Scaffold-free cartilage subjected to frictional shear stress demonstrates damage by cracking and surface peeling

Abstract

Scaffold-free engineered cartilage is being explored as a treatment for osteoarthritis. In this study, frictional shear stress was applied to determine the friction and damage behaviour of scaffold-free engineered cartilage, and tissue composition was investigated as it related to damage. Scaffold-free engineered cartilage frictional shear stress was found to exhibit a time-varying response similar to that of native cartilage. However, damage occurred that was not seen in native cartilage, manifesting primarily as tearing through the central plane of the constructs. In engineered cartilage, cells occupied a significantly larger portion of the tissue in the central region where damage was most prominent (18 ± 3% of tissue was comprised of cells in the central region vs 5 ± 1% in the peripheral region; p < 0.0001). In native cartilage, cells comprised 1-4% of tissue for all regions. Average bulk cellularity of engineered cartilage was also greater (68 × 10³ ± 4 × 10³ vs 52 × 10³ ± 22 × 10³ cells/mg), although this difference was not significant. Bulk tissue comparisons showed significant differences between engineered and native cartilage in hydroxyproline content (8 ± 2 vs 45 ± 3 µg HYP/mg dry weight), solid content (12.5 ± 0.4% vs 17.9 ± 1.2%), shear modulus (0.06 ± 0.02 vs 0.15 ± 0.07 MPa) and aggregate modulus (0.12 ± 0.03 vs 0.32 ± 0.14 MPa), respectively. These data indicate that enhanced collagen content and more uniform extracellular matrix distribution are necessary to reduce damage susceptibility. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: damage; depth-dependent cellularity; frictional shear; mechanical properties; scaffold-free engineered cartilage composition; tribology.

Figure 1. Tribological testing device configuration

A) Load application and measurement schematic with hemicylindrical mount. B) The flat mount utilizing a self-aligning platen. The hemicylindrical and flat mounts were interchangeable in the device.

Figure 2. Quantification of surface peeling (SP) in flat configuration

Representative stereomicroscope (A&B) and safranin O stained histological cross section (C&D) images of TE cartilage taken before (A&C), and after (B&D) exposure to frictional shear stress. Area exhibiting SP appeared mottled in stereomicroscope images (B). Damage was determined as the ratio of the area of SP (B, outlined in gray) to the total surface area (B, outlined in black). SP was also evident in histological cross sections (D, starting at the arrow and continuing the length of the section to the right). Internal cracking (arrowhead) is also seen in D. Scale bars in B and D also apply to A and C respectively.

Figure 3. Histomorphometry methods

Safranin O stained histological cross sections (A) were manually segmented in Photoshop to create a mask indicating the location of the tissue boundary (black border around tissue) and the lacunae (black circles) within the tissue (B). Masks were then analyzed with a custom Matlab program to divide the tissue into five equal regions (C) and calculate the number of cells and the area occupied by cells within each region.

Figure 4. Friction measurements in curved test setup

Friction force (A) and coefficient of friction (B) data for TE and native cartilage. C) Representative safranin O stained histological cross section of TE cartilage. Internal cracking (starting at the arrow and continuing left) is evident. Adhesive can be seen as the unstained region on the upper surface of the tissue (arrowhead). India ink was used to mark the adhered side of the tissue, and can be seen as black fragments near the upper surface. D) Representative toluene blue stained histological cross section of native cartilage. Minimal damage to the upper (adhered) surface of the specimen is shown (arrow).

Figure 5. Damage characterization of TE and thinned native cartilage in the curved setup

Safranin O stained histological cross sections of TE cartilage (A&B), native cartilage including the superficial zone (C&D), and native cartilage without the superficial zone (E&F), after exposure to frictional shear stress. The range of damage in each sample group is shown. A) Minimal cracking (arrow) observed in TE cartilage. B) Extensive cracking observed in TE cartilage. C) Narrowing of native cartilage including the superficial zone in the presumed loaded region (arrow), as opposed to the presumed unloaded region (arrowhead). D) Wear of native cartilage including the superficial zone. E) No apparent damage to native cartilage without the superficial zone. F) Minimal cracking (white space within the tissue) observed in native cartilage without the superficial zone. The dark lines running vertically through this sample section are sectioning artifacts (folds). All images are presented at the same scale.

Figure 6. Composition and biomechanical properties

Comparison of ECM composition by GAG (A) and HYP (B) content, cellularity (C), solid content (D), and the stiffness (E) of engineered constructs to native cartilage. Note the change in scale in graphs of A and B; *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 7. Depth-dependent cellularity

Patterns of cellularity varied between native cartilage and TE cartilage, and correspond to observed patterns of damage. Statistical significance was assessed within cartilage type by comparing each region to the 0-20% region (* and + for TE and native respectively), and by comparing each region between cartilage types (#). In each case, one symbol indicates p < 0.01, two indicates p < 0.001, and three indicates p < 0.0001.

Figure 8. SEM of engineered and native cartilage

Cross sections of engineered (A) and native (B) cartilage imaged by SEM. The cross sectional face of the engineered cartilage is shown between the black and white arrows. Both images are presented at the same scale. At this magnification native cartilage appeared to be a solid material with distinct lacunae, while engineered cartilage appeared less dense, with many more lacunae.