Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis, which occurs secondary to traumatic joint injury which is known to cause pathological changes to the osteochondral unit. Articular cartilage degradation is a primary hallmark of OA, and is normally associated with end-stage disease. However, subchondral bone marrow lesions are associated with joint injury, and may represent localized bone microdamage. Changes in the osteochondral unit have been traditionally studied using explant models, of which the femoral-head model is the most common. However, the bone damage caused during harvest can confound studies of microdamage. Thus, we used a novel patellar explant model to study osteochondral tissue dynamics and mechanistic changes in bone-cartilage crosstalk. Firstly, we characterized explants by comparing patella with femoral head models. Then, the patellar explants (n=269) were subjected to either mechanical or inflammatory stimulus. For mechanical stimulus 10% strain was applied at 0.5 and 1 Hz for 10 cycles. We also studied the responses of osteochondral tissues to 10ng/ml of TNF-α or IL-1β for 24hrs. In general the findings showed that patellar explant viability compared extremely well to the femoral head explant. Following IL-1β or TNF-α treatment, MMP13, significantly increased three days post exposure, furthermore we observed a decrease in sulfate glycoaminoglycan (sGAG) content. Bone morphometric analysis showed no significant changes. Contrastingly, mechanical stimulation resulted in a significant decrease sGAG particularly at 0.5Hz, where an increase in MMP13 release 24hrs post stimulation and an upregulation of bone and cartilage matrix degradation markers was observed. Furthermore, mechanical stimulus caused increases in TNF-α, MMP-8, VEGF expression. In summary, this study demonstrates that our novel patella explant model is an excellent system for studying bone-cartilage crosstalk, which responds well to both mechanical and inflammatory stimulus and is thus of great utility in the study of PTOA.
Traditionally, osteoarthritis (OA) has been associated mostly with degradation of cartilage only. More recently, it has been established that other joint tissues, in particular bone, are also centrally involved. However, the link between these two tissues remains unclear. This relationship is particularly evident in post-traumatic OA (PTOA), where bone marrow lesions (BMLs), as well as fluctuating levels of inflammation, are present long before cartilage degradation begins. The process of bone-cartilage crosstalk has been challenging to study due to its multi-tissue complexity. Thus, the use of explant model systems have been crucial in advancing our knowledge. Thus, we developed a novel patellar explant model, to study bone cartilage crosstalk, in particular related to subchondral bone damage, as an alternative to traditional femoral head explants or cylindrical core specimens. The commonly used osteochondral explant models are limited, for our application, since they involve bone damage during harvest. The specifics aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response and mechanical stimulation to determine the subsequent developments of PTOA. Lewis rats (n=48) were used to obtain patellar and femoral head explants which were harvested under an institutional ethical approval license. Explants were maintained in high glucose media (containing supplements), under sterile culture conditions. Initially, we characterised undamaged patellar explants and compared them with the commonly used femoral head. First, tissue viability was assessed using an assay of metabolic activity and cell damage. Second, we created chemical and mechanical damage in the form of IL-1B treatment, and mechanical stimulation, to replicate damage. Standard biochemical assays, histological assays and microstructural assays were used to evaluate responses. For chemical damage, explants were exposed to 10ng/ml of IL-1B for 24 hours at 0, 1, 3 and 7 days after harvesting. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles, for 7 days. Contralateral patellae served as controls. In both groups, sGAG, ADAMTS4, and MMP-13 were measured as an assessment of representative cartilage responses while ALP, TRAP and CTSK were assessed as a representative of bone responses. In addition to this, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out.Introduction and Objective
Materials and Methods
Osteoarthritis (OA) is a disease that affects both bone and cartilage. Typically, this disease leads to cartilage degradation and subchondral bone sclerosis but the link between the two is unknown. Also, while OA was traditionally thought of as non-inflammatory condition, it now seems that low levels of inflammation may be involved in the link between these responses. This is particularly relevant in the case of Post-Traumatic OA (PTOA), where an initial phase of synovial inflammation occurs after injury. The inflammatory mediator interleukin 1 beta (IL-1B) is central to this response and contributes to cartilage degradation. However, whether there is a secondary effect of this mediator on subchondral bone, Female Sprague Dawley rats (n=48) were used to obtain patellar explants, under an institutional ethical approval license. Patellae were maintained in high glucose media, under sterile culture conditions, with or without IL-1B (10ng/ml), for 7 days. Contralateral patellae served as controls. One group (n= 12) of patellae were assessed for active metabolism, using two both Live and Dead (L/D) staining and an Alamar Blue assay (AB). A second group (n=12) was used for tissue specific biochemical assays for both bone (Alkaline Phosphatase) and cartilage (sulfated proteoglycan and glycosaminoglycan (sGaG)). Finally, a third group (n=28) of explants were used for histologically analysis. Samples were decalcified, embedded in paraffin and sectioned to 7µm thickness, and then stained using H&E; and Safranin O with fast green. Additionally, toluidine blue and alkaline phosphatase staining were also performed. Our results demonstrate that our system can maintain good explant viability for at least 7 days, but that IL-1B reduces cell viability in patellar cartilage, as measured by both L/D and AB assays after 0, 2, 4 and 7 days in culture. In contrast, sGaG content in cartilage were increased by this treatment. Additionally, ALP, a marker of osteoblastic activity, was increased in IL-1B treated group 4 and 7 days, but was also showed some increase in control groups. Histological analyses showed that IL-1B treatment resulted in reduced proteoglycan staining, demonstrating the powerful effect of this factor in injury response over time. Thus, we conclude that IL-1B affects both bone and cartilage tissues independently in this system, which may have relevance in understanding bone-cartilage crosstalk after injury and how this is involved in PTOA development.
