ToF-SIMS and laser-SNMS analysis of apatite formation in extracellular protein matrix of osteoblasts in vitro

We have applied laser secondary neutral mass spectrometry (laser-SNMS) for examining different states of biomineralization in vitro. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to analyse fibronectin and collagen type I, the main matrix proteins involved in the mineralization process, in a model system. Primary osteoblast-like cells derived from bovine metacarpals were cultured for 5 weeks on clean smooth silicon substrates. For mass spectrometric investigations, cells and newly-formed minerals were cryofixed, freeze-fractured, and freeze-dried. The results indicate that extracellular enrichment of potassium typically occurs in the vicinity of single osteoblasts during the initial stages of mineralization. This interaction of potassium with matrix macromolecules may prevent uncontrolled apatite nucleation and control the transformation of the matrix into a nucleating system. Thus, monovalent cations seem to be involved in the regulation of mineral nucleation of the extracellular matrix. From the data obtained it can be concluded that ToF-SIMS and laser-SNMS are well suited for imaging trace element and molecule concentrations in biological samples as well as for identifying molecular fragments characteristic of matrix proteins. Copyright (C) 2004 John Wiley Sons, Ltd.