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Keywords:

  • osteoporosis;
  • corticosteroids;
  • biomechanics;
  • bone histomorphometry;
  • bone quantitative computed tomography

Abstract

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

Thus far, orthopedic research lacks a suitable animal model of osteoporosis. In OVX sheep, 6 months of steroid exposure reduced bone density and mechanical competence. Bone properties and bone formation did not recover for another 6 months. Therefore, steroid-treated OVX sheep may serve as a large animal model for osteopenic bone.

Introduction: The purpose of this study was to explore the effects of glucocorticoid treatment on cancellous bone density, microarchitecture, biomechanics, and formation of new bone.

Materials and Methods: Sixteen ovariectomized merino sheep received either a 6-month glucocorticoid treatment (GLU; 0.45 mg/kg methylprednisolone) or were left untreated (control). Cancellous bone biopsy specimens from the tibia were harvested 6 months after ovariectomy. After 12 months, the animals were killed, and biopsy specimens were obtained from the contralateral tibia and the lumbar spine. All biopsy specimens were scanned for apparent bone mineral density by peripheral quantitative computed tomography (pQCT) and tested mechanically in uniaxial compression. Three-dimensional bone reconstructions were obtained by microcomputed tomography. Formation of new bone was analyzed using histologies of the femoral condyles.

Results: After 6 months, mineral density (−19%) and mechanical competence (−45%) were reduced by glucocorticoid treatment (p < 0.1). BV/TV (−21%; p < 0.01) and trabecular thickness (−20%; p = 0.01) declined, whereas BS/BV increased (24%; p = 0.01). After 12 months, mineral density (−33%) and mechanical properties (−55%) were reduced even more profoundly (p < 0.05). Also, the structural parameters (BS/BV and Tb.Th.) still seemed to be affected by glucocorticoid treatment (p < 0.05). New bone formation, assessed by measurement of osteoid surface, was markedly reduced (−63%, p < 0.1) by glucocorticoid treatment. The differences between groups were generally more pronounced at the tibia and the femur than at the spine.

Conclusion: The effects of short-term high-dose steroid administration on bone mineral in this animal model were comparable with those observed in humans after long-term corticoid treatment. Reduction in bone quality and bone formation rate persisted after the cessation of steroid administration. Glucocorticoid treatment of ovariectomized sheep may therefore serve as a large animal model for steroid-induced osteopenia.


INTRODUCTION

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

One of the most important side effects of long-term glucocorticoid therapy is secondary osteoporosis. Bone loss occurs rapidly within the first 6 months and is greatest in cancellous bone. It is estimated that up to 50% of the patients chronically treated with glucocorticoids will suffer fractures.(1) These osteoporotic fractures are often difficult to stabilize because of the failure of fixation in weak osteoporotic bone. To improve orthopedic procedures and to develop new implants and fixation strategies, appropriate animal models are required. For orthopedic research, it is necessary to choose an animal model with adequate bone size and mechanical conditions that are comparable with humans.(2) In recent years, sheep have been used frequently for large animal models.(2,3) Bone remodeling(4,5) and cancellous bone structure(6) in sheep are similar to that of humans. Thus far, osteopenia in sheep has been induced by ovariectomy, which increases bone turnover,(7) reduces bone mass(5) and bone mineral density (BMD),(8–10) and decreases mechanical properties.(11) Unfortunately, the loss of BMD 12 months after ovariectomy of sheep is only moderate compared with the first year after menopause of women.(9,12) The reason that ovariectomy only causes slight effects on ovine bone is primarily caused by estradiol of nonovarian origin and calcium intake, number of lambs suckled, and seasonal effects.(7,12) Also, the decrease of bone strength after ovariectomy is not strong enough to cause spontaneous fractures in sheep.

There are only a few studies in which osteopenia in sheep has been induced by glucocorticoid administration. Histologies have shown that a 3-month glucocorticoid treatment resulted in a major bone formation deficit.(13,14) The combination of ovariectomy, glucocorticoid treatment, and calcium/vitamin D-restricted diet over a period of 7 months resulted in a decline in cancellous bone density and a reduction in vertebral compressive stiffness.(15,16)

The purpose of this study was to assess the effect of glucocorticoid administration in addition to ovariectomy. Moreover, we wanted to clarify whether there is any bone recovery after the termination of glucocorticoid treatment. We hypothesized that the combination of ovariectomy and glucocorticoid treatment results in a significant reduction of bone quality and bone formation, which persists after the cessation of glucocorticoid administration.

MATERIALS AND METHODS

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

Animal model

Sixteen merino ewes (age, 4–7 years old; mean weight, 88.1 ± 8.5 kg) were subjected to bilateral ovariectomies under general halothane anesthesia (Fluothane; Zeneca, Planckstadt, Germany). Postoperatively, antibiotics (ampicillin, 12.5 mg/kg; Ampi-Sleecol; Albrecht, Aulendorf, Germany) and analgesics (metamizole, 50 mg/kg; Novalgin; Hoechst, Unterschleiβheim, Germany) were administered subcutaneously. All animals were housed indoors under identical conditions and a constant photoperiod of 12 hours of light per day. They were fed grass hay and food pellets containing 0.9% calcium and 0.6% phosphorus (altromin 0133; Altromin, Lage, Germany). The sheep were divided into two groups. The first group was treated with glucocorticoids (GLU; n = 8), whereas the second group was left untreated (control; n = 8). Sheep in the GLU group received a daily dose of 0.45 mg/kg body weight methylprednisolone (MedrateSolubile 40; Pharmacia & Upjohn, Erlangen, Germany) by subcutaneous injection beginning 3 weeks after ovariectomy. The treatment was continued for 6 months. During the last 4 weeks, the dose was stepwise reduced to zero to avoid withdrawal symptoms connected with adrenocortical atrophy. To detect any signs of bone recovery after the cessation of glucocorticoid treatment, the animals remained untreated for another 6 months. After a total of 12 months, all animals were killed. All experimental procedures were in compliance with national regulations for the care and use of laboratory animals and were reviewed and approved by the Institutional Animal Care review board.

Biopsy specimens

Six months after the beginning of the study, cancellous bone biopsy specimens were obtained from all sheep. Under general halothane anesthesia (Fluothane; Zeneca), the cylindrical biopsy specimens (6 mm diameter, 7–9 mm height) were harvested from the medial aspect of the right tibia. The intake laid between the medial condyle and the tibial tuberosity 4 mm beneath the insertion of the joint capsule. After the death of the animals, cancellous bone biopsy specimens again were harvested from the contralateral tibia and from the vertebral body of the fourth lumbar vertebrae. The ends of the specimens were cut parallel with a precision diamond saw (EXAKT; PSI-Grünewald-Exakt, Laudenbach, Germany). The biopsy specimens were stored in 0.9% saline solution at room temperature to avoid drying of the specimens during the testing period, which lasted less than 6 h. All biopsy specimens were scanned for apparent BMD by peripheral quantitative computed tomography (pQCT; XCT960; Stratec, Pforzheim, Germany).(17)

Three-dimensional (3-D) bone reconstructions were obtained by μCT (Fan Beam μ-scope; Stratec, Pforzheim, Germany) at 30-μm spatial resolution. At the center of each biopsy, 150 slices were scanned at intervals of 30 μm. The scans were automatically reconstructed to a 3-D bone cylinder. Within that cylinder, a sphere of 4.5 mm diameter, encompassing a total volume of 48 mm3, was defined. The 3-D bone spheres were analyzed with 3-D image analysis software (analysis 3–0; SIS, Münster, Germany) for bone volume to tissue volume ratio (BV/TV), bone surface to bone volume ratio (BS/BV), trabecular thickness (Tb.Th.), trabecular number (Tb.N.), and trabecular separation (Tb.Sp.).

The mechanical properties of the bone biopsy specimens were assessed with a screw driven material testing machine (1445; Zwick, Ulm, Germany). The biopsy specimens were placed between two lubricated loading platens. One loading platen was fixed, and the other was equipped with a ball and a socket joint to minimize shear loading. Testing was performed in uniaxial compression at a maximum strain of 1% and a rate of 1 mm/min. The displacement was measured continuously by an extensometer (MT25; Haidenhain, Traunreut, Germany). The mechanical stress of the bone cylinders was obtained by calculating the ratio of maximum force by cross-sectional area of the specimen. Strain was calculated by the change of height dividing it by the height of the specimen before mechanical testing. Compression stiffness was defined as the ratio between stress and strain.

Femoral condyles

For bone histomorphometry, the condyles of the left femur were extracted after the death of the sheep. Histological slices were produced following a standardized protocol: (1) fixation in buffered 4% formalin; (2) dehydration by increasing alcohol concentrations (ethanol); (3) infiltration and embedding in methylmethacrylate (Polyscience, Eppelheim, Germany); (4) cutting of 80-μm-thick microtome sections (PSI-Grünewald-Exakt); and (5) staining with Paragon (Paragon C&C; New York, NY, USA), which resulted in blue trabeculae and dark blue osteoid.

The histological slices of the femoral condyles were examined under a light microscope (Axiophot; Zeiss, Oberkochen, Germany) in 25-fold magnification to define a region of interest including only cancellous bone. The region of interest was defined as a rectangle with a length of 9.5 mm and a height of 5.5 mm located 7 mm beneath the cartilage. The surface images were imported into an image analysis system (Soft Imaging Systems, Münster, Germany) and were automatically analyzed for bone volume to tissue volume ratio (BV/TV) and bone surface to volume ratio (BS/BV). The osteoid was defined manually (Fig. 1) and was analyzed for osteoid volume to bone volume ratio (OV/BV) and osteoid surface to bone surface ratio (OS/BS).

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Figure FIG. 1.. Histological slice of femur condyles after staining with Paragon. The picture shows trabeculae in light gray and newly formed bone in dark gray. The osteoid was defined manually by outlining the dark gray areas.

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Statistical analysis

Results are presented as mean ± SD. The Mann-Whitney U-test was used for the comparison between the GLU and the control group. Because this study was not hypothesis driven, an explorative data analysis was performed, and p values are provided for orientation purposes only.

RESULTS

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

During the 6-month treatment, the animals tolerated the glucocorticoids very well. However, during the abatement of the steroid administration, three of eight sheep developed joint pain and partial lameness, both being typical steroid withdrawal symptoms.(18) Two of these sheep were successfully treated with nonsteroidal anti-inflammatory pain medication (carprofen, 4 mg/kg; Rimadyl; Pfizer, Karlsruhe, Germany) by subcutaneous injection over a period of 3 days. The third sheep had to be killed because of the persistence of the symptoms and was excluded from further analysis.

Corticosteroid treatment resulted in a substantial reduction of BMD, bone quality, formation of new bone, and mechanical properties of bone. Differences were generally more pronounced at the appendicular skeleton than at the spine.

After 6 months of glucocorticoid administration, BMD of the tibial biopsy specimens decreased by 19% (p = 0.06) compared with control (Fig. 2). Changes in morphometric parameters were of similar magnitude (Table 1). Bone volume (BV/TV) declined by 21% (p = 0.08) and trabecular thickness (Tb.Th.) by 20% (p = 0.01). Bone surface (BS/BV) increased by 24% (p = 0.01). Trabecular number and trabecular separation, however, were not affected by glucocorticoid treatment. The greatest impact of the steroid administration was found for the mechanical properties of the bone specimens. Compared with control, the compressive stiffness of glucocorticoid-biopsy specimens from the tibia was reduced by 45% (p = 0.11) after 6 months (Fig. 3). After 12 months, the differences were even more pronounced, although glucocorticoid treatment had been terminated for 6 months. BMD at the tibia decreased by 34% compared with control (p = 0.05; Fig. 2). Also, bone structural parameters differed from the control group (Table 1) to a larger extent than at 6 months. Specifically, BV/TV was 28% (p = 0.14) and Tb.Th. was 25% (p = 0.02) lower, whereas bone surface was 26% (p = 0.02) larger in the glucocorticoid-treated group. Again, the number of trabeculae and trabecular separation were not affected. Mechanical competence of the bone specimens was markedly changed after 12 months (Fig. 3). Compressive stiffness of the glucocorticoid biopsy specimens was significantly reduced by 55% (p = 0.02).

Table Table 1. Histomorphometric Analysis of the Bone Biopsies After μCT Scanning and 3D Reconstruction (Mean ± SD)
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Figure FIG. 2.. Bone mineral density (mean ± SD) of the bone biopsy specimens from the tibia and the spine.

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Figure FIG. 3.. Compressive stiffness (mean ± SD) of the cylindrical biopsy specimens from the tibia and the spine.

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The effects of glucocorticoid administration at the spine were less pronounced than at the tibia; however, the trends were similar. BMD was reduced by 11% (p = 0.06), and structural parameters were slightly reduced (Table 1) after glucocorticoid administration. Mechanical competence, however, did not seem to be affected (Fig. 3).

At the femur, the histomorphometric parameters after 12 months differed markedly between the glucocorticoid-treated group and the control group (BV/TV: −23%, p = 0.03; BS/BV: +25%, p = 0.05; Table 2). The rate of newly formed bone was much lower after steroid treatment: osteoid volume (OV/BV) declined by 38% (p = 0.17) and osteoid surface (OS/BS) by 63% (p = 0.06).

Table Table 2. Histomorphometric Analysis of the Femoral Condyles (Mean ± SD)
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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

In this study, a 6-month high-dose glucocorticoid treatment of ovariectomized sheep resulted in substantial changes in bone mass, bone structure, mechanical properties, and formation of new bone. These changes were induced within the first 6 months of glucocorticoid administration and were maintained over another 6 months after the cessation of steroid treatment. The successful induction of osteopenia was demonstrated by densitometric, (histo-)morphometric, and biomechanical methods.

In previous studies, ovariectomy was performed in sheep to establish postmenopausal osteoporosis characterized by estrogen deficiency. Because of the short duration of many studies (3–12 months), BMD and histomorphometric and biomechanical parameters failed to differ significantly compared with control animals.(7,9,11,), 19 In two other studies, the histomorphometric and mechanical changes were statistically significant at the spine and at the ilium 24 months after ovariectomy.(5,20) Therefore, the ovariectomized sheep may serve as a large animal model of postmenopausal osteoporosis, 24 months after the ovariectomy is performed. For the investigation of new therapies and the evaluation of prosthetic devices, however, this model is costly and time consuming.

An alternative protocol to induce osteopenia in sheep is the administration of glucocorticoids. A 3-month glucocorticoid treatment in sheep was associated with a major bone formation deficit, a variation in histomorphometric bone parameters, and a reduction in serum osteocalcin.(13,14) In another study, sheep were treated with methylprednisolone over a period of 4 months. Both BMD and biomechanical results failed to reveal any differences between the steroid-treated group and the control group.(21) In our study, we combined ovariectomy and glucocorticoid treatment to obtain synergistic effects on bone. The treatment group was not compared with a control group with no treatment at all but to a group with ovariectomy alone. This particularly enables the examination of the additional effects of steroid administration. The analysis of glucocorticoid induced bone loss was performed both at load bearing sites and at the spine. Although both cortical and cancellous bone is lost in glucocorticoid-induced osteoporosis,(1) this study focused on cancellous bone, because bone loss occurs more rapidly in cancellous bone(22–24) because of a higher remodeling rate.

In our study, BMD, microarchitecture, and mechanical competence were altered after 6 months of glucocorticoid treatment and remained affected for another 6 months after the cessation of glucocorticoid treatment. At the tibia, the differences in BMD and most of the structural parameters between treated sheep and control animals ranged from 13% to 21% after 6 months and from 19% to 34% after 12 months. In humans, annual changes of cancellous BMD during menopause reach 3–10%.(25) In glucocorticoid-induced osteoporosis, the reduction can amount to 12% in the first year of treatment.(1) Therefore, the reduction of more than 30% in cancellous BMD in our sheep model can be considered as a substantial loss of bone. The loss of mechanical competence of the tibia biopsy specimens seemed to be even more dramatic. However, the effects on the mechanical properties of our bone specimens were possibly overestimated. Because of the anisotropy of cancellous bone the mechanical properties strongly depend on the loading direction.(26) In our study, the testing direction was medial-lateral and therefore perpendicular to the proximal-distal loading direction. In normal aging, and more seriously in osteoporosis, the horizontal trabeculae are preferentially lost.(27) Therefore, thinning and reduction of horizontal trabeculae after glucocorticoid treatment may have caused the profound decrease in compressive stiffness of the tibia biopsy specimens in the horizontal plane.

In our animal model, a profound depression of bone formation was seen in the histomorphometric analysis of the femur. The results of this study are in agreement with Chavassieux et al., who found a major bone formation deficit in sheep after a 3-month glucocorticoid treatment.(13,14) Bone loss after glucocorticoid treatment in humans is more pronounced in the axial skeleton than at peripheral sites.(1) In our animal study, the differences after 12 months in BMD and structural and biomechanical parameters were more marked at the tibia and the femur than at the spine. Because glucocorticoid-induced osteoporosis is at least partially reversible,(22) a higher remodeling rate at the spine may have caused an earlier bone recovery than at the appendicular skeleton. However, we have no data about density, structure, and biomechanics of vertebral cancellous bone at 6 months. In a pilot study of Lill et al.,(16) the structural parameters of vertebral bone biopsy specimens of two ovariectomized and glucocorticoid-treated sheep harvested after 6 months differed by up to 70% compared with the untreated control group. Compressive stiffness was reduced by 10%. The results of our study are in good accordance with another study of Lill et al.(15) They found a similar reduction in BMD at the tibia and the spine. The differences in bone morphometric parameters of the iliac crest biopsy specimens were more pronounced than in our work. This could be because of the fact that the sheep in their study were fed a calcium/vitamin D-restricted diet in addition to ovariectomy and steroid treatment. Moreover, their results were compared with a control group, which was untreated and therefore different from our own control group, which was ovariectomized.

Despite this successful induction of osteopenia in sheep, our animal model has several limitations. First, it remains unclear to what extent the ovariectomy contributed to the bone loss observed under glucocorticoid treatment. We did not investigate effects of glucocorticoid administration alone and therefore have no information whether similar results would have been obtained without ovariectomy. From the literature, it is known that the substitution of glucocorticoids results in a major bone formation deficit(13,14) but fails to reveal any changes in BMD or mechanical competence of bone.(21) It therefore seems likely that ovariectomy had some synergistic effects on bone with respect to bone mass and bone strength. Second, although the reduction of BMD in our study can be considered as a substantial loss of bone, the remaining bone mass is still higher than that in human osteoporotic bone. For this reason, spontaneous fractures neither occurred in our study nor are they described in the literature. Therefore, it would be more correct to describe the ovariectomized and glucocorticoid-treated sheep as a model of osteopenic than of osteoporotic bone.

The mechanisms of bone loss in our animal model are different than the mechanisms of bone loss in postmenopausal women. In postmenopausal osteoporosis, the increased rate of bone remodeling is caused by increased production of both osteoblasts and osteoclasts. Furthermore, the extension of the osteoclastic lifespan and the shortening of the osteoblastic lifespan lead to an overall imbalance in bone resorption and bone formation, with deeper resorption cavities and trabecular perforation.(28) In contrast, glucocorticoid excess has suppressive effects on osteoblastogenesis in the bone marrow and also promotes the apoptosis of osteoblasts and osteocytes. Moreover, the initial phase of rapid bone loss in glucocorticoid-induced osteoporosis is caused by an extension of the osteoclastic lifespan.(28,29) Therefore, our animal model may not serve as a model for postmenopausal osteoporosis. Our animal model is limited in that we used glucocorticoids to induce an artificial loss of bone. This limits the usefulness of this model for the first 6 months of our study. During that time, glucocorticoids not only would affect bone remodeling but also alter bone metabolism, delay wound healing, and suppress immune response to infections. However, clinical data indicate that the effects are only transient. It is known that the effects on bone are fully reversible after the termination of the steroid administration.(24) Therefore, this animal model of osteoporosis should not be used within the first 6 months of glucocorticoid administration. However, after termination of treatment, it is useful, because, on one hand, bone metabolism would return to normal and side effects would no longer occur, and on the other hand, the effects on BMD and structure and biomechanical competence still persist for at least 6 months.

Our study was explorative rather than hypothesis driven. A variety of assessment methods was used to explore the impact of glucocorticoid administration on bone. Because the group size was limited, the study was underpowered to determine the statistical significance for all the parameters assessed. In consequence, the presentation of the results should be considered as descriptive, and p values are provided for orientation purposes only.

The strengths of our study include the variety of parameters used to analyze the changes in bone and the long study duration, which enables the investigation of bone recovery after the cessation of steroid treatment. This large animal model of osteopenic bone allows the application of newly developed orthopedic devices of similar size and shape as used in humans. Bone loss was induced both at load bearing sites such as the tibia and the femur and at the axial skeleton. Both are potential target locations for the in vivo testing of orthopedic implants or surgical techniques. Moreover, our animal model may serve as a model for steroid-induced osteoporosis, in which pharmaceutical intervention for the treatment of glucocorticoid-induced osteoporosis can be studied. It should be noted, however, that sheep are ruminants and might differ from humans with respect to oral-based osteoporosis therapies.

In conclusion, we successfully induced osteopenia in sheep by a combination of ovariectomy and glucocorticoid treatment. This combination resulted in a profound reduction of bone density, bone quality, and mechanical competence of bone. Changes observed in our model occurred within 6 months and remained over another 6 months after termination of the treatment.

Acknowledgements

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

The authors thank Liselotte Müller-Molenar, Regina Kloos, Marion Tomo, and Patrizia Horny for excellent technical assistance and the SYNOS Foundation for financial support of the study.

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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