Cherubism Mice Also Deficient in c-Fos Exhibit Inflammatory Bone Destruction Executed by Macrophages That Express MMP14 Despite the Absence of TRAP+ Osteoclasts.

Currently, it is believed that osteoclasts positive for tartrate-resistant acid phosphatase (TRAP+) are the exclusive bone-resorbing cells responsible for focal bone destruction in inflammatory arthritis. Recently, a mouse model of cherubism (Sh3bp2KI/KI ) with a homozygous gain-of-function mutation in the SH3-domain binding protein 2 (SH3BP2) was shown to develop auto-inflammatory joint destruction. Here, we demonstrate that Sh3bp2KI/KI mice also deficient in the FBJ osteosarcoma oncogene (c-Fos) still exhibit noticeable bone erosion at the distal tibia even in the absence of osteoclasts at 12 weeks old. Levels of serum collagen I C-terminal telopeptide (ICTP), a marker of bone resorption generated by matrix metalloproteinases (MMPs), were elevated, whereas levels of serum cross-linked C-telopeptide (CTX), another resorption marker produced by cathepsin K, were not increased. Collagenolytic MMP levels were increased in the inflamed joints of the Sh3bp2KI/KI mice deficient in c-Fos. Resorption pits contained a large number of F4/80+ macrophages and genetic depletion of macrophages rescued these erosive changes. Importantly, administration of NSC405020, an MMP14 inhibitor targeted to the hemopexin (PEX) domain, suppressed bone erosion in c-Fos-deficient Sh3bp2KI/KI mice. After activation of the NF-κB pathway, macrophage colony-stimulating factor (M-CSF)-dependent macrophages from c-Fos-deficient Sh3bp2KI/KI mice expressed increased amounts of MMP14 compared with wild-type macrophages. Interestingly, receptor activator of NF-κB ligand (RANKL)-deficient Sh3bp2KI/KI mice failed to show notable bone erosion, whereas c-Fos deletion did restore bone erosion to the RANKL-deficient Sh3bp2KI/KI mice, suggesting that osteolytic transformation of macrophages requires both loss-of-function of c-Fos and gain-of-function of SH3BP2 in this model. These data provide the first genetic evidence that cells other than osteoclasts can cause focal bone destruction in inflammatory bone disease and suggest that MMP14 is a key mediator conferring pathological bone-resorbing capacity on c-Fos-deficient Sh3bp2KI/KI macrophages. In summary, the paradigm that osteoclasts are the exclusive cells executing inflammatory bone destruction may need to be reevaluated based on our findings with c-Fos-deficient cherubism mice lacking osteoclasts. © 2017 American Society for Bone and Mineral Research.

Stress analysis of irradiated human tooth enamel using finite element methods.

The objectives of this project were to use finite element methods to determine how changes in the elastic modulus due to oral cancer therapeutic radiation alter the distribution of mechanical stresses in teeth and to determine if observed failures in irradiated teeth correlate with changes in mechanical stresses. A thin slice section finite element (FE) model was constructed from micro CT sections of a molar tooth using MIMICS and 3-Matic software. This model divides the tooth into three enamel regions, the dentin-enamel junction (DEJ) and dentin. The enamel elastic modulus was determined in each region using nano indentation for three experimental groups namely - control (non-radiated), in vitro irradiated (simulated radiotherapy following tooth extraction) and in vivo irradiated (extracted subsequent to oral cancer patient radiotherapy) teeth. Physiological loads were applied to the tooth models at the buccal and lingual cusp regions for all three groups (control, in vitro and in vivo). The principal tensile stress and the maximum shear stress were used to compare the results from different groups since it has been observed in previous studies that delamination of enamel from the underlying dentin was one of the major reasons for the failure of teeth following therapeutic radiation. From the FE data, we observed an increase in the principal tensile stress within the inner enamel region of in vivo irradiated teeth (9.97 ± 1.32 MPa) as compared to control/non-irradiated teeth (8.44 ± 1.57 MPa). Our model predicts that failure occurs at the inner enamel/DEJ interface due to extremely high tensile and maximum shear stresses in in vivo irradiated teeth which could be a cause of enamel delamination due to radiotherapy.

Bivariate genome-wide association meta-analysis of pediatric musculoskeletal traits reveals pleiotropic effects at the SREBF1/TOM1L2 locus.

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% (95% CI: 34-52%) for TBLH-BMD, and 39% (95% CI: 30-48%) for TB-LM, with a shared genetic component of 43% (95% CI: 29-56%). We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5. Variants in the TOM1L2/SREBF1 locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that SREBF1 is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass.Bone mineral density and lean skeletal mass are heritable traits. Here, Medina-Gomez and colleagues perform bivariate GWAS analyses of total body lean mass and bone mass density in children, and show genetic loci with pleiotropic effects on both traits.

Bone muscle crosstalk targets muscle regeneration pathway regulated by core circadian transcriptional repressors DEC1 and DEC2.

Deletion of proprotein convertase Mbtps1 in bone osteocytes leads to a significant postnatal increase in skeletal muscle size and contractile function, while causing only a 25% increase in stiffness in long bones. Concerns about leakiness in skeletal muscle were discounted since Cre recombinase expression does not account for our findings, and, Mbtps1 protein and mRNA is not deleted. Interestingly, the response of normal skeletal muscle to exercise and the regenerative response of skeletal muscle to the deletion of Mbtps1 in bone share some key regulatory features including a preference for slow twitch muscle fibers. In addition, transcriptional regulators PPAR, PGC-1α, LXR, and repressors DEC1 and DEC2 all occupy central positions within these two pathways. We hypothesize that the age-dependent muscle phenotype in Dmp1-Cre Mbtps1 cKO mice is due to bone→muscle crosstalk. Many of the myogenic genes altered in this larger and functionally improved muscle are regulated by circadian core transcriptional repressors DEC1 and DEC2, and furthermore, display a temporal coordination with Dec1 and Dec2 expression consistent with a regulatory co-dependency. These considerations lead us to propose that Dmp1-Cre Mbtps1 cKO osteocytes activate myogenesis by increased release of an activator of muscle PPAR-gamma, for example, PGE2 or sphingosine-1-P, or, by diminished release of an inhibitor of LXR, for example, long-chain polyunsaturated fatty acids. We hope that further investigation of these interacting pathways in the Dmp1-Cre Mbtps1 cKO model will lead to clinically translatable findings applicable to age-related sarcopenia and other muscle wasting syndromes.

The Distribution of Carbonate in Enamel and its Correlation with Structure and Mechanical Properties.

The correlation of carbonate content with enamel microstructure (chemical and crystal structure) and mechanical properties was evaluated via linear mapping analyses using Raman microspectroscopy and nanoindentation. Mappings started at the outer enamel surface and ended in the inner enamel near the dentin-enamel junction (DEJ) in lingual and buccal cervical and cuspal regions. The carbonate peak intensity at 1070 cm-1 gradually increased from outer to inner enamel. Moreover, the phosphate peak width, as measured by the full width at half maximum (FWHM) of the peak at 960 cm-1, also increased, going from ~9 cm-1 in outer enamel to ~13 cm-1 in enamel adjacent to the DEJ, indicating a decrease in the degree of crystallinity of hydroxyapatite from outer to inner enamel. In contrast, Young's modulus decreased from 119±12 to 80±19 GPa across outer to inner enamel with a concomitant decrease in enamel hardness from 5.9±1.4 to 3.5±1.3 GPa. There were also significant correlations between carbonate content and associated crystallinity with mechanical properties. As carbonate content increased, there was an associated decrease in crystallinity and both of these changes correlated with decreased modulus and hardness. Collectively, these results suggest that enamel carbonate content and the associated change in the crystal structure of hydroxyapatite, i.e. degree of crystallinity, may have a direct effect on enamel mechanical properties. The combination of Raman microspectroscopy and nanoindentation proved to be an effective approach for evaluating the microstructure of enamel and its associated properties.

Separation of newly formed bone from older compact bone reveals clear compositional differences in bone matrix.

In long bone diaphyses, woven bone forms first and then transitions into a more mineralized compact bone tissue. Prior evidence suggests that the non-collagenous protein composition of woven bone may be distinct from that of more mature bone tissue, particularly with respect to a diverse group of phosphorylated, extracellular matrix proteins. To critically test this hypothesis, we developed an in situ approach to isolate newly formed bone from more mature bone within the same long bone, and combine this anatomical approach with Western blotting to make relative comparisons of 7 phosphorylated matrix proteins important for bone physiology and biomineralization. Interestingly, 75 kDa bone sialoprotein (BSP), 63 kDa osteopontin, and the 75 kDa form of bone acidic glycoprotein-75 (BAG-75) were enriched in primary bone as opposed to more mature cortical bone, while osteonectin, fetuin A, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein-1 (DMP-1) appeared to be equally distributed between these two bone tissue compartments. Analyses also revealed the presence of larger sized forms of osteopontin (and to a lesser degree BSP) mostly in newly formed bone, while larger forms of BAG-75 were mostly detected in more mature cortical bone. Smaller sized forms of DMP-1 and BAG-75 were detected in both newly formed and more mature bone tissue extracts, and they are likely the result of proteolytic processing in vivo. Intact DMP-1 (97 kDa) was only detected in unmineralized matrix extracts. These findings indicate that newly formed bone exhibits a non-collagenous matrix protein composition distinct from that of more mature compact bone even within the same long bone, and suggest that the temporal fate of individual non-collagenous proteins is variable in growing bone.

Eliminating exposure to aqueous solvents is necessary for the early detection and ultrastructural elemental analysis of sites of calcium and phosphorus enrichment in mineralizing UMR106-01 osteoblastic cultures.

The mechanism underlying the mineralization of bone is well studied and yet it remains controversial. Inherent difficulties of imaging mineralized tissues and the aqueous solubility of calcium and phosphate, the 2 ions which combine to form bone mineral crystals, limit current analyses of labile diffusible, amorphous, and crystalline intermediates by electron microscopy. To improve the retention of calcium and phosphorus, we developed a pseudo nonaqueous processing approach and used it to characterize biomineralization foci, extracellular sites of hydroxyapatite deposition in osteoblastic cell cultures. Since mineralization of UMR106-01 osteoblasts is temporally synchronized and begins 78 h after plating, we used these cultures to evaluate the effectiveness of our method when applied to cells just prior to the formation of the first mineral crystals. Our approach combines for the first time 3 well-established methods with a fourth one, i.e. dry ultrathin sectioning. Dry ultrathin sectioning with an oscillating diamond knife was used to produce electron spectroscopic images of mineralized biomineralization foci which were high-pressure frozen and freeze substituted. For comparison, cultures were also treated with conventional processing and wet sectioning. The results show that only the use of pseudo nonaqueous processing was able to detect extracellular sites of early calcium and phosphorus enrichment at 76 h, several hours prior to detection of mineral crystals within biomineralization foci.