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

ABSTRACT: 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.

SUBMITTER: Midura RJ

PROVIDER: S-EPMC3221780 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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

Midura Ronald J RJ Midura Sharon B SB Su Xiaowei X Gorski Jeffrey P JP

Bone 20110918 6

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 comb ...[more]

PMID: 21958842

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Project description:Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

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

Publications

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

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