Evidence is accumulating for the role of bone in the pathogenesis of osteoarthritis (OA). Previous studies have shown a generalised increase in bone mass and hypo-mineralisation in OA patients. However, the molecular and cellular mechanisms involved in the increased bone mass and matrix compositional profiles in OA, at distal skeletal sites to the articular cartilage, have not yet been well defined. This study examined whether gene expression of bone anabolic factors, trabecular bone architecture and matrix mineralisation are altered in human OA and non-OA hipbone. Intertrochanteric (IT) trabecular bone samples were obtained from 15 primary hip OA patients (mean age 65 [48–85] years) and 13 closely age- and gender-matched autopsy controls (mean age 63 [44–83] years). Semi-quantitative RT-PCR analysis revealed elevated mRNA expression levels of alkaline phosphatase (p < 0.002), osteocalcin (p < 0.0001), osteopontin (p < 0.05), collagen type-I α chains COL1A1 (p < 0.0001) and COL1A2 (p < 0.002), in OA bone compared to control, suggesting possible increases in osteoblastic biosynthetic activity and/or bone turnover at the IT region in OA. Interestingly, the ratio of COL1A1:COL1A2 mRNA was almost 2-fold greater in OA bone compared to control (p < 0.001), suggesting the potential presence of collagen type-I homotrimer at the distal site that may associate with hypomineralisation in OA individuals. Using a quantitative backscatter electron imaging technique, mineralisation profiles of IT trabecular bone indicated decreased mineralisation in the OA group compared to the control group (24.2 weight percent calcium [wt%Ca] versus 25.3 wt%Ca). Bone histomorphometric analysis found OA IT bone had increased surface density of bone and decreased trabecular separation compared to control bone. Taken together with a reported increase in diffuse microdamage in OA IT bone (Fazzalari et al. Bone 31:697–702, 2002), possibly due to hypomineralisation, these results are consistent with the altered bone material properties found in OA individuals. The finding of differential gene expression, altered mineralisation and architectural changes in OA bone, at a skeletal site distal to the active site of joint degeneration, supports the concept of systemic involvement of bone in the pathogenesis of OA.

Introduction Sheep are being used increasingly for spinal and other skeletal-related research. However, there is still limited information about the molecular pathways of bone remodelling in this species compared to rats or mice. It has been demonstrated in other animal models and in the human that the receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) play major regulatory roles in controlling osteoclast activity and their differentiation. We investigated the expression of RANKL and OPG in trabecular bone of an ovariectomised steroid-treated osteopaenic sheep model.

Methods Trabecular bone from the lumbar spine (LS) and proximal femur (PF) of ten osteopaenic ewes and four normal ewes were collected [1]. Total RNA was isolated and complementary DNA (cDNA) was synthesised. DNA encoding RANKL and OPG were sequenced and ovine specific primers were designed to amplify the cDNA by real time RT-PCR to generate products corresponding to mRNA encoding RANKL and OPG. The results were normalised to 18S RNA.

Results Total OPG expression (in trabecular bone) from the PF region was over two fold higher than the LS (P< 0.0001). The relative expression of OPG in the both LS and PF regions were significantly higher in the treated animals (steroid & oophorectomy) compared to controls (p< 0.05). The relative expression of RANK-L in the PF was significantly higher than in the LS (P< 0.0001). However, the relative RANK-L expression in the treated animals was not significantly different from the control animals in either region. The ratio of RANK-L:OPG in the PF and the LS was not significantly different but it was significantly reduced in the osteopaenic animals.

Discussion Based on this gene expression study and previous histomorphological data, it appears that trabecular bone loss is not due to increased osteoclastic activity but may rather due to lack of osteoblastic activity and function. Higher expression of OPG and RANK-L and greater bone loss compared to LS suggest that the rate of bone turnover is greater in the PF. Further investigation of the molecular pathways of bone loss in this animal model will increase its utility for osteoporosis research.

Introduction Bone loss and increased bone fragility may occur following long-term steroid therapy resulting in painful vertebral fractures. To fully understand the mechanisms underlying steroid-induced osteoporosis and to test potential spinal implants a large animal model is needed. The sheep is appropriate since bone remodelling is similar to humans and the vertebral body dimensions permit easy surgical manipulation. This study was undertaken to fully characterise the sheep as a model of steroid-induced vertebral osteoporosis and to evaluate its suitability for testing balloon kyphoplasty.

Methods Osteoporosis was induced in ten lactating ewes (mean age 8 years) by ovariectomy, weekly injection of 54mg dexamethasone (Dexafort, Intervet, Australia) and a diet containing 0.2% calcium for up to 6 months. Baseline and endpoint iliac crest biopsies were taken to measure static histomorphometric indices of bone formation and resorption. Control animals were not ovariectomised and not treated with steroids. Dual energy X-ray absorptiometry (DXA, Hologic QDR 1000+, USA) was used to monitor bone mineral density (BMD) in the lumbar spine (L2-L5) after 0, 3 and 6 months of steroid treatment. At each time interval sheep were killed by barbiturate injection and the entire lumbar spine (L1-L6) was processed for histology, quantitative histomorphometry, mechanical testing, micro-CT (computed tomography) and ex-vivo trials to inflate kyphoplasty balloon tamps.

Results After six months of treatment, BMD in the lumbar spine decreased by 29.5% from baseline. Trabecular bone volume of L2, L3 and L4 vertebrae (pooled) decreased by 31.4% (p< 0.05) and trabecular thickness decreased by 33.9%. Cortical bone thickness decreased by 43.9% (p< 0.05). The average load at which L1 yielded decreased by 67.4%. Static measurements of bone formation decreased by 68.3% and bone resorption increased 10 fold. Kyphoplasty balloon tamps were successfully inflated ex-vivo in vertebral bodies from treated animals.

Discussion Using DXA, cancellous bone histomorphometry and mechanical testing, this study has demonstrated significant trabecular and cortical bone loss in the sheep lumbar spine up to six months after ovariectomy and continuous steroid treatment. These changes are the result of increased resorption and decreased formation of bone. The successful inflation of bone tamps in osteoporotic vertebrae ex-vivo is a prerequisite for in-vivo studies to assess the safety and efficacy of the kyphoplasty procedure.

Introduction: Vertebral compression fractures are common in osteoporosis, resulting in spinal deformities, severe back pain and decreased mobility. Vertebroplasty and kyphoplasty procedures aim to restore the integrity of the deformed vertebral body by injection of biocompatible cement. To date, there have been no long-term studies of the bone-cement interaction in this setting. A reliable large animal model of vertebral osteoporosis would be useful to fully characterise the disease process, to assess potential treatment regimens and to investigate the biocompatibility of bone cements used in kyphoplasty and vertebroplasty. The aim of this pilot study was to develop such a model with ovariectomy, low calcium diet and continuous steroid treatment.

Methods: To induce osteoporosis, ten lactating ewes (mean age 8 years) were ovariectomised, injected weekly with 9 mg dexamethasone (Dexafort, Intervet, Australia) and fed low calcium diet. Weekly serum samples were taken to quantify generalised bone resorption (Type 1 collagen C-telopeptide [CTX], ‚-Cross Laps assay, Roche Diagnostics, Australia). Dual-energy X-ray absorptiometry (DEXA, Hologic QDR 1000+, USA) was used to monitor bone mineral density (BMD) in the lumbar spine (L3-L6) after 0, 2, 4, 6 and 9 months of treatment. At each time interval two sheep were killed by barbiturate injection. The entire lumbar spine (L1-L6) was processed for histology, quantitative histomorphometry, mechanical testing and micro-CT (computed tomography).

Results: CTX levels increased rapidly after two months (p< 0.05). Baseline BMD in the lumbar spine (0.87±0.06 g/cm2) decreased by 16.9±3.8% or 2.72 standard deviations (p< 0.001) after nine months of treatment. Structural parameters of cancellous bone also showed osteoporotic change. Trabecular bone volume of L2, L3 and L6 vertebrae (pooled) progressively decreased from 24.9±1.2% at two months to 16.5±0.47% at nine months (p< 0.05). Trabecular thickness decreased from 0.14±0.01mm to 0.09±0.01mm, (p< 0.05) and trabecular spacing increased from 0.42±0.03mm to 0.47±0.02mm in the same period. The compressive load at which the L1 vertebrae failed decreased by 39.4% after 9 months.

Discussion: This pilot study has demonstrated by DEXA, cancellous bone histomorphometry and mechanical testing, significant bone loss in the sheep lumbar spine up to nine months after ovariectomy and continuous steroid treatment. Assuming that the baseline BMD is representative of mature sheep, the changes in the lumbar spine could be interpreted as osteoporotic. Vertebral bone loss did not reach levels that would result in fracture. However, further work is underway using higher steroid doses to accelerate bone loss. This experimental model will be used to assess aspects of osteoporosis in general and vertebral augmentation procedures in particular.