Hello, I'm Samuel Adams, and the topic of this talk is intraarticular hematoma immediately after ankle fracture causes cartilage damage as partially attenuated by anti-inflammatory agents. I have no disclosures related to this presentation. My other disclosures are listed on the Academy website. So as foot and ankle surgeons, we see this quite often, going from an ankle fracture to ankle arthritis in sometimes a relatively quick time frame. And you can see here that in the ankle, we see a much, much higher instance of post-traumatic osteoarthritis. And part of this reasoning behind doing this study is to help us figure out why we see that so much in the ankle. Now, a lot of authors will describe or try to tell you that the reason we see that in the ankle is because the amount of damage that the cartilage sees at the time of injury, such as the talus abutting the fibula or the medial malleolus or the rest of the tibia during the injury. But I'm going to try to show you that this may not be the case and that there's another mechanism going on. Now we all learned that the first aspect of healing of any fracture is inflammation. And if you think about an ankle fracture, here you can see at least in two places where the medial malleolus or the fibula, where the inflammation that's occurring at the fracture site is also communicating with the ankle joint. And we've shown before that this fracture hematoma contains inflammatory cytokines and MMPs. And it's our argument that the cartilage that's not part of the fracture plane is really an innocent bystander. Here you can see an example of what a post-traumatic fractured ankle looks like. And you can see really there's diffuse cartilage damage and not just at the fracture lines. And so really this might be an indication where nature got it wrong in that the inflammation from the healing fracture is causing widespread joint damage. But no one has shown this yet. Previously we've demonstrated that the fracture hematoma that goes into the joint does contain inflammatory cytokines and matrix metalloproteinases, which degrade cartilage. We've also shown that this milieu and the cytokine composition changes over time. And initially, in this study that we performed, initially we've shown that the thing that hits the joint at first are inflammatory cytokines. And those are known to kind of create a cascade of inflammation and production of MMPs, which we see in the joint at about three to nine days after fracture. And then those MMPs end up causing cartilage degradation, as you can imagine. And in this study, we split it up into kind of clinically relevant time points where the zero to two days would be the ER visit or initially to go to the operating room to place an external fixator. The three to nine days would be a clinic visit. And then the greater than 10 days would be the definitive surgery. But as you can see here, that by 10 days, cartilage damage is potentially already occurring. And I'd ask you to think about your practices and see if you get to these ankle fractures within the first 10 days or even immediately. And the answer is probably no. So the purpose of this study was to determine if exposure of healthy cartilage to the inflammatory fracture hematoma actually does cause cartilage damage. And then to see if this cartilage damage can be mitigated by anti-inflammatory or anti-MMP agents. And actually, these two that are listed here that we used in this study are clinically available. IL-1RA is Kineret, and doxycycline is an antibiotic that's available. And so what we did is we took the fracture hematomas from about 50 patients that were collected for that previous study. And as in the methods of the previous study, we divided them into clinically relevant time points, 0 to 2 days, 3 to 9 days, and then 10 days out from fracture. And in this study, what we were able to do is obtain fresh human, and I think that's key here, is this is actually human cartilage. So human cartilage from tail eye donated by RTI Surgical. And what we did is we took a 5 millimeter biopsy punch and created cartilage discs. And then we took those cartilage discs and we cultured them in the fracture hematoma from these various time points. And then after culturing them for about 6 days, we then washed them, put them in a new well, and then added regular chondrocyte media. And we did that for about another 3 days. And during that 3-day period, we were testing for what these cartilage discs or the chondrocytes within the disc were producing and secreting out into the media. And then after this culture period, we analyzed what was secreted in the media. And we also did performed histology on the discs themselves for determining the amount of live or dead cells, and also for safranin O staining, which determines the proteoglycan content. And so here are the results of the secretion. And so again, imagine after being exposed to this fracture hematoma, we wanted to kind of see how sick were the chondrocytes within these discs. And so what did they produce? And if you look here, so the A's are the control groups. That just got regular chondrocyte media, so no fracture hematoma. B got the fracture hematoma from 0 to 2 days. C got the fracture hematoma from 3 to 9 days. And the D group got the fracture hematoma from greater than 10 days. And what you can see here is that exposing these cartilage discs and the chondrocytes within them to these fracture hematomas really upregulated their production of IL-6, IL-8, and then several of the MMPs. And again, these are all pro-inflammatory and cartilage breakdown markers. So these cartilage discs are becoming diseased when you expose them to the fracture hematoma. You can also see here that they produced the CTX2, which is a cartilage breakdown marker. And they had reduced SGAGs, which are glycosaminoglycans, which are helpful for the extracellular matrix. So all in all, what we see here is that exposure to the fracture hematoma does cause cartilage damage or cartilage sickness. And then these chondrocytes are then secreting these inflammatory cytokines that can then act on the adjacent cartilage and chondrocytes. But if you also look closely here, what we saw is that the group B, the 0-2 days, seemed to cause the most upsetting of the cartilage and the chondrocytes, or the most damage. And that will be important later on for the second part of this study. Now if you look at the cartilage discs after culturing, although these values here aren't significant, you can see a trend. So in that image on the left, the chondrocyte viability, exposure to the fracture hematoma from 0-2 days really killed a lot of the cells, took it down to less than 20% cell viability. And we did see also decreased cell viability with the other fracture hematoma groups. And then if you look at the image on the right, the safranin O staining, so you can see decreased staining with all of the fracture hematoma groups, although these were not significant values. But since we felt that the group B seemed to cause the most irritation or damage to the chondrocytes and also caused the most amount of chondrocyte death, we wanted to see if we could mitigate that response. And so we repeated the experiment, this time only using the fracture hematoma from the first 0-2 days after fracture, so that group B that we used prior. And to the fracture hematoma, we also added IL-1RA or doxycycline, and we wanted to see if this could mitigate those damaging results. And so here are the results of those group B cartilage discs, again, the fracture hematoma from 0-2 days. And then in addition, group E here is the addition of IL-1RA and group F is the addition of doxycycline. And what you can see here is the cartilage discs or the chondrocytes within that were exposed to these therapeutic agents end up becoming a lot less damaged and producing a lot less inflammatory cytokines and matrix metalloproteinases. And you can see here significant values in reduction of IL-6, IL-8, and several of the MMPs, as well as CTX-2, which again is a cartilage breakdown marker. So here we are showing that the addition of one of these therapeutic agents actually decreased the cartilage damage. And then if we add in these groups to the chondrocyte viability and the cartilage, the safranin O staining, you can see here that groups, again, E and F are the therapeutics. And when you add a therapeutic, you actually preserve the cell viability. Now again, these numbers aren't significant, but you can see a trend here that providing this therapeutic to the culture medium actually preserves the viability, as well as the proteoglycan content of the extracellular matrix. So this is the first study to show that intraarticular fracture hematoma can mitigate cartilage damage. Early fracture hematoma, again, those first two days after fracture likely causes the most damage. However, we really don't get to these patients at that time frame. We typically intervene around day 10 or later. This initial time point, and again, these samples were collected from patients presenting to the ER. And again, almost every ankle fracture either presents to the ER or an urgent care right away. And so is there a way that we can intervene at that time point, maybe with the labage or even eventually maybe injecting a therapeutic agent. And what we also showed in this study is that the anti-inflammatory and anti-MMP agents do reduce cartilage damage caused by that early fracture hematoma. And these are likely some agents that we can look at in the future to see if they can provide this same advantage clinically.

Samuel Adams

intraarticular hematoma

ankle fracture

cartilage damage

inflammatory cytokines