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1.  PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures 
Bone  2011;48(5):1066-1074.
Phosphatases are essential for the mineralization of the extracellular matrix within the skeleton. Their precise identities and functions however remain unclear. PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase involved in the generation of inorganic phosphate for bone mineralization. It is highly expressed at sites of mineralization in bone and cartilage. The bones of Phospho1−/− mice are hypomineralized, bowed and present with spontaneous greenstick fractures at birth. In this study we show that PHOSPHO1 is essential for mechanically competent mineralization that is able to withstand habitual load. Long bones from Phospho1−/− mice did not fracture during 3- point bending but deformed plastically. With dynamic loading nanoindentation the elastic modulus and hardness of Phospho1−/− tibiae were significantly lower than wild-type tibia. Raman microscopy revealed significantly lower mineral:matrix ratios and lower carbonate substitutions in Phospho1−/− tibia. The altered dihydroxylysinonorleucine/hydroxyllysinonorleucine and pyridoline/deoxypyridinoline collagen crosslink ratios indicated possible changes in lysyl hydroxylase-1 activity and/or bone mineralization status. The bone formation and resorption markers, N-terminal propeptide and C-terminal telopeptide of Type I collagen, were both increased in Phospho1−/− mice and this we associated with increased bone remodelling during fracture repair or an attempt to remodel a mechanically competent bone capable of withstanding physiological load. In summary these data indicate that Phospho1−/− bones are hypomineralized and, consequently, are softer and more flexible. An inability to withstand physiological loading may explain the deformations noted. We hypothesize that this phenotype is due to the reduced availability of inorganic phosphate to form hydroxyapatite during mineralization, creating an undermineralized yet active bone.
PMCID: PMC3078982  PMID: 21272676
Phospho1; biomineralization; mechanical and material properties; bone quality; hypomineralization
2.  Cholesterol Metabolism: the Main Pathway Acting Downstream of Cytochrome P450 Oxidoreductase in Skeletal Development of the Limb▿ †  
Molecular and Cellular Biology  2009;29(10):2716-2729.
Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.
PMCID: PMC2682028  PMID: 19273610
3.  Cyclooxygenase inhibition lowers prostaglandin E2 release from articular cartilage and reduces apoptosis but not proteoglycan degradation following an impact load in vitro 
This study investigated the release of prostaglandin E2 (PGE2) from cartilage following an impact load in vitro and the possible chondroprotective effect of cyclooxygenase-2 (COX-2) inhibition using non-steroidal anti-inflammatory drugs (NSAIDs).
Explants of human articular cartilage were subjected to a single impact load in a drop tower, and then cultured for 6 days in the presence of either a selective COX-2 inhibitor (celecoxib; 0.01, 0.1, 1.0 and 10 μM) or a non-selective COX inhibitor (indomethacin; 0.1 and 10 μM). The concentrations of PGE2 and glycosaminoglycans (GAGs), a measure of cartilage breakdown, were measured in the explant culture medium at 3 and 6 days post-impact. Apoptotic cell death was measured in frozen explant sections by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method.
PGE2 levels were increased by more than 20-fold in the medium of explants at both 3 (p = 0.012) and 6 days (p = 0.004) following impact, compared with unloaded controls. In the presence of celecoxib and indomethacin, the PGE2 levels were reduced in a dose-related manner. These inhibitors, however, had no effect in reducing the impact-induced release of GAGs from the cartilage matrix. Addition of celecoxib and indomethacin significantly reduced the number of trauma-induced apoptotic chondrocytes in cartilage explant sections.
In this study, a marked increase in PGE2 was measured in the medium following an impact load on articular cartilage, which was abolished by the selective COX-2 inhibitor, celecoxib, and non-selective indomethacin. These inhibitors reduced chondrocyte apoptosis but no change was observed in the release of GAGs from the explants, suggesting that the COX/PGE2 pathway is not directly responsible for cartilage breakdown following traumatic injury. Our in vitro study demonstrates that it is unlikely that COX-2 inhibition alone would slow down or prevent the development of secondary osteoarthritis.
PMCID: PMC2246251  PMID: 18096078
4.  Identification of hip fracture patients from radiographs using Fourier analysis of the trabecular structure: a cross-sectional study 
This study presents an analysis of trabecular bone structure in standard radiographs using Fourier transforms and principal components analysis (PCA) to identify contributions to hip fracture risk.
Radiographs were obtained from 26 hip fracture patients and 24 controls. They were digitised and five regions of interest (ROI) were identified from the femoral head and neck for analysis. The power spectrum was obtained from the Fourier transform of each region and three profiles were produced; a circular profile and profiles parallel and perpendicular to the preferred orientation of the trabeculae. PCA was used to generate a score from each profile, which we hypothesised could be used to discriminate between the fracture and control groups. The fractal dimension was also calculated for comparison. The area under the receiver operating characteristic curve (Az) discriminating the hip fracture cases from controls was calculated for each analysis.
Texture analysis of standard radiographs using the fast Fourier transform yielded variables that were significantly associated with fracture and not significantly correlated with age, body mass index or femoral neck bone mineral density. The anisotropy of the trabecular structure was important; both the perpendicular and circular profiles were significantly better than the parallel-profile (P < 0.05). No significant differences resulted from using the various ROI within the proximal femur. For the best three groupings of profile (circular, parallel or perpendicular), method (PCA or fractal) and ROI (Az = 0.84 – 0.93), there were no significant correlations with femoral neck bone mineral density, age, or body mass index. PCA analysis was found to perform better than fractal analysis (P = 0.019).
Both PCA and fractal analysis of the FFT data could discriminate successfully between the fracture and control groups, although PCA was significantly stronger than fractal dimension. This method appears to provide a powerful tool for the assessment of bone structure in vivo with advantages over standard fractal methods.
PMCID: PMC524177  PMID: 15469614
5.  High levels of fat and (n-6) fatty acids in cancellous bone in osteoarthritis 
Osteoarthritis (OA) is strongly linked with obesity and patients with osteoporosis (OP) have a low body mass index. Anecdotal evidence, clinical and laboratory, suggests that OA bone contains more fat. However, conversion of osteoblasts to adipocytes is reported in OP and this would suggest that the more porous OP cancellous bone would have a high fat content.
To test the hypothesis that OA bone contains more fat than OP bone.
Cores of cancellous bone were obtained from femoral heads of patients undergoing surgery for either OA or OP. Lipids were extracted using chloroform-methanol, weighed and expressed as a fraction of core mass and volume. A fatty acid analysis was performed using gas chromatography.
OA bone contained twice as much fat per unit volume of tissue as OP. Levels of n-6 fatty acids were elevated in OA, especially arachidonic acid (C20:4 n-6) which was almost double that found in OP.
These data support the hypothesis that lipids may play a significant role in the pathogenesis of OA and may provide part of the key to understanding why OA and OP lie at opposite ends of the spectrum of bone masses.
PMCID: PMC446208  PMID: 15207011
Osteoarthritis; Osteoporosis; Bone; Lipid; Fatty acids
6.  A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality 
Journal of Clinical Investigation  2001;107(7):899-907.
Osteoporosis is a common disease with a strong genetic component. We previously described a polymorphic Sp1 binding site in the COL1A1 gene that has been associated with osteoporosis in several populations. Here we explore the molecular mechanisms underlying this association. A meta-analysis showed significant associations between COL1A1 “s” alleles and bone mineral density (BMD), body mass index (BMI), and osteoporotic fractures. The association with fracture was stronger than expected on the basis of the observed differences in BMD and BMI, suggesting an additional effect on bone strength. Gel shift assays showed increased binding affinity of the “s” allele for Sp1 protein, and primary RNA transcripts derived from the “s” allele were approximately three times more abundant than “S” allele–derived transcripts in “Ss” heterozygotes. Collagen produced from osteoblasts cultured from “Ss” heterozygotes had an increased ratio of α1(I) protein relative to α2(I), and this was accompanied by an increased ratio of COL1A1 mRNA relative to COL1A2. Finally, the yield strength of bone derived from “Ss” individuals was reduced when compared with bone derived from “SS” subjects. We conclude that the COL1A1 Sp1 polymorphism is a functional genetic variant that predisposes to osteoporosis by complex mechanisms involving changes in bone mass and bone quality.
PMCID: PMC199568  PMID: 11285309

Results 1-6 (6)