Spatial mapping of the biomechanical properties of the pericellular matrix of articular cartilage measured in situ via atomic force microscopy

Abstract

In articular cartilage, chondrocytes are surrounded by a narrow region called the pericellular matrix (PCM), which is biochemically, structurally, and mechanically distinct from the bulk extracellular matrix (ECM). Although multiple techniques have been used to measure the mechanical properties of the PCM using isolated chondrons (the PCM with enclosed cells), few studies have measured the biomechanical properties of the PCM in situ. The objective of this study was to quantify the in situ mechanical properties of the PCM and ECM of human, porcine, and murine articular cartilage using atomic force microscopy (AFM). Microscale elastic moduli were quantitatively measured for a region of interest using stiffness mapping, or force-volume mapping, via AFM. This technique was first validated by means of elastomeric models (polyacrylamide or polydimethylsiloxane) of a soft inclusion surrounded by a stiff medium. The elastic properties of the PCM were evaluated for regions surrounding cell voids in the middle/deep zone of sectioned articular cartilage samples. ECM elastic properties were evaluated in regions visually devoid of PCM. Stiffness mapping successfully depicted the spatial arrangement of moduli in both model and cartilage surfaces. The modulus of the PCM was significantly lower than that of the ECM in human, porcine, and murine articular cartilage, with a ratio of PCM to ECM properties of approximately 0.35 for all species. These findings are consistent with previous studies of mechanically isolated chondrons, and suggest that stiffness mapping via AFM can provide a means of determining microscale inhomogeneities in the mechanical properties of articular cartilage in situ.

Figure 1

Light microscopy images illustrating testing regions in (A) human, (B) porcine, and (C) murine articular cartilage. The AFM cantilever is also shown. Murine articular cartilage exhibits the greatest cellularity of the species tested. This high cell density made it difficult to find test regions that were solely ECM in murine articular cartilage. Scale bar = 100 μm.

Figure 2

Stiffness mapping successfully depicted the spatial arrangement of moduli in PA ECM/PCM model systems. (A) Soft lithography was used to generate a mold of regularly spaced holes (20 μm diameter, 12 μm depth, 50 μm center-to-center distance) composed of stiff PA gel. (B) Holes were filled with a soft PA gel (green) to generate a soft inclusion within a stiff medium. Scale bar = 50 μm. (C) Stiffness map of model PCM showing height and calculated values of elastic moduli. (D) The elastic modulus of the stiff, outer material was fivefold higher than that of the soft, inner material (^∗p < 0.0001 as compared to respective ECM, # p < 0.001 as compared to macroscale). Moduli shown as mean ± standard deviation.

Figure 3

(A) SEM image of ECM/PCM in the middle/deep zones of porcine articular cartilage. The capsule-like PCM is structurally distinct from the surrounding ECM and localized around a cell-sized void in the tissue section. Magnification = 10,000×. Scale bar = 5 μm. (B) Porcine tissue sections were stained with fast green to indicate collagen fibers (green) and safranin-O to stain proteoglycans (red). Safranin-O staining is more intense in regions surrounding cell-sized voids, indicative of PCM. Scale bar = 50 μm. (C) Immunofluorescence illustrates the localization of type VI collagen to the pericellular matrix region around porcine chondrocytes. Scale bar = 50 μm.

Figure 4

Stiffness mapping of human, porcine, and murine articular cartilage. Representative test regions are shown. (A, D, and G) Representative stiffness maps of the PCM of articular cartilage showing the height and calculated values of elastic moduli. (B, E, and H) Contour maps of the PCM. For the purposes of this study, the PCM was defined as the region extending 1 μm from cell-sized voids. Voids and ECM regions are white. (C, F, and I) Histograms of calculated elastic moduli exhibit a bimodal distribution. The low modulus peak corresponds to points within the 1 μm PCM region (black). The high modulus peak corresponds to the local ECM within each scan region (white).