Microscale Diffusion Properties of the Cartilage Pericellular Matrix Measured Using 3D Scanning Microphotolysis

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Abstract
Chondrocytes, the cells in articular cartilage, are enclosed within a pericellular matrix (PCM) whose composition and structure differ from those of the extracellular matrix (ECM). Since the PCM surrounds each cell, molecules that interact with the chondrocyte must pass through the pericellular environment. A quantitative understanding of the diffusional properties of the PCM may help in elucidating the regulatory role of the PCM in controlling transport to and from the chondrocyte. The diffusivities of fluorescently labeled 70 kDa and 500 kDa dextrans were quantified within the PCM of porcine articular cartilage using a newly developed mathematical model of scanning microphotolysis (SCAMP). SCAMP is a rapid line photobleaching method that accounts for out-of-plane bleaching attributable to high magnification. Data were analyzed by a best-fit comparison to simulations generated using a discretization of the diffusion-reaction equation in conjunction with the microscope-specific three-dimensional excitation and detection profiles. The diffusivity of the larger molecule (500 kDa dextran) was significantly lower than that of the smaller molecule (70 kDa dextran), and values were consistent with those reported previously using standard techniques. Furthermore, for both dextran sizes, the diffusion coefficient was significantly lower in the PCM than in the ECM; however, this difference was not detected in early-stage arthritic tissue. We have successfully modified the SCAMP technique to measure diffusion coefficients within the small volume of the PCM using confocal laser scanning microscopy. Our results support the hypothesis that diffusivity within the PCM of healthy articular cartilage is lower than that within the ECM, presumably due to differences in proteoglycan content.