Real-time spectroscopic analysis of extracellular matrix produced by MC3T3-E1 preosteoblastic cells cultured under dynamic conditions

The deposition of extracellular matrix (ECM) produced by osteoblasts is one of the first steps in bone formation. Composition and structure of the ECM influence the development and strength of bone, as well as the onset of its mineralization. Since ECM is secreted onto the surface where the cells attach, Fourier transform infrared (FT-IR) attenuated total reflection (ATR), as a surface sensitive technique, provides a useful tool for its investigation, as ECM instead of the cells is predominantly detected by the IR beam. The purpose of the present study was to develop the FT-IR ATR technique so that real-time measurements of the ECM produced by MC3T3-E1 osteoblasts could be obtained in situ. Measurements were performed using polarized incident IR light to apply a procedure for solvent compensation which reduces the influence of culture medium on the evaluation of the amide I and II bands. The formation of ECM took place in a flow-through chamber under dynamic conditions by applying a constant flow of culture medium and was tracked over a time period of two weeks by evaluation of the integrated absorbance of amide I and II bands reflecting the amount and isotropic arrangement of amide bonds in the ECM. Cultures without ascorbic acid had a reduced protein concentration that enabled the analysis of cell-mediated matrix accumulation. Presence and proliferation of cells after two weeks of permanent flow-through of culture medium was shown by cell counting exhibiting a 67% increase in cell number as well as by crystal violet and live/dead staining. These results demonstrate the application of FT-IR ATR spectroscopy for monitoring matrix formation.