Ibrahim Ozbolat, PhD, Associate Professor, Tissue Engineer, area of expertise in 3D bioprinting, talks about new research being done to print 3D cartilage.
Interview conducted by Ivanhoe Broadcast news June 2017.
Our viewers have heard a lot about 3D printing, can you talk to me about how this is emerging as a field? What is 3D printing and what is 3D bioprinting?
Ozbolat: 3D printing is a technology where we can build objects layer by layer, adding each layer one by one and building very complex objects which can be obtained from digital images. So the 3D bioprinting is an extension of 3D printing in the area of tissue engineering, where we can actually print cells. It’s not really metals, it’s not plastics, it’s the living cells or many tissues that we can print them and localize them in wherever we want and then build complex tissue architectures. The deposition of living cells spatially, layer by layer, in order to create three-dimensional complex tissue architectures. And 3-D medical images from patients can be directly used; extracted data from these images can be sent to the 3D bioprinter, which is a machine. And the machine can treat printed cells based on the information that we get from the patients image dataset.
You are working right now on 3D cartilage, can you tell me why focus on cartilage?
Ozbolat: Cartilage is one of the projects that we have been involved. We have been engineering about eight, nine different tissue types; from pancreas to bone, from skin to composite tissues, tumor models … Cartilage is one of them and the main reason why we make cartilage is because osteoarthritis is a big problem. A lot of people suffer from this disease and people can’t really even walk. And then they have a very hard time doing activities in their daily life. So because of that, we want to make a cartilage made of patient’s own cells which can be implanted into patients in the future and fix their problem.
Can you take me step by step how this works in the lab? What exactly are you doing in the lab to get this to print?
Ozbolat: We use cells from animals and multiple human sources. So, with the animal cells, when we isoloate them; we culture them and then we grow them in large numbers. And then we have called bioink; the bioink is the biological version of the ink that is used in paper printers as you can see here. So it’s replaced with the bioink, which is the hydrogels in general. They are gels, like a contact lens is made of gel, it’s a very soft sub-straight that we have the cells loaded in them and then we load the bioink solutions in to a bioprinter nozzle, and we have various different bioprinting technologies. It can be extrusion based, droplet based, or laser based bioprinters that we can print the cells locally. And then we can create a complicated architecture with various different cell types; and particularly for cartilage, if we have only single cell type, then we can print that in a scalfold free manner, there’s no hydrogels. For cartilage, we made something very specific, not something that we use in general for other tissue types. We do that as biomaterial free or scaffold free. We don’t only use an exogenous material, it’s purely cellular. So what we do is we actually grow the cells in cylindrical fibers, we call them tissue strands. We make tissues of cartilage in a strand form, and then we extrude that fiber layer by layer, these are living tissues so they are not inert materials, and then they fuse into very large cartilage patch.
When you say a very large cartilage patch about how large are you talking about?
Ozbolat: It depends on what you want. We can make up to a centimeter scale, so five centimeters, but it depends on the number of cells that you can grow. For proof of concept demonstration, we did more than that but as long as we have the cells we can scale it up.
You had mentioned cartilage, because the nature of cartilage lends itself to this kind of printing, can you describe again why?
Ozbolat: The reason why cartilage is a perfect tissue type for this scaffold free bioprinting is because the cartilage doesn’t need any blood vessels and the cartilage cells can interact better if you don’t only put in exogenous material. So when you put the cells in exogenous materials the cell interactions are limited. Since we don’t use that exogenous material, we just have the cells assembled together. They can interact one to one so there is no confinement in between, and they can talk and they can deposit the cartilage specific proteins in a better way. We can significantly produce a huge difference, in the meantime the cells can grow and then turn out to be cartilage like properties in the first couple of weeks.
How long do the cells have to culture?
Ozbolat: It depends on what you want. Specifically for cartilage we use about 200 million cells. That’s huge. 200 million cells took us two months to expand that many cells. For other tissue types we don’t have to go to that number. It can be say 20/30 million cells.
What is next once you have these cartilage patches, down the road what would be the next step, what would be the translation?
Ozbolat: The next step is to recapitulate the need of organization of cartilage which has different zones. When you get a cross section of the cartilage, you see different zones, so the cells are oriented in a different way, you’ll have a different morphology of cells and then now we are recapitulating that organization. We just got a project and someone else now will take over the project, like a new student. He’ll be working on that and then the goal is to make that structural heterogeneity and mimic that. After doing that the next step, we’ll be translating that into the clinic, making everything humanized using human cells; should be stem cells. Because we can’t really grow cells in huge numbers if the patient is old. So, from young infants we can definitely grow that many cartilage cells but if the cells are older, if the patient is older, the cells will be old as well and then they lose their differentiation ability if you want to culture them in large numbers. When we have these stem cells from the operating room and then we differentiate them into cartilage-specific cells, then we do different zone architecture with the heterogeneity in the structural and biological properties. And then in order to test and study in large animals, we actually have sheep models that we’ll start soon. We have rabbit models that will be for another study but sheep model will give some better outcomes compared to small animals. And then after sheep, then we’ll move into clinical translation.
How far along do you think we are before we see this in humans?
Ozbolat: I think that’s a very difficult question for me all the time. I don’t really want to give exact numbers but I can say considering the research, perhaps it may take another four or five years. Then including the large animal studies, and then the clinical transition for humans, plus getting FDA approval may take, you know another five, seven years. I can say roughly from ten to fifteen years.
How would this work in humans, you print it and then implant and let it grow?
Ozbolat: Everything needs to be translated into clinic. So we can’t really have the patient cells coming into the lab and then we grow them, this won’t be allowed by FDA. We have the bioprinters, hopefully they’ll get into hospitals and OR rooms and then we have all the materials isolated from patients, and we can grow these cells in the hospitals and then print them and transplant it back to the patient.
So right there in the hospitals?
Dr. Ozbolat: Correct.
When you’re talking about a bioprinter, I’m looking at something that looks like a LaserJet printer in my office, is this what it actually looks like, a regular laser printer?
Ozbolat: We have different bioprinting mechanisms and one of them is inkjet printing. It’s two-dimensional inkjet printer that you use in your office. What we do is we extend that into 3-D. In your 2D printer, a paper printer, there’s a paper mechanism that you feed in the paper all the time. Instead of that paper-feeder mechanism, you have a three-dimensional motion stage where you can actually go in X, Y and Z. There is something being ejected from the top as the ink comes out from the cartridge from your printer, it’s the same mechanism. Instead of ink, there’s a bottle of cells that’s liquid. The liquid is ejected and then you deposit that on an X, Y, and Z motion stage. You can go in X and Y, you can control the positioning of this ejected droplet in X and Y in order to build in 3D, you also go down, and of course this is one of the mechanisms. We have extrusion, it’s very similar to extruding toothpaste from a tube, when you brush your teeth you extrude the paste, it’s the same technique. We have the cells in the solution, which are loaded in a syringe barrel and then we use the mechanical forces to extrude that like when we want to extrude the toothpaste. We use our hands to squeeze it, it’s the same principle. Instead of using hands we use a mechanical ram and we extrude that. In the meantime, during the extrusion the hydrogel cross-links and then it solidifies. It’s deposited in a form of a solid structure.
How long does it take?
Ozbolat: Imagine how big you want, the construct size and the mechanism that you use. For inkjet, it may take longer because you’re just injecting little droplets about like six or seven microns in diameter. But with the extrusion process you extrude fibers of like two hundred microns or so which takes shorter time. Perhaps you can build an object like say one centimeter in just like five, ten minutes. Again it depends on what you want to print. The resolution, the cross-linking time, the solidification time and number of cells that you want to print. If you want to print multiple cell types, then you need to move from one position to the other one. Then it takes some time too. So again, everything depends on the final goal and the way that you print them.
Was something like this even possible five years ago?
Ozbolat: Well bioprinting emerged early 2000 so it’s not that new. The first time that the cells were printed was in 2003. The cells were printed using a modified inkjet printer from HP. It was modified and the cells were printed, that was 2003. So right now we can print tissues. After fourteen years we are at the tissue level, we’re not just printing cells. Printing cells doesn’t mean you really print tissues, but now we can print tissues. And then in the next ten years timeframe when I look at the past, now, and the future, I can envision that we can be able to print very complicated organs in the next ten years.
END OF INTERVIEW
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