Implants of allogenic demineralized bone matrix were placed in distinct in vivo environments, i.e. calvarial (bony) and subcutaneous (soft tissue) sites. Detailed analyses of the biochemical components were performed. Quantitative levels of osteopontin (OPN), bone sialoprotein (BSP) and calcium phosphate (Ca-P) deposition within each implant environment varied as a function of new bone formation, and were substantially different in samples from calvarial and subcutaneous sites. Quantification of the extent of phosphorylation of affinity-purified OPN and BSP from such implants indicated that: (i) the number of mols of phosphoserine (P-Ser)/mol of affinity-purified OPN or BSP varied as a function of implant time and bone formation within both implant sites, and (ii) the ‘effective P-Ser concentration’ provided by the total OPN and BSP within each implant site varied and increased as a function of time, being approx. 5-fold higher for BSP in calvarial compared with subcutaneous implants. Peak levels of mols of P-Ser/mol of BSP coincided with maximum rates of Ca-P deposition in calvarial implants. Levels of OPN phosphorylation from both calvarial and subcutaneous implants also indicated fluctuations as a function of bone formation. Hence the present study, for the first time, provides direct evidence of natural variation in the extent of phosphorylation of both OPN and BSP as a function of time of mineralized tissue formation. Further evaluation of the data provides the first evidence of a direct and linear relationship between the rate of Ca-P deposition and the ratio of P-Ser-BSP/P-Ser-OPN for calvarial implants. Data for subcutaneous implants failed to provide such correlation. Overall, the present work demonstrates that the natural biological progression of the process of biomineralization follows strict criteria consistent with the anatomical location. Biomineralization fails to proceed in the same way in a soft tissue environment.
Abbreviations used: ECM, extracellular matrix; OPN, osteopontin; BSP, bone sialoprotein; Ca-P, calcium phosphate; mCKII, microsomal casein kinase II; DBM, demineralized bone matrix; P-Ser, phosphoserine; CBB, Coomassie Brilliant Blue R-250; ALP, alkaline phosphatase; PBST, PBS containing 0.05% (v/v) Tween 20.
These authors contributed equally to this work.
Present address: University of Southern California School of Dentistry, Los Angeles, CA, U.S.A.