| The mechanism for the calcification of bone has been studied intensively for at least the past 35 years. The incorporation of calcium phosphate mineral into the collagenous matrix of bone has been described to be driven directly by cellular activity, either by chondrocytes in calcifying cartilage or osteoblasts during normal bone turnover and remodeling. Non-collagenous matrix proteins or matrix vesicles derived from chondrocytes and osteoblasts are widely believed to play a prominent role in the nucleation of calcium phosphate mineral. However, the cell culture models used to study bone mineralization have been shown by some authors to be problematic due to the potential non-physiological precipitation of mineral under the culture conditions commonly used. This dissertation presents a new in vitro model for bone matrix mineralization in which a demineralized newborn rat tibia can be remineralized in cell culture medium with sufficient ionic calcium and phosphate and serum. This is the first time a bone matrix from an animal has been remineralized under conditions that require no cells and is driven by serum at concentrations as little as 1.5%. By powder X-ray diffraction and FTIR spectroscopy; the newly incorporated mineral is physically identical to that originally found in bone. Utilizing high speed centrifugation, gel filtration, dialysis, SDS PAGE, and protease digestion of serum, the component(s) responsible for nucleating mineralization of bone are shown to be macromolecular and proteinaceous. Treatment of serum with EDTA shows that mineral seeds may be present in serum and their stability may be attributed to their affinity for a nucleator in serum. Etidronate, a well characterized in vivo inhibitor of bone mineralization, also blocks serum initiated calcification. In addition to rat and fetal calf, the serum nucleator activity is shown to be present in teleosts and sharks, but is not found in invertebrates. The discovery of a serum nucleator of bone mineralization may contribute to the understanding of how normal bone mineralization is initiated. However, the systemic nature of the nucleator suggests an equally important role in pathologic conditions in which soft tissues such as arteries and heart vales may become calcified. |
