Disruption of articular cartilage extracellular matrix following mechanical trauma in an in-vitro model

INTRODUCTION: Traumatic joint injuries are known to leave lesions in articular cartilage which, left untreated, can progress to osteoarthritis. An in-vitro
model mimicking a traumatic injury is therefore essential to characterize the effects of cartilage degeneration over a span of time and validate the efficacy of
novel engineered solutions to treat those lesions. This study evaluates the consequences of a mechanical injury on osteochondral plugs by assessing the cartilage
mechanical properties, GAG release, and cellular viability versus an intact-control group. As the underlying integrity of cartilage is mainly provided by the
collagen network – which is imbedded with aggrecans (GAGs) – a mechanical injury is expected to damage the protein chains within the extra cellular matrix
(ECM), leaving a compromised structure. Expected alterations from the injury include reduced fibril modulus, increased hydraulic permeability of the ECM,
swelling of the cartilage, and GAG release from the matrix, among others.

METHOD: Osteochondral plugs (4 mm dia.) were harvested – with a trephine under constant irrigation – from equine femoral condyles a few hours
postmortem. Four plugs were extracted from each condyle and distributed into the following groups: mechanical test (MT) injured, MT control, no mechanical
test (no-MT) injured, and no-MT control. To mimic in-vivo conditions, osteochondral plugs were cultured in two separate compartments using a custom-made
holder to restrict crosstalk between cartilage and bone (see Fig. 1) – and incubated up to 9 days at 37°C and 5% CO₂ . Culture mediums were changed every 3
days and 1 ml aliquots were retained from the cartilage compartment to document GAG release with a DMMB colorimetric assay. Cartilage volume was
characterized by measuring thickness and diameter under a dissection microscope upon harvest (3 days pre-injury), on day 0 (injury day), and day 6 (final
timepoint). Mechanical injury was inflicted by a single unconfined compression of 50% deformation at a 100%/s deformation rate, as previously described in
the literature. Cellular viability was evaluated for all no-MT samples in the surface (first 100μm depth) and the transitional (100-200μm depth) regions of thin
cartilage slabs at day 6; a Live/Dead imaging kit (R37601, Invitogen) was utilized for the assessment. Mechanical tests were conducted on day 0 and day 6 in
a bath of PBS containing 2% antibiotic kept at 37±1°C. Unconfined compression (UC) consisted of a quasi-static 10% initial deformation followed by four
consecutive 2.5% deformations; cartilage stress was allowed to equilibrate after each deformation. Friction-on-glass was performed directly after UC by
rotating the cartilage on its axial axis at an effective speed of 43 deg/s for two revolutions (0°-720°). Stress-relaxation curves were curve-fitted to a fibrilreinforced poroelastic model to extract mechanical properties and the coefficient of friction (COF) was approximated as the ratio of the rotational force to the
loading force.

RESULTS: Experimental procedures were successfully conducted in a sterile manner with no sample contamination. Cellular viability showed distinct
alterations in the surface region of the cartilage between injured and control group with 40% more cell death 6 days post-injury (see Fig. 2). However, no
apparent discrepancies were observed in the transitional region. The injured group showed a decrease of fibril moduli with an increase of GAG release from
the cartilage ECM (see Fig. 3). Inversely, the ECM hydraulic permeability increased with GAG release. Furthermore, injured cartilage volume increased nearly
twice as much as the control group 6 days post-injury. The coefficients of friction were comparable between groups and timepoints and ranged between 0.2-
0.47, n=5.

DISCUSSION: The results tend to show mild signs of cartilage degeneration 6 days post-injury. The disruption in the ECM was highlighted by the lowered
strength of the collagen fibrils, the high GAG release, and the increase in hydraulic permeability of the cartilage tissue after the injury. Physically, the results
indicate that the mechanical trauma damaged the collagen fibers within the ECM which in turn triggered cartilage swelling and GAG release from the matrix.
GAG loss combined with cartilage swelling lead to an increase in ECM pore size which increased the hydraulic permeability of the cartilage. The differences
in chondrocyte viability between the cartilage surface and transitional region suggest that the degeneration was superficial and had not yet progressed into the
deeper tissue. Collectively, the results validate the proposed in-vitro model for cartilage degeneration. Forthcoming works shall focus on prolongating the
study to reach a more severe state of cartilage degeneration, studying the effect of harvesting plugs from specific regions on the femoral condyles (e.g., distal,
or proximal) to best normalize the data, and comparing the effect of different lubricants on cartilage properties.

SIGNIFICANCE: The significance of developing an in-vitro model that assesses the mechanical and biochemical properties of degenerated cartilage is to
provide a method for improved screening of potential novel engineered solutions and perform well-targeted pre-clinical studies.