title: 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.
authors.affiliation: Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. midurar@ccf.org
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Midura
authors.forename: Sharon B
authors.initials: SB
authors.lastname: Midura
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Su
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Gorski
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001857:Bone Matrix / Q000187:drug effects / Q000378:metabolism*; D002113:Calcification, Physiologic / Q000187:drug effects; D018483:Diaphyses / Q000166:cytology / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006244:Hardness / Q000187:drug effects; D008297:Male; D008970:Molecular Weight; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology*; D010521:Periosteum / Q000166:cytology / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013977:Tibia / Q000166:cytology / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D004492:Edetic Acid