ECTS2013 Poster Presentations Cell biology: osteoblasts and bone formation (50 abstracts)
Mineralizing properties of DMP1 studied in vitro with cellular and acellular 3D collagen model systems mimicking the bone tissue
Jérémie Silvent 1 , Nadine Nassif 1 , Thierry Azaïs 1 , Christophe Hélary 1 , Sidney Delgado 1 , Fabrice Soncin 2 , Marie Madeleine Giraud-Guille 1 & Jean-Yves Sire 1
1Université Pierre et Marie Curie, Paris, France; 2Institut de Biologie de Lille, Lille, France.
Bone is a complex structure associating cells to an extracellular organic phase, including collagen and non collagenous proteins (NCPs), in close association with apatite mineral platelets. Although bone has given rise to extensive studies, the exact part played by NCPs in nucleating or inhibiting the mineral phase remains controversial. The present study aimed to better understand the functions of a major mineralizing protein, dentin matrix phosphoprotein 1 (DMP1), an acidic, highly phosphorylated protein secreted during dentin, and bone formation.
In a first step, in order to identify a correlation between the expression of various NCP genes and apatite crystal deposition, we performed a 60 days cell culture experiment using primary human osteoblasts seeded on dense 3D collagen matrices mimicking the osteoid tissue. We show that i) the cells displayed features characteristic of osteoblasts in vivo (mineralization, protein, and gene expression) and ii) DMP1 expression correlated with the first hydroxyapatite nucleation at day 21.
In a second step, in order to target conserved motifs that could be involved in the mineralization process, we underwent an evolutionary analysis of mammalian DMP1. Among various evolutionary conserved motifs we identified in the C-terminal region several new motifs rich in acidic residues. This region was predicted to play a role in the mineralization process.
In a third step, we tested the possible function of these highly conserved motifs using a recombinant DMP1 peptide, designed in the C-terminal region, which comprised also two collagen binding sites. The recombinant was added at two concentrations (2.5 and 25 μg/ml) to a dense collagen-matrix system mimicking the compact bone matrix. In the two conditions, our first data strongly suggest that DMP1 is involved in aggregating the mineral phase inside the collagen fibrils and in inhibiting ectopic mineralization.
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Bone Abstracts
ISSN 2052-1219 (online)
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