Summary

Ex vivo injury models allow to study the mechanisms of PTOA and preventive biologic treatments

Abstract

Introduction

Acute cartilage injury contributes to the onset of post-traumatic osteoarthritis (PTOA). To understand the underlying mechanisms that drive the progression of cartilage/joint injury to PTOA we developed various ex vivo models that utilize either mechanical impaction to create cartilage injury when cartilage is still attached to subchondral bone and/or the treatment with high doses of proinflammatory cytokines known to be elevated immediately after injury. Unlike various animal models, this approach uses normal intact joints from human organ donors. It allows mimicking joint injury and studying immediate cellular responses to injury by chondrocytes, osteoblasts, and synovium cells. We also developed a model of focal cartilage defects to investigate chondrocytes regenerative ability.
Materials and Methods.
For all our PTOA modelling studies we have been using fresh human knee and ankle joints collected through the Gift of Hope Organ and Tissue Donor Network (Itasca, IL). Donors of all ages (ranging from 19 to 90 years old) and both genders are utilized. The following ex vivo models of cartilage injury have been developed, validated and studied: 1) Cartilage damage created by mechanical impaction using the pneumatic pressure controlled impactor with the transferred impulse of 1Ns. In this case, fresh human normal tali (cartilage with bone attached) are used; 2) Cartilage explants treated with high dose interleukin-1ß (IL-1ß); 3) co-culture of damaged (impacted or IL-1ß-treated) cartilage with normal intact synovium; 4) a donut-shaped cartilage explants, 10 mm in outer diameter and 6 mm central hole, have been created to model a chondral defect; and 5) co-culture of osteochondral plugs (impacted or cytokine-treated) and synovium.

Results

Similar to ACL or impact injuries in patients we found that the first events occurring in all these models are chondrocyte death by necrosis and apoptosis and activation of inflammatory cascade. Cell death by apoptosis, if not arrested, propagates longitudinally and horizontally to the areas adjacent, but not immediately affected by injury. We identified a number of anti-catabolic treatments capable of inhibiting chondrocytes death. These include a non-ionic surfactant P188, osteogenic protein-1 (OP-1), IL-1 receptor antagonist (IL-RA), tumor necrosis factor antagonist (anti-TNF), pan-caspase inhibitors Z and Q, and dexamethasone. Out of all these agents the most effective were P188, OP-1, anti-TNF and dexamethasone. Cell viability under these treatments was about 2-fold higher than in the corresponding control (P<0.05). Interestingly, normal synovium obtained from the same joints was also able to reduce cell death when co-cultured with injured (impacted, P<0.05, or IL-1ß, P<0.01, treated) chondrocytes. Importantly, both, the biological and normal synovium, treatments were able to not only prevent cell death, but also protect extracellular matrix from degradation. The effect of synovium was lost in co-cultures with osteochondral plugs. In the chondral defect model, we demonstrated an age-related ability of chondrocytes to migrate into the defect area and fill it with the extracellular matrix.

Conclusions

The results of these studies confirm the utility of ex vivo models for studying human joint injuries and the mechanisms of development of PTOA. Further, they provide an excellent platform for testing various biologic approaches as potential treatments for preventing PTOA.