 Our final presentation is by Peter Riri. He's a fourth year medical student at UT Southwestern in Dallas. He grew up in Provo and went to BYU. And he's going to be talking about a novel model for studying fibroblast migration through fibrin matrices. Okay. So, as he said, my name is Peter Riri. I'm a native Utah and I can report that everything is bigger in Texas. The ophthalmologists are bigger. They're huge and everything. I've had a really good time here. This is a really amazing center. I was talking to my dad. He was an ophthalmology resident here in the 60s. And he was actually a visiting resident because there was no program here in the 60s. He was from the University of Iowa. And I guess they didn't have any faculty. And he said this local community ophthalmologists would come in and help out the visiting residents. So it's really amazing to see how much has changed in that amount of time. So I'm going to shift a little bit from my talk from the clinical side to more of the basic science. This is some research I did during medical school with a bioengineering PhD at UT Southwestern. I saw some of, when I was looking at some research I wanted to do, I saw the pictures he was taking with a confocal microscope and I thought, man, those looked really cool. So that's kind of what drew me to it. So my presentation is on a novel model for studying fibroblast migration through fibrin matrices. So some background. We know that cells migrate and will change their morphology and remodel their surrounding environment in response to different chemokines, growth factors. And we also know that the extracellular matrix provides a very important role in that process as well. It provides physical and chemical signals to the cells and also the substrate through which the fibroblast migrate during wound repair. So the purpose of our study, a big picture we're trying to answer, how do these processes relate to corneal wound healing and understanding the molecular steps involved with wound healing is essential to maybe someday preventing excessive scar formation which can really impair vision. So again, the goal of this study was to develop and assess a new model for assessing corneal fibroblast migration into fibrin which is a key event in following corneal injury. So the name of the model we came up with is a nested 3D matrix model. What I'd do is I'd take these compressed collagen discs that were populated with human corneal fibroblasts and then embed them in a cell-free either collagen or fibrin matrix. I would then culture the cells for three and seven days with PDGF which is a growth factor known to cause fibroblast migration. And then I would stain and image the cells with 3D confocal microscopy to assess the cell connectivity and cytoskeletal organization. So some of the greedy details we don't need to go over now. Here's a diagram of what I do. Take that big buck of collagen that has the cells and press it down and then punch out a button if anybody's worked with collagen before. It's very floppy and hard to work with. So a lot of effort to get that embedded within another matrix which is that outer pink thing around the disc that didn't have any cells and then pour the media on top. And that's all sitting on a glass side that allowed for imaging. So here's a confocal image of a confocal microscope. The advantage of a confocal microscope is that it provides higher resolution images than your typical conventional light microscope because it blocks out all of the out-of-focus information that's coming in. It also allows for optical sectioning so you don't have to have the tissue specimen so there's no need for processing or sectioning like in pathology. So here's some pictures I took just of the plain matrices of collagen on the left and fibrin on the right which is much of a difference. The fibrils look about the same. So we found that only samples that had been treated with the PDGF had cells that had moved out of the inner button and that's what we expected. The others were negative controls. Also the treatment with the PDGF caused the cells to interact and remodel. They'd pull on the matrix around them which is also what we expected. Some unexpected things. We found that the corneal fibroblast with PDGF migrated more rapidly into collagen when compared to the fibrin and also the pattern of the migration was very different. The corneal fibroblast migrating into collagen developed these dendritic processes and they moved independently whereas the cells migrating into fibrin had a more fusiform morphology and they formed these interconnected bridges out into the fibrin working together. And also imaging of the samples showed increased migration and more remodeling of the surrounding matrix which makes sense as the cells had more time. So here are some of the images from three days. On the left we have I'll explain kind of what's going on here. This is that button that I made and this is the surrounding outer matrix and here are the cells migrating out into the outer matrix of fibrin. These are multiple cells as we said when they migrate into the fibrin they work together to build these bridges out into the fibrin. And then on the right here we have the cells migrating into collagen where there are more cells have migrated out they're doing it individually and here the arrow is pointing to an area where they're really beginning to remodel that outer matrix. So here's kind of how we would determine if it was more than one cell or not. These two are different stains. The top here we have a stain for the cytoskeleton and the bottom here is a stain for the nucleus and when you overlay these pictures you get something like this which shows multiple cells working together going out and pulling on that fibrin. Here are some pictures from seven days. On the left again we have cells migrating out into the fibrin and again they're forming these interconnected bridges working together whereas on the right again here's the button and the cells are migrating out in the collagen there's more cells that have migrated out and they're working individually. So our conclusions that we could draw is overall it appears that this is a useful model for studying matrix remodeling during migration through both fibrin and collagen matrices and this model provides some unique insights and how the migrating cells perceive and respond to their 3D environment. The pattern as I've said between the fibrin and collagen was very different. The cells migrating into the collagen did it individually and made these dendritic processes whereas the cells migrating into fibrin worked together and built these interconnected meshworks. This collective migration interestingly is also observed during healing of full thickness corneal wounds in vivo. So this work continued and I was lucky enough to be part of a paper that was published this June of this year in the Journal of Experimental Eye Research. We added some things to the study that I did such as a time lapse microscopy where we get these movies of the cells actually moving out and pulling on the matrix and doing working together and we also redid the experiments using dermal fibroblasts instead of corneal fibroblasts and saw that the response was not unique to corneal fibroblasts. The spreading and migration patterns were consistent in both rigid and compliant substrates so the difference in the extracellular matrix stiffness wasn't causing the findings that we were finding. And most importantly, these results demonstrated for the first time that the extracellular matrix alone can induce a switch to collective versus individual migration. We know that different growth factors and things can cause cells to do that but this first time it shows that the actual matrix, the cells know what type of matrix they're in and that causes them to behave differently. And it's thought that similar processes to this may also influence cell behavior not only during wound healing but also in development, tumor invasion and repopulation of bioengineered tissues. Different growth factors we're going to try and then also coming up with a taking this model now and try doing, creating a wound to the button and seeing how the cells respond to a wound. Well, we're tired.