Strength of cancellous bone trabecular tissue from normal, ovariectomized and drug-treated rats over the course of ageing☆
Introduction
Osteoporosis is caused by an imbalance in the normal bone remodeling process whereby excessive osteoclast resorption occurs without adequate new bone formation by osteoblasts. The trabecular struts become thin and eventually resorb altogether leading to a reduction in bone strength and an increased likelihood of bone fracture [1]. These fractures are generally attributed to the reduction in the bone mass and trabecular connectivity. Hormone therapy (HT) drugs and bisphosphonates maintain bone mass and architecture by inhibiting osteoclast activity and thereby reducing bone resorption [2]. Although this reduces fracture susceptibility [3], even with continuous treatment, fractures still arise [4]. It has previously been proposed that osteoclastic bone resorption serves as a repair mechanism to remove aged and damaged bone tissue [5], [6], [7] and a concern with some anti-resorptive drugs is that inhibiting the remodeling process prevents the normal turnover of bone tissue. This may allow microdamage to accumulate in the bone tissue, so that, although bone mass and architecture are maintained, bone strength at the tissue level may be impaired. In order to assess the efficacy of anti-resorptive drug treatments for improving the mechanical performance of osteoporotic bone, measurements of the mechanical behaviour of bone tissue during osteoporosis and drug treatment are required.
Measurement of the effects of a decline in bone mass and architecture during osteoporosis on the mechanical behaviour of bone has been performed using whole bone testing [8], [9], [10], [11], [12], or testing of volumes of cancellous bone [13], [14], [15], [16]. Using ovariectomy to induce bone loss in the rat, it has been found, for example, that the compressive strength of rat vertebral bodies and the bending strength of rat femora are decreased following ovariectomy [8], [9], [10]. However, significant decreases in trabecular bone volume occurred in these experiments and thus it cannot be determined whether changes were due only to the reduction in bone mass or whether a reduction in tissue strength also contributed. A number of studies have assessed the mineral content of bone tissue during osteoporosis; some reveal that the mineral content is unchanged or slightly lower in the osteoporotic bone tissue [17] and others reveal an increase in the mineral content and a lack of collagen [18], [19], [20], [21], [22], [23], [24], [25]. Such microstructural changes strongly suggest that a change in the mechanical behaviour at the tissue level will occur during osteoporosis. However, the mineral content and mechanical properties of individual trabecula from ovariectomized bone are unknown so such ideas remain conjecture.
The efficacy of drug treatments has been assessed and it has been found, for example, that treatment with tibolone [8], [9], [10], estrogen, and other anti-resorptive drugs such as risedronate, alendronate, and incandronate [11], [12], [13], [14], [15] increases the structural strength of trabecular bone [13], [14], [15] and the whole bone strength [8], [9], [10], [11], [12]. However, it has also been found that treatment with high doses of certain anti-resorptive drugs is associated with an increase in spontaneous fractures of the thoracic spinous process, ribs and pelvic fractures [12], [25], [26]. As treatment in the current study, tibolone was used. Tibolone is a tissue-selective compound with estrogenic effects on bone, vagina and brain. In a 2.5-mg dose, it has been used for the treatment of climacteric complaints and prevention of bone loss. Tibolone offers substantial protection against bone fractures, similar to conventional HT, as demonstrated in the Million Women Study [27]. While previous studies [8], [9], [10], [11], [12], [13], [14], [15] suggest that the structural mechanical behaviour is maintained using anti-resorptive drugs, they do not determine the properties of the mineralized tissue itself.
A number of researchers have performed experiments on uniform samples machined from trabecular tissue, which eliminated the error of structural irregularity [28], [29], and on single trabeculae [30], [31], [32], [33], [34], [35] from healthy bone. Test methods applied have included 3/4-point bending [28], [29], buckling [30], [31], cantilever beam tests [32], microtensile testing [33], [34], [35] and ultrasonic measurement [34]. Reported values for the elastic modulus of an individual trabecula from these studies have varied widely, with values ranging from 0.75 GPa–20 GPa. The small size of test specimens and irregularity of their geometry have limited the accuracy of such test methods, and a reliable, consistent test method is required if the mechanical behaviour of trabecular bone tissue is to be accurately determined.
The objective of our study was to test the hypothesis that the material properties of single trabeculae will differ for normal, ovariectomized and drug-treated rat bones over the course of ageing. If this hypothesis were to be confirmed, it would strongly suggest that, in addition to the well-known effects of osteoporosis and drug treatment on bone mass, there are also important processes ongoing that significantly affect bone strength at the tissue level.
Section snippets
Animal experimentation
Three groups of 44-week-old female Wistar rats were treated as follows: (1) a control group was sham-operated and treated with vehicle (placebo-treated), (2) a group was ovariectomized (OVX) as a model of osteoporosis and treated with vehicle and (3) an OVX group was treated orally with tibolone (2 mg/kg, day). All treatments were carried out for 54 weeks, i.e. from the 44th week to the 98th week of life. All animal procedures were approved by the animal Ethics Committee.
Bones were harvested
Results
There was no significant difference found between the elastic moduli of control group between 0, 4, 14, 34 and 54 weeks (P = 0.98) (Fig. 3). No significant difference was found between the elastic moduli of control group at 0 weeks and the OVX group at 4, 14, 34 and 54 weeks (P = 0.28). However, the OVX tissue was stiffer than the control bone tissue at 14 weeks (5.11 ± 3.89. vs. 2.67 ± 2.06; P = 0.05) and 34 weeks (3.66 ± 1.95. vs. 2.61 ± 0.79; P = 0.05). The elastic modulus of OVX was higher
Discussion
We believe this is the first study to quantify the effect of estrogen deficiency and drug treatment on the mechanical properties of bone tissue from individual trabeculae. We found that ovariectomy in rats leads to an increase of the mechanical properties of the mineralized tissue that constitutes trabecular bone compared to normal. We also found that no such increase occurs when OVX animals were treated with tibolone. Also the properties of trabeculae of tibiae of control-treated normal rats
Conclusions
In conclusion, in this study, we found a difference in the mechanical properties of rat trabecular bone tissue from normal, OVX and OVX tibolone-treated bone over the course of ageing. By measuring the tensile strength and elastic modulus of individual trabeculae, we have found that the bone tissue of trabeculae remains constant over a long period of 54 weeks in the tibia of placebo-treated normal and tibolone-treated ovariectomized rat but becomes significantly stronger and stiffer in bone
Acknowledgments
Financial support provided by the European Commission Framework 6 program project MIAB, “Mechanical Integrity and Architecture of Bone”. Funding source: European Commission funded project “Mechanical Integrity and Architecture of Bone”. The MTS Titron was purchased by a grant from the Health Research Board of Ireland.
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Presented in part at the Orthopaedic Research Society, San Francisco, USA, March, 2004. Awarded an ASBMR Harold B Frost Young Investigator Award at the 35th International Sun Valley Workshop On Skeletal Tissue Biology, Idaho, USA, 2005.