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Research Papers

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

[+] Author and Article Information
Holly A. Leddy, Susan E. Christensen1

Departments of Surgery and Biomedical Engineering, Duke University Medical Center, Durham, NC 27710

Farshid Guilak2

Departments of Surgery and Biomedical Engineering, Duke University Medical Center, Durham, NC 27710guilak@duke.edu

1

Leddy and Christensen contributed equally to this work.

2

Corresponding author.

J Biomech Eng 130(6), 061002 (Oct 08, 2008) (8 pages) doi:10.1115/1.2979876 History: Received February 27, 2008; Revised May 15, 2008; Published October 08, 2008

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.

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Copyright © 2008 by American Society of Mechanical Engineers
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Figures

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Figure 1

The intensity of the photobleached line decreases over the course of a SCAMP experiment. Throughout this time frame, the intensities are similar for an experimental data set (top) and its corresponding theoretical simulation (bottom).

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Figure 2

The average fluorescence intensity across a SCAMP line is plotted for a range of diffusion coefficients, D(μm2 s−1), and bleaching rate constants, k(s−1). Higher diffusion coefficients and lower bleaching rate constants lead to slower bleaching.

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Figure 3

The average line intensity decreases over time due to photobleaching in SCAMP experiments. The simulation provides excellent fits to the data (R2=0.98).

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Figure 4

The point-spread function was measured by assembling z-stack images of subresolution fluorescent microspheres. x-y is the imaging plane. The clear asymmetry varies greatly from the theoretical point-spread function, which would show rotational symmetry along the z-axis. An intensity of 1.0 represents a voxel maximally detectable by the microscope; an intensity of 0.0 represents a voxel minimally detectable by the microscope.

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Figure 5

The bleach (excitation) matrix was determined by photobleaching along a 12-pixel line in an agarose gel, which contained immobilized fluorophore, and then by imaging the matrix at very low laser power. x-y is the imaging plane; x is the line-scan direction. A higher intensity corresponds to a greater degree of bleaching that occurs at a given point in space.

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Figure 6

(a) The percentage error in the estimate of D decreases as more time points are fitted (n=11/bar; mean+SEM). (b) The percentage error in the estimate of D decreases as the signal-to-noise ratio increases (n>100/bar; mean+SEM). The data sets being fitted were simulations of known D, covering the range of expected values, to which random noise with a standard deviation of 1, 2, or 3 was added.

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Figure 7

The relative size of the SCAMP photobleaching region and the PCM. (a) A DIC image, (b) a fluorescence image stained for type VI collagen, and (c) an overlay of the two show a porcine chondrocyte with the surrounding PCM defined by the presence of type VI collagen (staining as described in Youn (28)). A porcine chondrocyte is typically 14 μm in diameter and surrounded by a 2-μm-thick PCM. (d) When positioned appropriately, a rectangular region containing the maximal excursion of a bleached region (3.3×1.9×5.7 μm3) fits within this PCM.

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Figure 8

The bleaching rate constant, k(s−1), decreases significantly with increasing depth into the tissue (top, linear regression p=0.00005), while the diffusion coefficient, D(μm2 s−1), does not change significantly (bottom, linear regression p=0.1). The line and equation (top) show the significant fit of the bleaching rate constant versus ln(depth) (R2=0.44).

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Figure 9

Diffusion coefficients for the ECM (dark bars) and PCM (light bars) of porcine cartilage for 70 kDa and 500 kDa dextrans (mean±SEM). There was a significant effect of both dextran size and matrix with no significant interactive effect (ANOVA, p<0.00001). Diffusion coefficients were significantly lower in the PCM than in the adjacent ECM (p=0.0001) and were significantly lower with the 500 kDa dextran than with the 70 kDa dextran (p=0.02).

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