Nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell.

Avian (and formerly dinosaur) eggshells form a hard, protective biomineralized chamber for embryonic growth-an evolutionary strategy that has existed for hundreds of millions of years. We show in the calcitic chicken eggshell how the mineral and organic phases organize hierarchically across different length scales and how variation in nanostructure across the shell thickness modifies its hardness, elastic modulus, and dissolution properties. We also show that the nanostructure changes during egg incubation, weakening the shell for chick hatching. Nanostructure and increased hardness were reproduced in synthetic calcite crystals grown in the presence of the prominent eggshell protein osteopontin. These results demonstrate the contribution of nanostructure to avian eggshell formation, mechanical properties, and dissolution.

Molecular dynamics simulation of mechanical behavior of osteopontin-hydroxyapatite interfaces.

Bone is characterized with an optimized combination of high stiffness and toughness. The understanding of bone nanomechanics is critical to the development of new artificial biological materials with unique properties. In this work, the mechanical characteristics of the interfaces between osteopontin (OPN, a noncollagenous protein in extrafibrillar protein matrix) and hydroxyapatite (HA, a mineral nanoplatelet in mineralized collagen fibrils) were investigated using molecular dynamics method. We found that the interfacial mechanical behavior is governed by the electrostatic attraction between acidic amino acid residues in OPN and calcium in HA. Higher energy dissipation is associated with the OPN peptides with a higher number of acidic amino acid residues. When loading in the interface direction, new bonds between some acidic residues and HA surface are formed, resulting in a stick-slip type motion of OPN peptide on the HA surface and high interfacial energy dissipation. The formation of new bonds during loading is considered to be a key mechanism responsible for high fracture resistance observed in bone and other biological materials.

Spheroid-forming subpopulation of breast cancer cells demonstrates vasculogenic mimicry via hsa-miR-299-5p regulated de novo expression of osteopontin.

The growth of cancer cells as multicellular spheroids has frequently been reported to mimic the in vivo tumour architecture and physiology and has been utilized to study antitumour drugs. In order to determine the distinctive characteristics of the spheroid-derived cells compared to the corresponding monolayer-derived cells, we enriched multicellular spheroid-forming subpopulations of cells from three human breast cancer cell lines (MCF7, MCF10AT and MCF10DCIS.com). These spheroid-derived cells were injected into female athymic nude mice to assess their tumorigenic potential and were profiled for their characteristic miRNA signature. We discovered that the spheroid-derived cells expressed increased levels of osteopontin (OPN), an oncogenic protein that has been clinically correlated with increased tumour burden and adverse prognosis in patients with breast cancer metastasis. Our studies further show that increased OPN levels are brought about in part, by decreased levels of hsa-mir-299-5p in the spheroid-forming population from all three cell lines. Moreover, the spheroid-forming cells can organize into vascular structures in response to nutritional limitation; these structures recapitulate a vascular phenotype by the expression of endothelial markers CD31, Angiopoeitin-1 and Endoglin. In this study, we have validated that hsa-mir-299-5p targets OPN; de novo expression of OPN in turn plays a critical role in enhancing proliferation, tumorigenicity and the ability to display vasculogenic mimicry of the spheroid-forming cells.

Role of phosphate groups in inhibition of calcium oxalate crystal growth by osteopontin.

Osteopontin (OPN) inhibits the growth of calcium oxalate monohydrate (COM) and other crystal phases in a phosphorylation-dependent manner. In the present study, the role of OPN phosphate groups in adsorption to, incorporation into and inhibition of COM crystals was studied by comparing OPN isoforms differing in phosphorylation. OPN isoforms purified from rat bone (bOPN), which contains 10 phosphates, and cow milk (mOPN), which contains 25 phosphates, were compared with rat recombinant OPN (rOPN), which is not phosphorylated. Using fluorescence-labeled proteins and confocal microscopy, we show that mOPN and rOPN, like bOPN, adsorb preferentially to the edges between {100} and {121} faces of preformed COM crystals, and to a lesser extent to the {100} and {121} faces. Using scanning electron microscopy, we show that growth of COM in the presence of bOPN or mOPN results in a 'dumbbell' morphology, whereas crystals grown with rOPN are only slightly affected. COM crystals grown in the presence of low concentrations of fluorescence-labeled bOPN incorporate the protein into the crystal lattice. In crystals imaged in the {010} plane, incorporation of bOPN results in a cross-shaped pattern of fluorescence, consistent with preferential adsorption to {100}/{121} edges throughout the growth process.

The influence of surface mineral and osteopontin on the formation and function of murine bone marrow-derived osteoclasts.

The phosphorylated glycoprotein osteopontin (OPN) is involved in the regulation of biomineralization under normal and pathological conditions. Its actions include inhibiting apatite crystal growth and promoting the formation and function of mineral resorbing cells, including osteoclasts (OCL). The purpose of this study was to develop stable apatitic mineral surfaces and determine their influence on OCL formation and mineral resorption from bone marrow macrophages derived from OPN wild-type (OPN+/+) and OPN deficient (OPN-/-) mice. We demonstrated that these mineral coatings were stable and supported bone marrow-derived macrophage differentiation to OCL under our culture conditions. Macrophages harvested from OPN-/- mice had a greater capacity to form OCL than macrophages from OPN+/+ mice when allowed to differentiate on tissue culture plastic. In contrast, when allowed to differentiate on a mineral surface, no difference in OCL formation was observed. Interestingly, OPN+/+ OCL were more efficient at mineral dissolution than OPN-/- OCL, and this difference was observed regardless of differentiating surface. Our results suggest that mineralized substrates as well as ability to synthesize OPN both control OCL function in our model system. The exact nature of these effects may be dependent on variables related to mineral substrate presentation.