Characterization of biological mineralization in vitro

Abstract

Mineralization is an essential requirement for normal skeletal development, which is generally accomplished through the function of two cell types, osteoblasts and chondrocytes. Soft tissues do not mineralize under normal conditions, but under certain pathological conditions some tissues like articular cartilage and cardiovascular tissues are prone to mineralization. The aim of this study was to gain more insight in the processes that take place during the initiation of biological or cell-mediated mineralization and the effectors involved in these processes. Therefore, the mouse embryonal carcinoma-derived ATDC5 cell line was used in our study. It was hypothesized that serum, which is known to contain mineralization inhibitors such as fetuin, inhibits the early stages of mineralization in the cell culture system. Therefore, it was tested whether excluding serum from the medium shortened the cell culture period to induce mineralization, making this model system more suitable for investigation. This resulted in the development of large (sub millimetre size range) mineralizing structures (LMS) in the medium in 2 hours. LMS were shown to contain whole cells, which were embedded in hydroxyapatite and observed to have a stretched morphology. In the presence of serum no LMS were formed, but multiple small mineralized structures were observed inside the cell after 24 hours. Taken together, the results show that excluding serum in the cell culture system enhances rapid crystal growth and suggest that cell-mediated mineralization may start intracellularly. To investigate whether ATDC5 cells themselves or factors released by ATDC5 cells nucleate and/or remodel hydroxy-apatite in the absence of serum, the effect of conditioned medium from ATDC5 cells on the formation of apatite crystal was investigated. It was found that soluble factors released by the ATDC5 cells have the ability to affect the formation of the calcium-phosphate crystal. This suggest that soluble factors released by ATDC5 cells play a role in the growth phase of LMS formation once in the initial phase the crystal nucleus has been formed. Since an imbalance of mineralization may lead to pathological conditions, the ATDC5 cell culture system was also used to test the effect of several agents implicated in bone growth and development as well as pathological mineralization, including the gaseous substance nitric oxide (NO). Therefore, the effect of an NO donor drug, sodium nitroprusside (SNP) on cell mediated mineralization was investigated. It was found that 100 ?M SNP inhibits mineralization. However, the inhibition was not affected by inhibitors of guanylyl cyclase nor mimicked by a cGMP analog. Furthermore, sodium nitroprusside did not inhibit phosphate uptake nor inhibited apoptosis in the ATDC5 cells. Therefore, we subsequently investigated the effect of SNP on mineralization to elucidate the mechanism of action. It was shown that the iron moiety of sodium nitroprusside, rather than nitric oxide, inhibits mineralization.