Mechanical Loading and Matrix Metalloproteinase

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DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to develop a high-resolution force loader for applying precise loads to connective tissues, and to elucidate the role of mechanical stress and strain to rheumatoid joints. A turnover of collagens, predominant components in soft connective tissues, is at least in part regulated by families of proteolytic enzymes, and the matrix metalloproteinases (MMPs) have been considered as the influential group of proteinases in bone and soft connective tissues. In analyzing the role of mechanical stress and strain in matrix degradation, precise control of mechanical loads is important since the expression and the activities of MMPs depends heavily upon intensities, gradients, and duration of shear and strain. In this revised 2-year bioengineering research project, a specific aim is to design and fabricate a state-of-the-art piezo-electric device that can be utilized with inverted fluorescence and optical microscopy. Life scientists will be able to characterize cellular morphology and molecular dynamics in real-time under mechanical stimuli. The proposed piezo-electric device will be able to apply precise deformation of 100-500 um to cultured cells at 0.1-100 Hz sinusoidal or impulsive waveforms in varying directions. The device will be validated by reproducing the previous results showing that gentle cyclic stain alters the expression and the activities of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human synovial cells. In determining the expression and activities of MMPs and TIMPs, we will use RT-PCR, Western blotting, zymography as well as in vitro and in situ fibril degradation assays. The proposed project will contribute to understanding effects of mechanical stimuli on cellular morphology as well as molecular activities. The device can be used to address fundamental questions about how mechanical forces affect cells in real time as well as to elucidate the mechanisms of inflammation and degradation of connective tissue under mechanical stimuli.