Paper 10: Characterization of Bone in Osteochondral Allografts Utilizing Inflammatory-Mediated Cellular Death Pathways

OBJECTIVES: Osteochondral allografts are used to treat chondral and osteochondral defects. While prior studies have correlated cell death within the chondral component of osteochondral allografts directly with clinical outcomes, cell death within the subchondral and trabecular bone has not been well characterized. While prior studies have demonstrated a role for apoptosis in chondral cell death in osteochondral allografts, none have examined the role of pyroptosis and necroptosis in osteochondral allografts. These pathways are of particular relevance because persistent inflammation associated with osteochondral allografts on imaging is correlated with poor patient-reported outcomes. We report here a study examining cell death and mechanism in osteochondral allografts. METHODS: Fresh human talus allografts were cultured for 14, 28, or 42 days at 4(o)C in standard clinical media. Osteochondral plugs were taken at the specified time points and fixed in 10% formalin. Cell death was measured in the cartilage, subchondral bone, and trabecular bone with TUNEL staining. The mechanism of cell death was further examined with staining for apoptosis (activated caspase 3), necroptosis (MLKL), and pyroptosis (cleaved gasdermin D). Percentage of cells staining positive was calculated in each zone (cartilage, subchondral bone, trabecular bone). Each time point included 4-5 replicates. Appropriate statistical comparisons were made between groups. RESULTS: Cell death was significantly greater in the trabecular bone and subchondral bone than the cartilage. There was an increase in cell death over time in cartilage as well as subchondral bone. Apoptosis was the most prevalent mechanism of cell death, but necrosis and pyroptosis were present in all areas of bone and cartilage. CONCLUSIONS: This study suggests that a component of osteochondral allograft clinical outcomes may be related to viability of the bony component of the allograft. There is significantly greater cell death in the bone versus the cartilage and a significantly greater percentage of the cell death is through pyroptosis, an inflammatory-mediated pathway. Culture media and surgical strategies that limit the effect of inflammatory cell death in the bone component of osteochondral allograft may help to improve postoperative incorporation and control persistent inflammation that can complicate surgical outcomes.