Cartilage Assessment Requires a Surface Characterization Protocol: Roughness, Friction, and Function

Abstract

The surface of articular cartilage is integral to smooth, low-friction joint articulation. However, the majority of cartilage literature rarely includes measurements of surface characteristics and function. This may, in part, be due to a shortage of or unfamiliarity with fast, nondestructive, and, preferably, noncontact methods that can be applied to large cartilage surfaces for evaluating cartilage surface characteristics. A comprehensive methodology for characterizing cartilage surfaces is useful in determining changes in tissue function, as for example, in cases where the quality of cartilage grafts needs to be assessed. With cartilage storage conditions being an area of ongoing and active research, this study used interferometry and tribology methods as efficient and nondestructive ways of evaluating changes in cartilage surface topography, roughness, and coefficient of friction (CoF) resulting from various storage temperatures and durations. Standard, destructive testing for bulk mechanical and biochemical properties, as well as immunohistochemistry, were also performed. For the first time, interferometry was used to show cartilage topographical anisotropy through an anterior-posterior striated pattern in the same direction as joint articulation. Another novel observation enabled by tribology was frictional anisotropy, illustrated by a 53% increase in CoF in the medial-lateral direction compared to the anterior-posterior direction. Of the storage conditions examined, 37°C, 4°C, -20°C, and -80°C for 1 day, 1 week, and 1 month, a 49% decrease in CoF was observed at 1 week in -80°C. Interestingly, prolonged storage at 37°C resulted in up to an 83% increase in the compressive aggregate modulus by 1 month, with a corresponding increase in the glycosaminoglycan (GAG) bulk content. This study illustrates the differential effects of storage conditions on cartilage: freezing tends to target surface properties, while nonfreezing storage impacts the tissue bulk. These data show that a bulk-only analysis of cartilage function is not sufficient or representative. The nondestructive surface characterization assays described here enable improvement in cartilage functionality assessment by considering both surface and bulk cartilage properties; this methodology may thus provide a new angle to explore in future cartilage research and tissue engineering endeavors.

Keywords: anisotropy; articular cartilage; osteochondral allografts; storage; surface characterization; tribology.

FIG. 1.

Schematic diagram of the cartilage explants obtained from juvenile bovine femoral condyles and the subdivisions used for mechanical, biochemical, and histological assays. Wavy, banded lines represent collagen fibers. This schematic illustrates the wide range of assays used to comprehensively examine the effects of storage. Histo, histology; IHC, immunohistochemistry; Pyr, pyridinoline crosslinks analysis. Color images are available online.

FIG. 2.

Surface topography impacts tribology measurements. (A) A topographical image of a representative sample's surface taken by interferometry reveals a striated pattern in the direction of articulation. (B) Tribology measurements performed in the A–P and M–L directions indicate that the M–L CoF is 53% greater than in the A–P direction, providing further support for the anisotropy of articular cartilage surfaces. The asterisks denotes significant differences compared to the control condition based on Fisher's LSD post hoc test (* denotes p < 0.05). A–P, anterior–posterior; CoF, coefficient of friction; M–L, medial–lateral. Color images are available online.

FIG. 3.

Surface properties are unaffected by storage at nonfreezing temperatures. Using interferometry measurements, significant differences in (A) roughness were detected at subphysiological temperatures. No significant differences were observed in (B) alignment compared to the control samples (37°C at 1 day). However, tribological analysis showed that (C) CoF is significantly reduced at both −20°C and −80°C at 1 week. The asterisks denotes significant differences compared to the control condition based on Fisher's LSD post hoc test (* denotes p < 0.05). Color images are available online.

FIG. 4.

IHC of explants from the femoral condyle. Antilubricin shows expression of the protein predominantly in the superficial zone of cartilage in the positive control (1 day at 37°C). No lubricin is detected in nonarticulating costal cartilage surfaces (negative control). Even after a month of storage, the surface layer of lubricin is unaffected by temperature, however, cellular expression of lubricin is only maintained at 37°C. Color images are available online.

FIG. 5.

The (A) Young's modulus and (B) ultimate tensile strength values significantly differed for a few groups compared to controls, but there is no clear trend to the changes. (C) Collagen per DW and (D) pyridinoline crosslinks per DW, both of which are associated with the tensile properties of cartilage, similarly lacked any trends, despite the presence of a few significantly different groups. The asterisks denotes significant differences compared to the control condition based on Fisher's LSD post hoc test (* denotes p < 0.05). DW, dry weight. Color images are available online.

FIG. 6.

Compressive properties and GAG content increased in samples stored at physiological temperature. A significant increase was found in the (A) aggregate modulus and (B) SM values of samples stored at 37°C beginning after 1 week. (C) GAG normalized by DW also increased after a month in samples stored at 37°C. The compressive properties and GAG content were not adversely affected by storage at 4°C or lower. The asterisks denotes significant differences compared to the control condition based on Fisher's LSD post hoc test (* denotes p < 0.05). GAG, glycosaminoglycan; SM, shear modulus. Color images are available online.

FIG. 7.

H&E, picrosirius red, and safranin-O stains of samples stored at 37°C. Safranin-O histological stains indicated that the GAG content of cultured cartilage explants increased over time. H&E, hematoxylin and eosin. Color images are available online.