In workpackage (WP) 1 we used our novel joint injury model to examine knee joints by MRI and histological analyses for microdamage. Our experience with this process allowed us to qualitatively determine when a test has been successful due to the change in load/displacement readout during the test. However, MRI imaging can definitely assess ACL status. We showed, using sagittal MRI images that all injured groups had MRI confirmed ACL ruptures. We then used T2 weighted fat-supressed imaging to determine the extent of subchondral bone damage (specifically in the form of BMLs) caused by injury. The injured knees display increased signal in the subchondral compartments of both femur and tibia bones. We used histology to assess levels of bone microdamage caused by ACL injury. We found significantly increased level of microdamage in the injured knees, as measured by crack density, and that cracks and BMLs were significantly correlated.
The primary aim of WP2 was to build on WP1 by developing our ability to evaluate the level of co-localization of subchondral microdamage with osteoclast activity which occurs after ACL injury. In carrying out this work we determined that a system of microdamage-mediated bone turnover exists in the subchondral compartment, and that these changes do indeed correlate with subsequent cartilage degeneration. Specifically, we tested a potent bisphosphonate (ZOL) at two different time points post- injury. In addition a fluorochrome bone labeling agent (Calcein) was also given on the day of injury and 3 days prior the end of the experiment. Injury resulted in increased uptake of calcein in the subchondral region. Early ZOL administration blocked calcein uptake almost completely, whereas late administration brought uptake back up to control levels. Finally, we assessed the effect of ZOL treatment, at two different time points after injury, on articular cartilage health and thus PTOA development. We showed that in comparison with Vehicle treatment, ZOL treatment prevented proteoglycan loss to a significant, and moderate level, respectively. This suggests an osteoclast-independent mechanism may be responsible for this aspect of the injury/disease paradigm.
The primary aim of WP3 was to build on the advances made in the previous WPs by developing and characterizing bisphosphonate-loaded alginate microparticles for injectable delivery to the knee joint, targeting subchondral bone, after knee injury to prevent PTOA. This enhances efficacy of the drug, reducing the number of administrations of the drug and minimizing off-target effects. Alginate-based microparticles are excellent candidates for IA drug-delivery because of their hydrophilic nature, biocompatibility, and physical architecture. We made poly(lactic-co-glycolic) (PLGA) microparticles to encapsulate ZOL. Their size and release profile was controlled by adjusting spray drying and material parameters. Release of ZOL as a function of time was measured. We also tested whether the ZOL agent could be reliably and reproducibly delivered by intra-articular (IA) injection to the joint. Here we made use of a ZOL analogue which was tagged with a fluorescent moiety in order to trace the location of the drug in the joint after delivery. Specifically, we wished to test whether ZOL would penetrate the osteochondral junction (from the articular side), to reach the subchondral bone compartment beneath the joint and thus have the intended therapeutic effect on local osteoclastic activity.
As an overview, our results show that increased subchondral microdamage in the injured knees, is related to BMLs and localized remodeling. Furthermore inhibition of this process by bisphosphonates in microparticle carriers is a feasible drug delivery approach.These data can be exploited in terms of a novel early treatment after joint injury, early data has been disseminated at research meeting, journal publications and public health seminars.
