Effects On Articular Cartilage Mechanical Properties Following A Non-Invasive Anterior Cruciate Ligament Rupture In Rat Model

Purpose

Post-traumatic osteoarthritis (PTOA) occurs after physical injury to a joint. To mimic this disease, the most widely published in vivo model is ACL surgical transection. However, this procedure does not simulate the normal development of this disease, as it is an invasive process cutting through the skin and joint capsule, which affects the native biological response to knee injury. Moreover, there is a compelling need to detect PTOA at an early stage due to its progressive pathogenic mechanism. An early sign of cartilage degeneration is swelling which leads to lower mechanical properties of the cartilage. Hence, the aim of this study is first to replicate a non-invasive ACL rupture (Maerz-2015) and secondly to investigate the early effects of a ligamentous injury on cartilage mechanical properties using 3D indentation mapping.

Five 14-weeks old Lewis rats were used in this study and housed at the INRS-LNBE animal facility following Canadian guidelines for animal care and experimentation. Their hindlimb joints were assigned to three different groups (Control, Rupture and Contralateral). For one animal, both joints were left uninjured and used as Control (n=2). The remaining four animals were subjected to the non-invasive ACL rupture using an adapted mechanical loading protocol with a custom fixture and testing system (Mach-1v500cs, Biomomentum). Only their right hindlimbs were subjected to the ACL rupture (n=4 part of the Rupture group), while their left hindlimbs were left uninjured (n=4 part of the Contralateral group). On the day of injury, the right knee of anesthetized animals was flexed on a bottom platen while the paw was maintained in a top platen. The hindlimb was then subjected to a preconditioning followed by a rapid vertical displacement of 2 mm at 8 mm/s. This rapid loading caused an anterior subluxation of the tibia over the distal femur which results in ACL failure. Euthanasia of animals was performed 4 weeks post-injury; Femoral distal ends and tibial plateaux were collected (n=20 with 10 tibial plateaus and 10 femoral condyles) and cartilage was assessed mechanically by indentation and thickness mapping using a mechanical tester (Mach-1v500css, Biomomentum). Mapping grids were defined for each surface and normal indentation mapping was performed at each position based on the surface orientation (amplitude=0.05mm at 0.05mm/s). The resulting force was measured with a spherical indenter (r=0.5mm) fixed onto a multiaxial loadcell (17N). The spherical indenter was then replaced with a needle probe and moved towards the surface at each position of the same grid. Cartilage thickness was estimated using the displacement between the initial contact force and the sudden force increase when the needle tip transitions from the softer cartilage to the stiffer bone. The Instantaneous Modulus at each position was obtained by fitting the load-displacement curve to an elastic model in indentation using the measured thickness.

Animal health technicians noted that animals were walking on tiptoes for a few days following injury. At necropsy, ACL rupture was confirmed on the right knee of all injured animals while an intact ACL was observed on their left hindlimbs as well as both hindlimbs of the control animal. Instantaneous modulus and cartilage thickness mappings of the femoral condyles and tibial plateaus were generated for all groups (Fig. 1). The cartilage thickness measured for control samples were in line with the literature. In comparison, the cartilage was thicker in the entire femoral condyles in the Rupture group suggesting cartilage swelling (Fig. 2A). On the other hand, only the medial and lateral posterior regions of the contralateral femoral condyles showed an increase in thickness (Fig. 2A). The cartilage thickness in tibial plateaus did not show any significant difference between all groups (Fig. 2B). The medial-anterior regions of the femoral condyles and the tibial plateaus of the Rupture group have a significant decline in their mechanical properties (Fig. 2C&D). Similarly, the medial-anterior region of the tibial plateau also showed a significant decrease in instantaneous modulus in the Contralateral group (Fig. 2D).

The Mach-1 system was successfully used to induce a non-invasive ACL rupture and mechanically characterize early post-traumatic cartilage degeneration through a unique 3D indentation and thickness mapping technique. Results show significant differences between the Control and Rupture group related to cartilage swelling and a decrease in instantaneous modulus in the medial anterior regions of both femoral and tibial knee surfaces.