Matrix Recruitment and Calcium Sequestration for Spatial Specific Otoconia Development

Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca2+ for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca2+ in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the null mice leads to drastically reduced matrix-Ca2+. In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca2+-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca2+ for efficient CaCO3 crystallization, thus uncover a critical process governing spatial specific otoconia formation

Study of the maturation of the organic (type I collagen) and mineral (nonstoichiometric apatite) constituents of a calcified tissue (dentin) as a function of location: A Fourier transform infrared microspectroscopic investigation

International audienceFourier transform infrared microspectroscopy (FTIRM) was used to investigate the organic and mineral phases of a calcified tissue (dentin) as a function of its location from predentin toward enamel, Thin dentin slices (decalcified or not) were fixed in formaldehyde and embedded in glycolmethylmethacrylate (GMA), Fixation did not denature collagen, and GMA did not interact with organic or mineral constituents of dentin, The nu (x)nu (3) PO4 domain was studied in particular in order to estimate mineral maturity and amide I, II, A, and B to obtain data on protein conformation. The results showed that dentin apatite became increasingly mature (stoichiometric) from the mineralization front toward the enamel, especially through loss of HPO42- groups and vacancies. Moreover, collagen fibrils became less and less hydrated, suggesting that intrafibrillar mineralization partially dehydrated the collagen. Combined study of the organic and mineral fractions of calcified tissues may help clarify their relationships in physiological and pathological tissues

Quantitative Immunogold Labeling of Bone Sialoprotein and Osteopontin in Methylmethacrylate-embedded Rat Bone

Methylmethacrylate (MMA) embedding of undecalcified bone is routinely employed for histomorphometric analyses. Although MMA-embedded bone has been used for immunolabeling at the light microscopic level after removal of the resin, there are no such reports for electron microscopy. The aim of the present study was to determine whether MMA embedding can be used for ultrastructural immunolabeling and how it compares to LR White (LRW), an acrylic resin frequently used for immunocytochemistry of bone. Rat tibiae were fixed by vascular perfusion with aldehyde and embedded either in MMA or LRW resin. Thin sections were processed for postembedding protein A-gold immunolabeling with antibodies to rat bone sialoprotein (BSP) and osteopontin (OPN). The density of gold particles over bone was quantified. The density and distribution of immunolabeling for BSP and OPN respectively, were comparable between MMA and LRW. These results indicate that MMA performs as well as LRW for the ultrastructural immunolabeling of noncollagenous bone matrix proteins. </jats:p

Calcium-deficient apatite: A first in vivo study concerning bone ingrowth

Biphasic calcium phosphate (BCP) materials are increasingly used to restore bone loss in surgery. Calcium-deficient apatites (CDA), the precursors of BCP, are closer in structure to biological apatites and can be associated with therapeutic agents to form drug-delivery systems. The purpose of this first hi vivo study of CDA was to evaluate the osteoconductive properties of two composites, consisting of 40-80 mum granules carried by a cellulose-derived polymer, used to fill critical size bone defects in rabbit femoral ends. Animals were sacrificed 2 or 3 weeks after implantation. Histomorphometric analysis of scanning electron microscopy implant surface files was performed using gray level threshold that distinguish between bone or materials (white) and noncalcified tissue (black). Quantitative results for new bone formation showed no significant differences between the composites or the implantation periods. However, nearly all of the CDA disappeared early while supporting more extensive bone colonization than biphasic calcium phosphates implanted in the same conditions. (C) 2003 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 402-408, 2003

Development of a monoclonal antibody against dentin phosphophoryn : a tool to study odontoblastic activity

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Dental Caries and Enamelin Haplotype

International audienceIn the literature, the enamelin gene ENAM has been repeatedly designated as a possible candidate for caries susceptibility. Here, we checked whether ENAM variants could increase caries susceptibility. To this aim, we sequenced coding exons and exon-intron boundaries of ENAM in 250 children with a severe caries phenotype and in 149 caries-free patients from 9 French hospital groups. In total, 23 single-nucleotide polymorphisms (SNPs) were found, but none appeared to be responsible for a direct change of ENAM function. Six SNPs had a high minor allele frequency (MAF) and 6 others were identified for the first time. Statistical and evolutionary analyses showed that none of these SNPs was associated with caries susceptibility or caries protection when studied separately and challenged with environmental factors. However, haplotype interaction analysis showed that the presence, in a same variant, of 2 exonic SNPs (rs7671281 and rs3796704; MAF 0.12 and 0.10, respectively), both changing an amino acid in the protein region encoded by exon 10 (p.I648T and p.R763Q, respectively), increased caries susceptibility 2.66-fold independent of the environmental risk factors. These findings support ENAM as a gene candidate for caries susceptibility in the studied population