UC San Diego

Our long-term goal is to understand the biochemical mechanism responsible for the calcification of collagen fibrils in normal bone formation. Previous studies in our lab have shown that purified type I collagen and demineralized bone matrix both calcify rapidly when incubated in serum in the absence of cells [1-4]. Therefore, the calcification of collagen is due to the presence of a serum calcification activity. Molecules smaller than a 6kDa protein can diffuse into the fibril matrix and can directly impact mineralization. While molecules larger than a 40kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril matrix [5]. Therefore, without a size-excluding matrix, inhibitors of apatite growth prevent us from studying the calcification activity in serum. We developed a simple method where we can examine the calcification activity in serum without the need of a size excluding matrix. Diluting serum 50- fold into a system with temperature, pH and calcium and phosphate levels comparable to blood enables us to isolate the serum calcification activity by reducing inhibitors of apatite growth down to concentrations where they can no longer prevent mineral formation. With this dilution method, we gain more insight into the calcification activity in serum and how this activity relates to bone metabolism