grant from JRF. That's our only relevant disclosure. So osteochondroallografts are commonly used to treat large chondral defects of more than two centimeters squared. And the survivorship at 15 to 20 years is about 75 percent. Failure of these grafts is correlated with MRI cystic changes and persistent clefts characteristic of inflammation, and that's from previous studies. So cell death in osteochondroallografts has been previously characterized primarily within cartilage, not within the bone component. The characterization has been mainly with live dead staining, which stains non-fixed samples. Things that we do know from these previous studies are that cell death increases with culture time, and that increased cell death is associated with a worse clinical outcome. So our understanding of cell death mechanisms has advanced in the last 10 years, thanks to research really in cancer. So we know about apoptosis, and we've known about it for a while, but there are other mechanisms of innate cell death that are associated with inflammation, pyroptosis, necroptosis, and then panoptosis, which is really a combination of all three. These are characterized by activation of different enzymes and different proteins. So caspase-3 activation is characteristic of apoptosis, pyroptosis is characterized by cleaved gas dermin, and then MLKL activation is characteristic of necroptosis. So these are assays you can look at to see if these are activated. We devised a simple experiment, really looking at three specific aims and hypotheses. So the first one we wanted to look at using fixed samples, which hadn't really been done before, to look at cell death and the bony component compared to the cartilage component of osteochondralografts. And then we wanted to look at activation of inflammatory cell death pathways, and then to see if there was any dramatic correlation of cell death or activation of these inflammatory pathways with culture time or temperature. So we used human osteochondralografts taken from taluses, and we stained for markers of necroptosis, pyroptosis, and apoptosis, as well as a late-stage cell death marker tunnel staining. We did this on fixed decalcified sections, so we were able to see the bone as well. So here is tunnel staining of an osteochondralograft, and you can see here in green is the tunnel positive cells that are marked by degrading DNA characteristic of irreversible cell death, while the blue stains cell nuclei. So intense blue staining is DAPI, and you can see that the cell death is significantly greater within bone than cartilage by about a five-fold difference. This is the staining for the presence of inflammatory cell death mediated markers. So MLK activation, you can see, was active both within cartilage and within bone. So this is characteristic of pyroptosis, and then the cleavage of gasdermin D was present within bone and cartilage as well, characteristic of necroptosis, and then caspase-3 activation staining was also positive, which is characteristic of apoptosis. So really all three activated in both bone and cartilage. And then we didn't notice any dramatic variation with temperature and time. I'll note that our study was underpowered to detect dramatic differences, to detect subtle differences. What we were really looking for here was whether there was a dramatic difference in terms of, you could see it by naked eye, of activation within one component or another. So in summary, I would say the big things that we found were that the overall cell death was much more prominent in bone than in cartilage. Inflammatory cell death pathways are activated in both bone and cartilage, and we didn't see any dramatic effect of time or temperature variation within these, but we were underpowered really to detect that. I think the clinical implications of this are using a thin backing of bone and trying to remove as much of the cellular component can help to decrease the inflammation that you may see from that component of your osteochondral allografts. And then in future studies, it would be interesting to see if there's an ability of an inhibitor of these inflammatory cell death pathways to limit inflammation and improve outcome. I'd like to thank some of the co-authors on this study. So Dr. Rodeo from championing the study from the beginning and helping to get funding and really promoting it forward. Lily, who is one of the technologists within Dr. Rodeo's lab, who's fantastic, and really her technical ability made the sectioning of these osteochondral allografts possible. And then Krishna Anand, who helped a lot with the analysis and interpretation of the results. Thanks. Okay.

osteochondroallografts

chondral defects

cell death mechanisms

inflammatory cell death pathways

graft inflammation