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. 2012 Nov 7;9(76):2997-3007.
doi: 10.1098/rsif.2012.0314. Epub 2012 Jun 6.

Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage

Rebecca E Wilusz  1 , Louis E DeFrate, Farshid Guilak
Affiliations

Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage

Rebecca E Wilusz et al. J R Soc Interface. .

Abstract

The pericellular matrix (PCM) is a narrow region that is rich in type VI collagen that surrounds each chondrocyte within the extracellular matrix (ECM) of articular cartilage. Previous studies have demonstrated that the chondrocyte micromechanical environment depends on the relative properties of the chondrocyte, its PCM and the ECM. The objective of this study was to measure the influence of type VI collagen on site-specific micromechanical properties of cartilage in situ by combining atomic force microscopy stiffness mapping with immunofluorescence imaging of PCM and ECM regions in cryo-sectioned tissue samples. This method was used to test the hypotheses that PCM biomechanical properties correlate with the presence of type VI collagen and are uniform with depth from the articular surface. Control experiments verified that immunolabelling did not affect the properties of the ECM or PCM. PCM biomechanical properties correlated with the presence of type VI collagen, and matrix regions lacking type VI collagen immediately adjacent to the PCM exhibited higher elastic moduli than regions positive for type VI collagen. PCM elastic moduli were similar in all three zones. Our findings provide further support for type VI collagen in defining the chondrocyte PCM and contributing to its biological and biomechanical properties.

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Figures

Figure 1.
Figure 1.
(a) Phase contrast and (b) fluorescence images illustrating the selection of PCM and ECM testing regions in the deep zone of porcine cartilage as seen during AFM testing. Localization of type VI collagen immediately surrounding cell-sized voids was used to identify the PCM in IF-labelled samples. The AFM cantilever is shown in (a) and outlined in (b), with the red circle indicating the approximate location of the spherical tip. Scale bars, (a,b) 20 µm. (Online version in colour.)
Figure 2.
Figure 2.
An image analysis was performed in Mathematica to align stiffness maps and IF images for PCM data analysis. The inner edge of cell-sized voids was detected in (a) topographical maps and (b) IF images using (c,d) edge detection. The topographical map was translated in the x- and y-directions to minimize the distance between it and the IF image (e). Stiffness maps and IF images were cropped for alignment as needed (shown overlaid in f). Aligned IF images were thresholded and converted to binary masks to determine regions of positive IF-labelling (g). IF-positive masks and elastic moduli contour maps were analysed in Matlab to extract PCM moduli from each scan region (h). Scale bars, 5 µm. (Online version in colour.)
Figure 3.
Figure 3.
Stiffness mapping of the PCM in the (a,d) superficial, (b,e) middle and (c,f) deep zones of articular cartilage. (a–c) Representative IF images showing the distribution of type VI collagen around cell-sized voids in each zone. (d–f) Representative contour maps of calculated elastic moduli of PCM scan regions in each zone. The spatial distribution of type VI collagen co-localized with softer modulus regions within each scan. Scale bars, 5 µm. (Online version in colour.)
Figure 4.
Figure 4.
Elastic moduli of ECM and PCM regions measured in the middle/deep zone of unlabelled (black) and IF-labelled (white) cartilage sections. No difference in moduli was observed with IF-labelling in either region (p = 0.39 for ECM, p = 0.97 for PCM). ECM elastic moduli were significantly greater than PCM moduli in both unlabelled and IF-labelled cartilage sections (a: p < 0.001). Moduli presented as mean ± s.e. (N = 3, n = 17).
Figure 5.
Figure 5.
Representative scatter plots of elastic modulus versus relative fluorescence intensity in PCM scan regions in the (a) superficial, (b) middle and (c) deep zones. Regions positive for type VI collagen are designated in green and regions lacking type VI collagen are designated in black. Regions with higher relative fluorescence in IF-positive regions for type VI collagen exhibited lower elastic moduli. (Online version in colour.)
Figure 6.
Figure 6.
Box plots of coefficient of determination (r2) for linear regression of elastic modulus versus relative fluorescence intensity for each PCM scan region in the (a) superficial, (b) middle, (c) deep zones and (d) across all zones. Separate regression analyses were performed on PCM regions alone, ECM regions alone and the full scan region (PCM + ECM). Median r2 value is designated by the horizontal line within each box.
Figure 7.
Figure 7.
Elastic moduli of ECM (black) and PCM (white) regions measured in the superficial, middle and deep zones of articular cartilage. PCM elastic moduli exhibited zonal uniformity (p = 0.21). ECM elastic moduli in all zones were greater than their respective PCM moduli (a: p < 0.05). Superficial zone ECM elastic moduli were greater than middle and deep zone ECM moduli (b: p < 0.05). Moduli presented as mean ± s.e. (N = 5, n = 20 per zone).
Figure 8.
Figure 8.
Outward stiffness progression of elastic moduli from the PCM outer edge to the ECM in the superficial (open diamonds), middle (filled squares) and deep (open circles) zones. In the superficial zone, PCM moduli were less than moduli in all regions beyond the PCM outer edge (a: p < 0.05) and elastic moduli reached ECM values 2.5 μm from the PCM outer edge (*p > 0.05 when compared with ECM). In the middle zone, PCM moduli were less than moduli in regions greater than 1.0 μm from the PCM outer edge (b: p < 0.05) and elastic moduli reached ECM values 1.5 μm from the PCM outer edge (#p > 0.05 when compared with ECM). In the deep zone, PCM moduli were less than moduli in regions greater than 1.5 μm from the PCM outer edge (c: p < 0.05) and elastic moduli reached ECM values at the PCM outer edge (&p > 0.05 when compared with ECM). Moduli presented as mean ± s.e. (N = 5, n = 20 per zone).
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