The glycosylation profile of osteoadherin alters during endochondral bone formation.

Endochondral bone formation involves the dynamic interplay between the cells and their extracellular environment to facilitate the deposition of a calcified matrix. Numerous molecules are involved within this process, including collagens and non-collagenous proteins, and their post-translational modifications have been shown to effect their biomolecular interactions. Osteoadherin (OSAD), a keratin sulfate (KS)-substituted small leucine-rich proteoglycan has been isolated from mineralized tissues and is considered to be a mineralized tissue-specific protein. However, to date, information is limited concerning the dynamic expression and role of this proteoglycan during bone formation and the biomineralization process. The current study aimed to examine the dynamic expression of this protein throughout mouse metatarsal long bone development, from the cartilage anlagen (E15) to the fully formed bone (Adult). Using quantitative gene expression analysis we observed that OSAD was produced with the onset of mineralization and the formation of the ossification center. This finding was reflected in the localization studies, using both light and electron microscopy, and showed that initial OSAD localization was restricted to the endosteal surfaces of the diaphysis and forming metaphysis. Furthermore, we analyzed protein extracts, both mineral and non-mineral associated fractions, and showed that OSAD was substituted with varying patterns of glycosylation during bone development. Sequential enzymatic digestions of the non-mineral bound protein extracts demonstrated that OSAD lacked any KS chains throughout all development stages. Whereas, in the mineral bound fractions, with long bone maturation the substitution with KS became more apparent with development. Therefore, it can be concluded that different pools of OSAD are produced during endochondral bone formation and these may have specific roles in directing the mineralization process.

Low-dose radiation with 80-kVp computed tomography to diagnose pulmonary embolism: a feasibility study.

BACKGROUND: Mounting collective radiation doses from computed tomography (CT) implies an increased risk of radiation-induced cancer in exposed populations, especially in the young. PURPOSE: To evaluate radiation dose and image quality at 80-kVp CT to diagnose acute pulmonary embolism (PE) compared with a previous study at 100 and 120 kVp with all other scanning parameters unchanged. MATERIAL AND METHODS: A custom-made chest phantom with a 12 mg I/mL-syringe was scanned at 80/100/120 kVp to evaluate relative changes in computed tomographic dose index (CTDI(vol)), attenuation, image noise, and contrast-to-noise ratio (CNR). Fifty patients underwent 80 kVp 16-row detector CT at 100 "Quality reference" mAs. A total of 350 mg I/kg were injected to compensate for increased CNR at 80 kVp, while 300 mg I/kg had been used at 100/120 kVp. CTDI(vol), dose-length product (DLP), and estimated effective dose were evaluated including Monte Carlo simulations. Pulmonary artery attenuation and noise were measured and CNR calculated. Two radiologists evaluated subjective image quality using a four-grade scale. RESULTS: Switching from 120 to 80 kVp in the phantom study decreased radiation dose by 67% while attenuation and noise increased 1.6 and 2.0 times, respectively, and CNR decreased by 16%. Switching from 120 to 80 kVp in the patient studies decreased estimated effective dose from 4.0 to 1.2 mSv (70% decrease) in median while pulmonary artery attenuation and noise roughly doubled from 332 to 653 HU and from 22 to 49 HU, respectively, resulting in similar CNR (13 vs. 12). At 80 kVp all examinations were regarded as adequate (8%) or excellent (92%). CONCLUSION: Switching from 120 to 80 kVp CT without increased mAs but slightly increased iodine dose may be of special benefit to diagnose PE in younger individuals with preserved renal function where the primary aim is to minimize radiation dose and reaching levels below that of scintigraphy.

Osteoadherin accumulates in the predentin towards the mineralization front in the developing tooth.

BACKGROUND: Proteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization. CONCLUSIONS: These studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

Substitution of bovine dentine sialoprotein with chondroitin sulfate glycosaminoglycan chains.

Dentine sialoprotein (DSP) represents 5-8% of all non-collagenous proteins present in the tooth, but, together with dentine phosphoprotein, has been shown to be vital for correct tooth formation. Recently, the existence of a highly glycosylated form of porcine DSP has been reported and it was shown to possess glycosaminoglycan (GAG) chains. The current investigation confirms that this is also the case for bovine DSP and has further characterized these carbohydrates. Dentine sialoprotein was purified from bovine dentine extracts by anion exchange chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectroscopy. An increase in molecular mass was observed, from 120 kDa to greater than 250 kDa, with a corresponding rise in anionic strength. Cellulose acetate electrophoresis and western blotting indicated the presence of chondroitin sulfate GAG chains within these dentine fractions. Further examination using sequential digestion with chondroitinase AC and N-glycosidase cleaved the samples first to 95 kDa and then to 80 kDa, respectively, confirming a high level of glycosylation. These results support the classification of bovine DSP as a proteoglycan, and that the carbohydrate substitutions may contribute to the functional properties of DSP.