Resolving curling and swelling in cartilage and other deformation-prone tissue explant models: a scalable, simple...

Ex vivo swelling and curling can undermine reproducibility in compression-based injury models.

We developed a simple, regression-guided flattening step that computes explant-specific loads from each explant's swelling state to restore native thickness and flatness prior to loading. Using adult articular cartilage, we tested the hypothesis that mitigating initial deformation amplifies injury model readouts and permits standardized injury of the deformation-prone superficial zone (SZ), which many models exclude.

Discs spanning 250-1200 µm exhibited curling; 250 µm SZ discs were chosen for calibration. Post-equilibration thickness (T₂) strongly predicted the load required to restore native thickness (LT₀), and nonlinear regression yielded a practical load-prediction equation, refined with an expanded dataset (n = 129 discs). Applying the predicted flattening load immediately before standardized compression (50%-65% strain, 100%·s^-1) did not affect chondrocyte viability on its own. When combined with injury, it amplified canonical readouts - lower viability and higher apoptosis - with effects concentrated in the upper superficial zone, notably within the top 44  μm at 24 h and with apoptosis persisting to 96 h. Click-chemistry EdU labeling revealed injury-induced proliferation: SZ-wide S-phase entry, most pronounced at 96 h in adult, mechanically mature cartilage without exogenous growth factors.

Together, regression-guided flattening restores native geometry, standardizes the mechanical stimulus, increases readout reliability, and enables inclusion of the clinically important SZ. Because deformation artifacts are common across hydrated soft tissues, and prior pre-compression practices were uncalibrated, this framework establishes a cartilage-based proof of concept for regression-guided flattening that may be extendable to other deformation-prone tissues through tissue-specific recalibration and experimental validation, thereby providing a more standardized and generalizable route to faithful ex vivo injury studies.