Human articular cartilage is orthotropic where microstructure, micromechanics, and chemistry vary with depth and split-line orientation

Abstract

Objective: Quantitative, micrometer length scale assessment of human articular cartilage is essential to enable progress toward new functional tissue engineering approaches, including utilization of emerging 3D bioprinting technologies, and for improved computational modeling of the osteochondral unit. Thus the objective of this study was to characterize the structural organization, material properties, and chemical composition of human skeletally mature articular cartilage with respect to depth and defined morphological features: normal to the articulating surface, parallel to the split-line, and transverse to the split-line.

Method: Three samples from the lateral femoral condyles of 4 healthy adult donors (55-61 years old) were evaluated via histology, second harmonic generation, microindentation, and Raman spectroscopy. All metrics were evaluated as a function of depth and direction relative to the split-line.

Results: All donors presented with intact and healthy tissue. Collagen fiber orientation varied significantly between testing directions and with increasing depth from the articular surface. Both compressive and tensile modulus increased significantly with depth and differed across the middle and deep zones and depended on orthogonal direction relative to the split-line. Similarly, matrix components varied with both depth and direction, where chondroitin sulfate steadily increased with depth while collagen prevalence was highest in the surface layer.

Conclusions: Microscale measurements of human articular cartilage demonstrate that properties are both depth-dependent and orthotropic and depend on the underlying tissue structure and composition. These findings improve upon existing knowledge establishing more accurate measurements, with greater degree of depth and spatial specificity, as inputs for tissue engineering and computational modeling.

Keywords: Anisotropy; Articular cartilage; Indentation; Raman; SHG.

Fig. 1

Schematic of sample location and test setup show separate slices were harvested for histological analysis and all other characterization. Three separate cubes were harvested from each donor and split-line orientation was referenced when shaping the cubes so the three faces evaluated were normal (face 3), parallel (face 2), and perpendicular (face 1) to the split-line.

Fig. 2

Representative images showing donors had healthy articular cartilage A) MRI, B) gross dissection, C) Mankin scoring for each donor with inset representative histology slice (representative images from donor C170243).

Fig. 3

Collagen fiber orientation varied with depth and showed directional dependence relative to the split-line. A) SHG and OrientationJ overlay with insets highlighting fiber orientation analysis across zones B) continuous fit from face 1 and face 2 across the full depth of the articular cartilage (shaded regions represent confidence of fit) showing differences in collagen fiber orientation with depth between faces and C) binned zones with 0–20% representing surface, 20–60% middle, 60–90% deep zones (box plot with data outliers removed for visualization). (# denotes significant difference within zone between faces; S, M, and D denot significant difference with the surface, middle, and deep zone respectively within face).

Fig. 4

Modulus is dependent on both depth and testing direction, and anisotropy indicates cartilage as a zonally orthotropic material. A) compressive modulus across all three zones with directional dependence in the middle and deep zones, B) directionally and depth dependent tensile modulus values in the deep zone, C) compressive modulus anisotropy in the surface and middle zone, and D) tensile modulus anisotropy in the middle and deep zones (box plot with data outliers removed for visualization). (# denotes significant difference within zone between faces; S, M, and D denote significant difference with the surface, middle, and deep zone respectively within face; % denotes significant difference from the isotropic value of 1).

Fig. 5

Representative heat maps of Raman spectroscopy results highlighting depth-dependent distributions of DNA, collagen, ChS, and NCP.

Fig. 6

Samples exhibited varying distributions of Raman spectroscopy results for DNA, ChS, collagen, and NCP with depth and direction relative to the split-line. A) Mean results as a function of depth (confidence interval shaded), B) ratio of ChS to collagen by zone (box plot with data outliers removed for visualization). (# denotes significant difference within zone between faces; S, M, and D denote significant difference with the surface, middle, and deep zonerespectively within face).