Susan M. Rackard

Biomechanical properties across trabeculae from the proximal femur of normal and ovariectomised sheep.

The elastic behaviour of trabecular bone is a function not only of bone volume and architecture, but also of tissue material properties. Variation in tissue modulus can have a substantial effect on the biomechanical properties of trabecular bone. However, the nature of tissue property variation within a single trabecula is poorly understood. This study uses nanoindentation to determine the mechanical properties of bone tissue in individual trabeculae. Using an ovariectomised ovine model, the modulus and hardness distribution across trabeculae were measured. In both normal and ovariectomised bone, the modulus and hardness were found to increase towards the core of the trabeculae. Across the width of the trabeculae, the modulus was significantly less in the ovariectomised bone than in the control bone. However, in contrast to this hardness was found not to differ significantly between the two groups. This study provides valuable information on the variation of mechanical material properties in healthy and diseased trabecular bone tissue. The results of the current study will be useful in finite element modelling where more accurate values of trabecular bone modulus will enable the prediction of the macroscale behaviour of trabecular bone.

Effects of ovariectomy on bone turnover, porosity, and biomechanical properties in ovine compact bone 12 months postsurgery

Compact bone makes up approximately 80% of the human skeleton by mass; but there are little data available on the effects of increased bone turnover on compact bone mechanical and material properties. This study addresses this question by measuring intracortical remodeling, resorption cavity number, and porosity in an ovariectomized (OVX) sheep model, and measures changes in biomechanical properties. Thirty‐eight sheep were divided into two groups. Group 1 were controls (n = 19), and Group 2 were ovariectomized (OVX; n = 19). Fluorochrome dyes were administered intravenously to both groups at five time points over 12 months post‐OVX to label sites of bone turnover. At 12 months post‐OVX all animals were euthanized. Samples were harvested from the left metatarsal and were analyzed for intracortical bone turnover at five time points, the number of resorption cavities, and the level of intracortical porosity. The effects of these parameters on bone biomechanical properties were then measured. Bone turnover was increased in the OVX group at 6, 9, and 12 months (p < 0.05). Resorption was also higher in the OVX group at 12 months (p < 0.05). Furthermore, porosity was significantly increased in the OVX group at 12 months (p < 0.05). Stiffness and yield strength were reduced in the OVX group compared to controls (p = 0.05). Ultimate compressive strength and work to fracture did not differ between groups. These findings provide new insights into the mechanisms and effects of increased bone turnover on bone material and microstructural properties.

Effects of estrogen deficiency and bisphosphonate therapy on osteocyte viability and microdamage accumulation in an ovine model of osteoporosis.

It has been proposed that osteocyte viability plays an important role in bone integrity, and that bone loss in osteoporosis may be partially due to osteocyte cell death following estrogen depletion. Osteoporosis treatments such as bisphosphonates can inhibit osteocyte apoptosis which in turn may also reduce remodeling. Consequently, microcracks in bone which are normally repaired by bone remodeling may accumulate. This study used an ovine model of osteoporosis to examine the effects of estrogen depletion and bisphosphonates on osteocyte apoptosis and microdamage accumulation. Skeletally mature ewes were randomly assigned into two equal groups; ovariectomy (OVX) and a non‐treatment group (control). Half of these animals were sacrificed 12 months post‐OVX. Twenty months post‐OVX, a number of OVX animals were randomly selected and each received a supra‐pharmacological dose of the bisphosphonate, zoledronic acid (Zol). This group and all the remaining animals were sacrificed 31 months post‐OVX. A compact bone specimen was removed from the left metacarpal of each animal; half was used for osteocyte apoptosis detection and the remainder for microdamage analysis. Estrogen deficiency resulted in significant increases in the levels of osteocyte apoptosis while zoledronic acid significantly reduced the level of apoptosis in osteocytes. Zoledronic acid treatment resulted in the formation of more microcracks. However, these cracks were shorter than in control or OVX groups which may provide one explanation as to why increased damage levels following bisphosphonate treatment have not lead to increased fractures. This study also provides additional evidence of the importance of estrogen in preserving the osteocyte network.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

Effects of High Bone Turnover on the Biomechanical Properties of the L3 Vertebra in an Ovine Model of Early Stage Osteoporosis

Study Design. Investigations of the effects of high bone turnover on the L3 vertebra were carried out, using an ovariectomized (OVX) ovine model of early stage osteoporosis. Objective. To assess the contribution of bone turnover to the biomechanics of L3. Summary of Background Data. Clinically, dual energy x-ray absorptiometry (DEXA) is used to measure bone mineral density (BMD). However, this can only predict 60% to 70% of bone strength; the remainder is due to bone quality. There is currently little information available on how strength is affected by changes in bone quality parameters, particularly bone turnover. Turnover can be assessed clinically using biochemical markers; however, this provides systemic values, whereas localized values are required to predict site-specific fracture risk. Methods. Thirty-eight sheep were assigned to 2 groups (control, n = 19; OVX, n = 19). Both groups were intravenously administered a fluorochrome dye on the day of surgery and 3, 6, 9, and 12 months thereafter, to label sites of bone turnover. After 12 months, animals were killed and the spinal columns harvested. L3 vertebrae were scanned using DEXA. Bone turnover was quantified using epifluorescence microscopy, and microarchitecture was assessed by microCT scanning. Compressive testing was used to characterize the mechanical properties of the vertebrae. Results. BMD and microarchitecture were unchanged in OVX compared with controls. However, bone turnover, as measured by fluorochrome labeled sites, was significantly increased in the OVX group in cortical and trabecular compartments. As a consequence, the biomechanical properties were significantly reduced in that group. Conclusion. These findings show that OVX resulted in changes in bone turnover, which reduced biomechanical properties in a model of early stage osteoporosis. These differences were present despite microarchitecture or BMD remaining unchanged. In the future, the ability to assess site-specific bone turnover would greatly enhance the accuracy with which fracture risk could be predicted.

The effects of estrogen deficiency and bisphosphonate treatment on tissue mineralisation and stiffness in an ovine model of osteoporosis

While much research has been dedicated to understanding osteoporosis, the nature of mineral distribution and the mechanical property variation in diseased bone is poorly understood. The current study aimed to determine the effect of estrogen deficiency and bisphosphonate therapy on bone tissue properties using an ovine model of osteoporosis. Skeletally mature animals (4+ years) were divided into an ovariectomy group (ovx, n=20) and a non treatment control group (control, n=20). A zoledronic acid treated group was also included in which animals were estrogen deficient for 20 months prior to receiving treatment (Zol, n=4). Half of the control and ovx groups were euthanized 12 or 31 months post-operatively and all Zol animals were euthanised at 31 months. Individual trabeculae were removed from the proximal femur and were analysed at specific locations across the width of the trabeculae. The mineral content was measured using quantitative backscatter electron imaging and the modulus was measured using nanoindentation. The spatial distribution of tissue modulus and mineral content in bone from ovariectomised animals was similar to control. However, ovariectomy significantly reduced the overall mineral content and tissue modulus relative to the control group after 12 months. Interestingly, significant differences were not maintained 31 months post-OVX. Treatment with zoledronic acid increased the mineral content and tissue modulus relative to both the ovariectomised and control groups. Zoledronic acid was also found to alter the mineral and modulus gradients normally associated with healthy bone tissue. The current study provides evidence that both estrogen deficiency and zoledronic acid therapy significantly alter mineral content and the mechanical properties of trabecular tissue.

Subchondral trabecular structural changes in the proximal tibia in an ovine model of increased bone turnover

Ovariectomized (OVX) sheep are now considered to be useful models for a variety of metabolic bone disorders. The specific aim of this study was to determine the effects of ovariectomy on the structural parameters and material density of the subchondral bone of the ovine tibial plateau as measured by microcomputed tomography (MicroCT). Twenty‐three sheep were examined in this study; 10 of the sheep underwent ovariectomy (OVX), and the remainder (n = 13) were kept as controls (CON). These animals were then sacrificed at 12 months post‐operatively. Three‐dimensional analyses were performed of osteochondral samples (15 mm deep) which were obtained from the medial tibial plateau using MicroCT. Bone volume fraction of the subchondral trabecular bone was reduced in the ovariectomized sheep as compared to control animals (0.439 vs. 0.483, P = 0.038). Trabeculae were also significantly thinner in the OVX group (0.220 vs. 0.252 mm, P = 0.010), with reduced connectivity density (7.947 vs. 11.524 mm−3, P = 0.014). There was a trend towards lower numbers of individual trabeculae present in the OVX group as compared to controls, but this did not reach significance (2.817 vs. 3.288 mm−1, P = 0.1). There was also increased trabecular separation in the OVX group, which again fell short of significance (0.426 vs. 0.387 mm, P = 0.251). There was no difference in hydroxyapatite concentration (HA) between the two groups (929 vs. 932 mgHA cm−3, P = 0.687). In conclusion, significant alterations of the trabecular architecture under the tibial plateau were observed following 12 months of oestrogen‐deficiency in this ovine model. Despite these marked morphological and structural density differences, the material densities were equal in the two groups.

Variation of trabecular microarchitectural parameters in cranial, caudal and mid-vertebral regions of the ovine L3 vertebra.

The lumbar vertebrae are major load‐bearing structures within the spinal column. The current understanding of the microstructure of these bodies and their full role in load‐bearing is incomplete. There is a need to develop our understanding of these issues to improve fracture prediction in musculoskeletal diseases such as osteoporosis. The lumbar vertebrae consist primarily of trabecular bone enclosed in a thin cortical shell, but little is known about how microstructural parameters vary within these structures, particularly in relation to the trabecular compartment. The specific aim of this study was to use micro‐computed tomography to characterize the trabecular microarchitecture of the ovine L3 vertebra in cranial, mid‐vertebra and caudal regions. The L3 vertebra was obtained from skeletally mature ewes (n = 18) more than 4 years old. Three‐dimensional reconstructions of three pre‐defined regions were obtained and microarchitectural parameters were calculated. Whereas there was no difference in bone volume fraction or structural model index between regions, trabecular number, thickness, spacing, connectivity density, degree of anisotropy and bone mineral density all displayed significant regional variations. The observed differences were consistent with the biomechanical hypothesis that in vivo loads are distributed differently at the endplates compared with the mid‐vertebra. Thus, a more integrative approach combining biomechanical theory and anatomical features may improve fracture risk assessment in the future.

Examination of osteoarthritis and subchondral bone alterations within the stifle joint of an ovariectomised ovine model

The exact relationship between osteoporosis and osteoarthritis is still a matter for debate for many. The ovariectomised ewe is frequently used as a model for osteoporosis, resulting in significant alterations in bone morphometry and turnover in both trabecular and subchondral bone after 1 year. This study examines whether ovariectomy has any impact on development of osteoarthritis within the ovine stifle joint at the same time point. In addition, we investigate whether there are any significant correlations present between articular cartilage degeneration and alterations in microstructural parameters or turnover rates in the underlying bone. Twenty‐two sheep were examined in this study; 10 of the sheep underwent ovariectomy and 12 were kept as controls. Five distinctive fluorochrome dyes were administered intravenously at 12‐week intervals to both groups, to label sites of bone turnover. All animals were then sacrificed 12 months postoperatively. Although most specimens showed some evidence of osteoarthritis, no measurable difference between the two study groups was detected. Osteoarthritis was associated with a thinning of the subchondral plate, specifically the subchondral cortical bone; however, whereas previous studies have suggested a link between trabecular thinning and osteoarthritis, this was not confirmed. No correlation was found between osteoarthritis and bone turnover rates of either the subchondral trabecular bone or bone plate. In conclusion, despite the fact that ovariectomy results in marked morphological and structural changes in the ovine stifle joint at 1‐year postoperatively, no evidence was found to suggest that it plays a direct role in the aetiology of osteoarthritis.

Subchondral osteopenia and accelerated bone remodelling post-ovariectomy - a possible mechanism for subchondral microfractures in the aetiology of spontaneous osteonecrosis of the knee?

Osteopenia and subchondral microfractures are implicated in the aetiology of spontaneous osteonecrosis of the knee (SPONK). The ovine tibia shows significant alterations of the trabecular architecture within the subchondral bone of the medial tibial plateau post‐ovariectomy (OVX), including reduced trabecular bone volume fraction. We hypothesise that accelerated subchondral bone resorption may also play a role in increasing microfracture risk at this site. Twenty‐two sheep were examined in this study; 10 of the sheep underwent OVX, while the remainder (n = 13) were kept as controls (CON). Five fluorochrome dyes were administered intravenously at 12‐week intervals via the jugular vein to both groups, to label sites of bone turnover. These animals were then killed at 12 months post‐operatively. Bone turnover was significantly increased in the OVX group in both trabecular bone (2.024 vs. 1.047 no. mm−2; P = 0.05) and within the subchondral bone plate (4.68 vs. 0.69 no. mm−2; P < 0.001). In addition to the classically described turnover visible along trabecular surfaces, we also found visual evidence of intra‐trabecular osteonal remodelling. In conclusion, this study shows significant alterations in bone turnover in both trabecular bone and within the subchondral bone plate at 1 year post‐OVX. Remodelling of trabecular bone was due to both classically described hemi‐osteonal and intra‐trabecular osteonal remodelling. The presence of both localised osteopenia and accelerated bone remodelling within the medial tibial plateau provide a possible mechanism for subchondral microfractures in the aetiology of SPONK. Further utilisation of the OVX ewe may be useful for further study in this field.