A novel osteochondral explant model to study bone and cartilage responses to damage in PTOA

Abstract

Purpose: Osteoarthritis (OA) is a progressive joint disease, which was traditionally associated with degradation of articular cartilage, but is now known to affect other joint tissues including subchondral bone. Indeed, changes in subchondral bone have been shown to precede the process of cartilage degradation and may contribute to it. One specific example of this is the presence of bone marrow lesions (BMLs) in acute joint injury, and their potential role in development of post-traumatic osteoarthritis (PTOA). This process of bone-cartilage cross-talk can be challenging to study in vivo due to its complexity, thus multi-tissue explant model systems are crucial. Traditional osteo-chondral explant models (femoral head, or cylindrical cored specimens) are useful in capturing certain aspects of this process but are limited in that bone damage is necessarily created, during the process of isolation. Thus, in this study we used a novel bone-cartilage explant model, where no bone damage was created during harvest. Then, experimental damage was introduced by either chemical or mechanical means, and the subsequent effects on cartilage and bone tissue status were determined. Methods: First, we characterised the rat patella (n=12) as an osteo-chondral explant system, with no initial bone damage, by comparing with the state-of-the-art femoral head explant in terms of baseline viability assays. The baseline viability of both explant models was evaluated using biochemical, histological and microstructural assays. Then samples were subjected to either chemical or mechanical damage and the individual tissue damage responses were assessed. For chemical damage, explants were exposed to 10ng/ml of IL-1β for 0, 1, 3 and 7 days. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles. In both cases, Sulfated glycosaminoglycan (sGAG), and MMP-13 were measured as an assessment of representative cartilage responses while and alkaline phosphatase (ALP) was assessed as a representative bone response. Furthermore, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out. Results: Our results demonstrate that the patellar explant is an excellent ex vivo model system to study bone-cartilage crosstalk, and one which does not induce any bone damage at the time of tissue harvest. We also established culture conditions to maintain viability in these explants in standard media for up to 28 days in culture (data not shown). In the histological sections, haematoxylin and eosin were used to show general morphology of patella explant and femoral head (Fig. 1C). Rat patellae exposed to IL-1β showed that both proteoglycan content and bone metabolism markers were significantly increased (p<0.05) after 7 days of treatment when compared with the controls (Fig.1 A-B). To confirm this finding, qualitative immunohistochemical staining showed chondrocytes increased expression of MMP13 after treatment with IL-1β. Mechanically compressed patellae showed a decrease in compressive moduli from day 3 to day 7 (Fig. 2A), this suggested that subchondral bone remodelling may have taken place as a compensatory mechanism to repair microdamage. To fully validate these findings additional bone microstructural parameters need to be assessed by computer tomography (μCT) (data not shown). In addition to this, increase in MMP13 release was observed three days after samples were mechanically damaged when compared with the control (Fig. 2B). Conclusions: In summary, these initial Results showed that patella explant is a useful model to study bone cartilage cross-talk. We used this system to study the effects of chemical and mechanical damage, to both tissue types. The aim of this project was to establish a system to recapitulate the subchondral bone damage associated with joint injury and PTOA. We used a novel patellar explant model, which does not involve the creation of basal bone damage during isolation. IL-1β was used as a surrogate for bone damage and resulted in cartilage and bone specific changes. It is well known that cartilage has poor reparative potential and overloading has catabolic effects in the osteochondral tissue. In summary, this study demonstrates that the explant model can be useful for bone-cartilage crosstalk in a model of PTOA.