4.6/ mm3. In the man, cortical thickness increased from 600 to 700 μm after treatment and connectivity density increased from 2.1 to 3.8/mm3.

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Subperiosteal and endocortical surfaces

To investigate the mechanism underlying the anabolic effect on cortical bone, we analyzed dynamic indices of bone formation on the subperiosteal (outer) and endocortical (inner) surfaces as well as the eroded perimeter and wall width of bone packets on the endocortical surface. The latter measurement could not be performed on the periosteal surface because packets could not be identified reliably at that site. There were no significant changes in dynamic indices of bone formation on either the subperiosteal or the endocortical surface (data not shown). However, on the endocortical surface there was a significant increase in wall width of the packets and a significant decrease in eroded perimeter (Figs. 3 and 4).

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Figure FIG. 3. Wall width of (A) packets and (B) eroded perimeter on the endocortical surface before and after PTH treatment.

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Figure FIG. 4. A bone packet on the endocortex of a 52-year-old man (A) before treatment and (B) after treatment. The section in panel B happens to be stained more intensely than that in panel A and therefore appears darker. The dashed lines demarcate the reversal lines. The packet in the posttreatment biopsy specimen is more than twice as wide as that in the pretreatment biopsy specimen. Measurement of all packets revealed an average increase in wall width of 58% in this patient (original magnification ×13).

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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

This study has provided two novel observations regarding the mechanism of action of daily PTH treatment in patients with osteoporosis. First, we obtained evidence that PTH exerts anabolic action on human cortical bone with no increase in cortical porosity. Second, we observed an increase in 3D trabecular connectivity in the majority of patients.

For many years it has been held that the anabolic action of PTH occurs predominantly in cancellous bone and there still is a concern that PTH treatment may have a deleterious effect on cortical bone.(30) On the contrary, this study indicates that PTH has an anabolic effect on human cortical bone, at least in the axial skeleton. Indeed, we observed a greater anabolic effect on cortical bone than on cancellous bone. An increase in cortical thickness would be predicted to contribute substantially to bone strength,31-33) even at skeletal sites such as the vertebra where cancellous bone traditionally has been thought to play the dominant role in determining strength.

The anabolic action on cortical bone is consistent with previous studies in animals, including rats,34-36) rabbits,(37) and dogs.(38) In animals, PTH stimulates bone formation on both the periosteal and the endosteal surfaces.34-38) In this study we obtained evidence for an anabolic effect on the endosteal surface, where we observed an increase in the packet wall width. Greater PTH-induced stimulation of bone formation on the endosteal than the periosteal surface of the cortex has been observed in some animal studies.(36, 39, 40) Our finding that PTH exerts anabolic action on the endosteal surface of cortical bone in humans also is consistent with a recent preliminary report by Cann et al.(41) who used 3D quantitative CT in vivo to examine envelope-specific responses to PTH in the femur.

The positive effect of increased endocortical wall width on cortical thickness may have been enhanced by a reduction in resorption on that surface because we observed a marked decrease in eroded perimeter. This is a surprising observation because we might have expected PTH to increase the eroded perimeter secondary to an increase in activation frequency. This is what is seen in cancellous bone.(4, 14) However, there are some data in the literature to support our observation. In aged rats, osteoclast perimeter on the endocortical surface of the vertebral body was normal during PTH administration and increased dramatically after PTH withdrawal.(36) In adult beagles, Zhang et al.(42) noted that PTH treatment increased the number of cortex to node trabecular struts, most likely because of reduced resorption on the endocortical surface.

We observed an increase in endocortical wall width in both the men and the women, but a statistically significant increase in cortical thickness was seen only in the women. There are several plausible explanations for this. First, the women were treated twice as long as the men. It is possible that with longer treatment the increase in endocortical wall width would result in an increase in cortical width. Bone density measurements indicated that men in whom PTH treatment was continued after 18 months continued to gain bone mass at the hip.(43) Second, the women were treated with a combination of PTH and estrogen and there may be additive effects of the two agents. Previously, this has been indicated in animal studies and there are some data to suggest it also may be true in humans.(44)

With the use of micro-CT, we observed an increase in the connectivity density of cancellous bone in the majority of patients after PTH treatment. The contribution of this 3D index of trabecular connectivity to bone strength is, as yet, unclear. There are few data available for humans and almost none in patients with osteoporosis. As far as we are aware, this is the first study in which 3D connectivity has been measured before and after treatment of patients with osteoporosis. Based on measurements of normal cancellous bone, Kabel et al.(45) concluded that connectivity density contributed little to its mechanical properties but conceded that this may not be the case in disease states. Inclusion of connectivity density with bone volume in a regression analysis significantly improved the prediction of vertebral bone strength.(46) Moreover, Kinney and Ladd(47) concluded that recovery of mechanical function in patients with osteoporosis is dependent on preservation or restoration of trabecular connectivity. Increases in trabecular connectivity after PTH treatment have been shown previously in animals,(48, 49) and also patients with mild primary hyperparathyroidism display greater trabecular connectivity than age- and sex-matched controls.(19, 50, 51)

The mechanism underlying the increase in connectivity density in this study is unclear. There were modest improvements in trabecular number in men and in trabecular thickness in both men and women. Therefore, it is possible that trabecular elements that were separated by short distances became connected or reconnected. Another possibility is an increase in trabecular perforation(52) or intratrabecular tunneling, which would result in an increase in the number of connections in 3D space. Intratrabecular tunneling has been reported previously in monkeys treated with daily PTH injections.(53)

Cancellous bone area and volume were not significantly increased after PTH treatment. This is consistent with the most recent paired biopsy study(14) but differs from earlier studies(4, 13) in which a substantial increase in cancellous bone area was observed. The current observation and that of Hodsman et al.(14) also seem inconsistent with the established increments in spinal BMD. A likely explanation is the high intraindividual variability in cancellous bone area at the iliac crest,(54) which makes it difficult to show statistically significant differences with small sample sizes. However, it also should be noted that several studies in intact, large animals (dogs and rabbits) have not shown increases in cancellous bone area in the iliac crest and lumbar spine after PTH treatment.(37, 38, 41) In one of the studies, an increase in cortical bone area occurred in the absence of an increase in cancellous bone area.(37) It also is possible that part of the increase in spinal BMD in humans treated with PTH is caused by an increase in the thickness of the cortical shell. Cosman et al.(55) previously reported that in patients with osteoporosis, cortical width in the bone biopsy specimen was the best predictor of spinal BMD.

In conclusion, this paired biopsy study indicates that daily treatment with PTH exerts anabolic action on both cortical and cancellous bone in the human iliac crest. Cortical porosity was unchanged and there was an increase in 3D trabecular connectivity in most patients. Although limited by the small sample size, the data suggest that PTH is not only capable of increasing bone mass in patients with osteoporosis but also of correcting the microarchitectural defects in cortical and cancellous bone that increase skeletal fragility. If confirmed in larger studies, these findings would provide an explanation at a structural level for the recent demonstration that PTH treatment for only 18 months reduces the incidence of spine and nonspine fractures in patients with osteoporosis.(12)

Acknowledgements

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

We thank Rhône-Poulenc (now Aventis Pharma, Bridgewater, NJ, USA) for supplying synthetic PTH(1-34). This work was supported by the National Institutes of Health (NIH) grants AR 39191, DK 42892, and DK 4631, the Food and Drug and Administration (FDA) grant FD-R001024, and Biomeasure Inc. (Milford, MA, USA).

REFERENCES

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