Differential gene expression of bone anabolic factors and trabecular bone architectural changes in the proximal femoral shaft of primary hip osteoarthritis patients

Previous studies have shown a generalised increase in bone mass in patients with osteoarthritis (OA). Using molecular histomorphometry, this study examined the in vivo expression of mRNA encoding bone anabolic factors and collagen type I genes (COL1A1, COL1A2) in human OA and non-OA bone. Bone samples were obtained from the intertrochanteric (IT) region of the proximal femur, a skeletal site distal to the active site of disease, from individuals with hip OA at joint replacement surgery and from autopsy controls. Semi-quantitative reverse transcription-polymerase chain reaction analysis revealed elevated mRNA expression levels of alkaline phosphatase (p < 0.002), osteocalcin (OCN) (p < 0.0001), osteopontin (p < 0.05), 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 twofold greater in OA bone compared to control (p < 0.001), suggesting the potential presence of collagen type I homotrimer at the distal site. Insulin-like growth factor (IGF)-I, IGF-II, and transforming growth factor-β1 mRNA levels were similar between OA and control bone. Bone histomorphometric analysis indicated that OA IT bone had increased surface density of bone (p < 0.0003), increased trabecular number (Tb.N) (p < 0.0003), and decreased trabecular separation (Tb.Sp) (p < 0.0001) compared to control bone. When the molecular and histomorphometric data were plotted, positive associations were observed in the controls for OCN/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) versus bone tissue volume (r = 0.82, p < 0.0007) and OCN/GAPDH versus Tb.N (r = 0.56, p < 0.05) and a negative association was observed for OCN/GAPDH versus Tb.Sp (r = -0.64, p < 0.02). These relationships were not evident in trabecular bone from patients with OA, suggesting that bone regulatory processes leading to particular trabecular structures may be altered in this disease. The finding of differential gene expression, as well as architectural changes and differences in molecular histomorphometric associations between OA and controls, at a skeletal site distal to the active site of joint degeneration supports the concept of generalised involvement of bone in the pathogenesis of OA

Application of in vivo micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis

The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/13/6/R210Introduction: Osteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT). Methods: Male Wistar rats received a single intra-articular injection of low-dose MIA (0.2 mg) in the right knee joint and sterile saline in the left knee joint. The animals were scanned in vivo by micro-CT at two, six, and ten weeks post-injection, analogous to early, intermediate, and advanced stages of OA, to assess architectural changes in the tibial subchondral bone. The articular cartilage changes in the tibiae were assessed macroscopically and histologically at ten weeks post-injection. Results: Interestingly, tibiae of the MIA-injected knees showed significant bone loss at two weeks, followed by increased trabecular thickness and separation at six and ten weeks. The trabecular number was decreased at all time points compared to control tibiae. The tibial subchondral plate thickness of the MIA-injected knee was increased at two and six weeks and the plate porosity was increased at all time points compared to control. At ten weeks, histology revealed loss of proteoglycans, chondrocyte necrosis, chondrocyte clusters, cartilage fibrillation, and delamination in the MIA-injected tibiae, whereas the control tibiae showed no changes. Micro-CT images and histology showed the presence of subchondral bone sclerosis, cysts, and osteophytes. Conclusions: These findings demonstrate that the low-dose MIA rat model closely mimics the pathological features of progressive human OA. The low-dose MIA rat model is therefore suitable to study the effect of therapeutic drugs on cartilage and bone in a non-trauma model of OA. In vivo micro-CT is a non-destructive imaging technique that can track structural changes in the tibial subchondral bone in this animal model, and could also be used to track changes in bone in preclinical drug intervention studies for OA treatments.Geetha Mohan, Egon Perilli, Julia S Kuliwaba, Julia M Humphries, Ian H Parkinson and Nicola L Fazzalar

The ratio of messenger RNA levels of receptor activator of nuclear factor kB ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

© by the American Society for Bone and Mineral Research.The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor κB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.Nicola L. Fazzalari, Julia S. Kuliwaba, Gerald J. Atkins, Mark R. Forwood, and David M. Findla

Temporal changes in bone composition, architecture, and strength following estrogen deficiency in osteoporosis

Using an ovariectomized (OVX) ovine model, we provide an analysis of the timing of changes in bone following estrogen deficiency. The expression of genes known to regulate osteoclastogenesis, matrix production, and mineralization, as measured by real-time RT-PCR, was significantly increased by 12 months; and increased expression was maintained through to 31 months post-OVX compared to controls. FTIR spectroscopy confirmed that mineralized crystals were less mature than in controls 12 months post-OVX and were even less so by 31 months. The mineral-to-matrix ratio was significantly reduced by 31 months, while the ratio of mature to immature collagen cross-linking was initially increased at 12 months and subsequently reduced at 31 months post-OVX. In contrast, trabecular number, thickness, and separation were unchanged at 12 months. Significant reductions in trabecular number and thickness and a significant increase in trabecular separation were observed 31 months after OVX. Most notably perhaps these combined changes led to a significant reduction in the compressive strength of trabecular bone after 31 months. The results indicate that there is an initial increase in bone turnover, which is accompanied by a change in bone composition. This is followed by a continued increase in bone resorption and relative reduction in bone formation, leading to deterioration in bone microarchitecture. Ultimately, these cumulative changes led to a significant reduction in the compressive strength of bones following 31 months of estrogen deficiency. These findings provide important insight into the time sequence of changes during osteoporosis

MALDI mass spectrometry imaging of N-glycans on tibial cartilage and subchondral bone proteins in knee osteoarthritis

Magnetic resonance imaging (MRI) is a non-invasive technique routinely used to investigate pathological changes in knee osteoarthritis (OA) patients. MRI uniquely reveals zones of the most severe change in the subchondral bone (SCB) in OA, called bone marrow lesions (BMLs). BMLs have diagnostic and prognostic significance in OA, but MRI does not provide a molecular understanding of BMLs. Multiple N-glycan structures have been observed to play a pivotal role in the OA disease process. We applied matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of N-glycans to formalin-fixed paraffin-embedded (FFPE) SCB tissue sections from patients with knee OA, and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was conducted on consecutive sections to structurally characterize and correlate with the N-glycans seen by MALDI-MSI. The application of this novel MALDI-MSI protocol has enabled the first steps to spatially investigate the N-glycome in the SCB of knee OA patients.6 page(s

Stability of RNA isolated from human trabecular bone at post-mortem and surgery

Copyright © 2005 Elsevier B.V. All rights reserved.To determine the reliability of gene expression studies in human post-mortem bone, it is important to evaluate the stability of RNA isolated from such tissues as a function of the post-mortem interval. The stability of total RNA and bone-specific mRNA species was examined in bone samples obtained from routine autopsies and at surgery. The optimal temperature for any storage and transport of the bone before RNA isolation was shown to be 4 °C, and RT-PCR analysis is the preferred technique for the analysis of gene expression in post-mortem bone as it tolerates partial RNA degradation. For gene expression studies in bone, post-mortem cases, with a post-mortem interval of less than 48 h, should be selected, and the time that bone is stored after retrieval at autopsy or surgery should be kept to a minimum. Overall, our findings indicate that with appropriate storage and handling, RNA can be reliably isolated from human bone obtained at post-mortem and surgery to study ex vivo the pattern of gene expression in healthy individuals and in patients with musculoskeletal diseases such as osteoporosis and osteoarthritis.Julia S. Kuliwaba, Nicola L. Fazzalaria, and David M. Findlayhttp://www.elsevier.com/wps/find/journaldescription.cws_home/506068/description#descriptio

Temporal changes in bone composition, architecture, and strength following estrogen deficiency in osteoporosis

Using an ovariectomized (OVX) ovine model, we provide an analysis of the timing of changes in bone following estrogen deficiency. The expression of genes known to regulate osteoclastogenesis, matrix production, and mineralization, as measured by real-time RT-PCR, was significantly increased by 12 months; and increased expression was maintained through to 31 months post-OVX compared to controls. FTIR spectroscopy confirmed that mineralized crystals were less mature than in controls 12 months post-OVX and were even less so by 31 months. The mineral-to-matrix ratio was significantly reduced by 31 months, while the ratio of mature to immature collagen cross-linking was initially increased at 12 months and subsequently reduced at 31 months post-OVX. In contrast, trabecular number, thickness, and separation were unchanged at 12 months. Significant reductions in trabecular number and thickness and a significant increase in trabecular separation were observed 31 months after OVX. Most notably perhaps these combined changes led to a significant reduction in the compressive strength of trabecular bone after 31 months. The results indicate that there is an initial increase in bone turnover, which is accompanied by a change in bone composition. This is followed by a continued increase in bone resorption and relative reduction in bone formation, leading to deterioration in bone microarchitecture. Ultimately, these cumulative changes led to a significant reduction in the compressive strength of bones following 31 months of estrogen deficiency. These findings provide important insight into the time sequence of changes during osteoporosis