Soluble factors released by ATDC5 cells affect the formation of calcium phosphate crystals

During biomineralization the organism controls the nature, orientation, size and shape of the mineral phase. The aim of this study was to investigate whether proteins or vesicles that are constitutively released by growing ATDC5 cells have the ability to affect the formation of the calcium phosphate crystal. Therefore, subconfluent cultured ATDC5 cells were incubated for 1 h in medium without serum. Subsequently, medium was harvested and incubated for 24 h in the presence of additional Pi. This resulted in the formation of flat mineralizing structures (FMS), consisting of complex irregularly shaped flat crystals, which occasionally contained fiber-like structures (∼ 40 μm in size). Without pre-incubation of medium with cells, only small punctate (dot like) calcium phosphate precipitates were observed. The formation of FMS was shown to be caused by soluble factors released by subconfluent ATDC5 cells. Proteomic analysis by mass spectrometry showed that FMS contained a specific set intracellular proteins, serum proteins, and extracellular matrix proteins. Bulk cytosolic proteins derived from homogenized cells or serum proteins did, however, not induce the formation of FMS. Conditioned medium from HeLa, CHO K1, RAW 264.7 and MDCK cells was also capable to form FMS under our experimental conditions. Therefore the formation of FMS seems to be caused by specific soluble factors constitutively released by ADTC5 and other cells. This in vitro model system can be used as a tool to identify factors that affect the shape of the biomineral phase.