Intro (Nina Jablonski): Evolution involves more than the survival of the fittest. It's also about the survival of the most cooperative and mutually beneficial relationships are critical to the survival of every species. Welcome to The Symbiotic Podcast, where we will explore the collaborative side of life and work to consciously evolve science itself.

Cole Hons: Greetings, fellow Homo sapiens, and welcome to The Symbiotic Podcast. I'm Cole Hons, and today I've got three of my colleagues from Penn State here with me to talk about the work that they're doing involving bioprinting. Very exciting episode. First, on my left here, Ibrahim Ozbolat, is Hartz Family Associate Professor of Engineering Science and Mechanics here at Penn State. His major research interest is bioprinting and tissue engineering. Daniel Hayes is an associate professor of biomedical engineering at Penn State with an emphasis on nanomaterials, macromolecules, and composite structures for applications ranging from regenerative medicine to lab-on-a-chip technologies. And finally, we have Dino Ravnic, an associate professor in Penn State's Department of Surgery. He directs the plastic surgery research laboratory at the Milton S. Hershey Medical Center, which focuses on the creation of engineered tissue that is suitable for microsurgical implantation. Welcome, gentlemen, and thanks so much for coming down to be on our podcast today.

So first of all, I'd like to know a little bit about each of you, about your professional background, your research interests, and your expertise a little deeper than just in bio. Would somebody like to start on that? Ibrahim?

Ibrahim Ozbolat: Sure. I'm a mechanical engineer by training. I also received a Bachelor's Degree in Industrial Engineering with specializing in manufacturing in general. But later, with my PhD, I started to work on tissue engineering and 3D printing together. So then I established a lab in Iowa. I was an assistant professor before I started my associate professor faculty position here at Penn State. So there in Iowa, I did a lab that was focusing on bioprinting. And then we started to build different tissue bioprinting technologies and we focused on only a few different tissues at the time. So after I moved to Penn State, I expanded the lab significantly. And now, we've been working on various bioprinting projects and we double up, about 10 different tissues types from pancreas to skin, to bone, and cartilage.

Cole: Fantastic. Dan, what about you?

Daniel Hayes: Sure. I'm actually a Penn State grad, so I graduated from Ibrahim's department, Engineering Science, Mechanics, that's where I got my PhD. The focus of my work there was largely on nanomaterials and nanomanufacturing. After that, I moved to Louisiana State University and began working on biomedical applications of nanomaterials, nanofabrication, nanosensors. And then 2016, I came back to Penn State as a member of the Biomedical Engineering program. I also have an appointment in Huck and an appointment in the Material Research Institute. And now, we're focused on using spatial and temporal control to do nanomaterial drug delivery, to control differentiation of stem cells to make complex tissues.

Cole: Fantastic, thank you. And Dino, the surgeon on the team.

Dino Ravnic: Oh, I'm also a graduate of Penn State. I finished the Penn State general surgery residency program. After that, I completed plastic surgery training in Indiana University, and then reconstructive microsurgery fellowship at Sloan Kettering in New York City. My clinical practice primarily deals with reconstruction of cancer and traumatic defects across, really, the entire body. I'm fortunate to have somewhat of a science background that interrupted my surgical training for some basic science research. And most recently, I also have a master's in stem cells and regenerative medicine. And so our lab really looks at utilizing human progenitor cells and stem cells for the creation of vascularized tissues. And so it integrates, I think, pretty well with what these guys are doing.

Cole: Great, thank you. Wow. You guys do some amazing stuff. So I'd like to learn a little bit about the focus of your collaboration and what roles you each play. Where did it kind of kick off and what are you working on?

Ibrahim: So I think we can do a little bit history about how we got together, right. So I joined in 2015, and Dan in 2016, a year later. And Dino was already at Penn State when I came here. So I got an invitation from Dino to give a seminar talk in Hershey, and my interest was to bring more clinical perspectives into our research problems. So I have a lot of things that I was interested in translating into clinics, and one them is integrated bioprinting that we're going to discuss today a little bit. So in order to realize the clinical translation of bioprinting after, we definitely need surgeons and we definitely need people from other disciplines, like Dan in drug delivery or gene delivery purposes.

So I got together with Dino, we talked about how we can work together in creation of tissues and particle [inaudible 00:06:00], repairing defects on the live subject and surgical settings. And at the time, Dan joined us and then we got together. I mean, we were working on gene delivery, which is something that we already had a project, with the plasmid DNA. But he was not really doing plasmid DNA at the time. He was more focusing on micro RNA that he'll describe in details. And then I see that it's more beneficial and clinically translatable compared to plasmid DNA technology. And now, we start to work together. We published articles with Dan's group, with Dino's group. We share materials all the time. I focus more in bioprinting side, Dan is more in gene delivery or gene aging side. And Dino is giving us a lot of idea about the surgical implications. And then he also comes here and participates in surgeries quite often.

Cole: Thank you. Do you guys want to chime in a little bit about that collaboration?

Daniel: Sure. Ibrahim did a great job of summing it up. My interest in the collaboration is in developing these complex tissues. I mean, talk about intraoperative printing and making interfaces between bones, adipose, skin. That is right in the area that my group is focused in, but we don't have any knowledge or access about 3D printing. So that is not the strength of our group. But we do look at the gene regulation and how can we take undifferentiated progenitor cells or stem cells, and convince them to becomes these three or four different types of tissues in close proximity. There's a lot of complexity in the crosstalk between those tissues as they develop. And so how do you get from an undifferentiated mass of cells into three mature tissues at a very, very confined space? And that's one of the nice parts about the 3D printing in general, but particularly intraoperative printing, is it allows us to take these cells that we've pushed down these differentiation pathways, and get them in very close proximity, control where putting them so it can create these types of structures.

Cole: Yes. And for our listeners, let's define what intraoperative bioprinting is in particular.

Daniel: I'm going to kick that over to Ibrahim, he's the expert.

Ibrahim: So the intraoperative bioprinting is a process where the bioprinting process is performed directly on a live subject in a surgical setting. So this was previously done in some extent, not really in very deep and extensive manner, by a few groups in the world only. And particularly for skin regeneration. And the process is called in situ bioprinting, and some people call in vivo bioprinting, but none of these terms could explain the process precisely. And then since we had some clinical translation names, Dino came up with a very good name, "Intraoperative bioprinting, hey, let's call this."

Cole: Oh, you did that phrase?

Ibrahim: The name came from him, so he's the father of intraoperative bioprinting term, not the process, but the name.

Cole: Interesting.

Ibrahim: But nowadays, we're trying to change the name. Because people use in situ, in vivo, and now, we call it intraoperative, interchangeably. And hopefully, in the next couple of years, everybody call that process as intraoperative bioprinting, which is 3D printing of tissues directly on an animal. In the future, on a patient, to repair the defects.

Cole: Got it. I didn't realize that a member of your team had coined that phrase. Very cool. Dino, would you like to chime in on the collaborative nature of what it means to you to work with these gentlemen in terms the work that you're doing? What kind of opportunities arise from that for you?

Dino: Yeah. I mean, reconstructive surgery is very, very broad, and a lot of the solutions can be suboptimal and a lot of them require really complex reconstruction. So especially when kind of form and function are not [inaudible 00:10:32] restored, you always look for better opportunities. And I think the one thing with engineered tissues, both intraoperative bioprinting and other ways is really to bring this to the patient. And I really look at it primarily, at the end level of how we could utilize these tissues, how they could be made better, to be clinically translated. We'll say the one thing, anytime you're implanting human tissues, we really need human materials to start with. So what we've been doing is, we've been taking, essentially, spare parts or excess tissue from our surgical patients that we've been processing to isolate stem cells out of, and then we've been shuffling it to these guys here to do further differentiation, both for printing applications and driving them down differentiation pathways. And so that's something, I think, that it would allow the entire process to be basically, personalized from both the cell sourcing to the actual, the bioprinting and application process.

Cole: Wow, that's fascinating. So I have to ask, then, if you get these cells, you kind of harvest these cells from your patients and hand them off to these guys. Then where do you do you guys pick up? What do you do with those cells when you get them? What does that look like?

Daniel: When they come to our group, as Ibrahim had indicate, we look at the genetic regulation. So it's a process called post-transcriptional gene regulation. If you think about the central dogma biology, DNA to RNA, RNA to protein, well that RNA to protein step, we can interfere with the process using interfering RNA and change the expression of the gene. And we've identified a number of pathways that if we push on them in certain ways, we can drive stem cells down differentiation pathways so that you go down an osteogenic pathway, or adipogenic pathway, or an endotheliogenic pathway. And that allows us to control their future fate. And so that's what we look at. When we get the cells from Dino, we start looking at how we can manipulate them to go down these pathways that we're interested in to create these complex tissues.

Cole: Amazing. And then Ibrahim, you're trying to put it into the bioprinter?

Ibrahim: Sure. I think I'm at the last step. So after the cells are all transfected, and basically, he defines where these cells should go in terms of the final cell type, then we spatially, by controlling the locations of these cells, we print them layer-by-layer into the designated points. And then, they go to the formation of right tissue type or the targeted tissue type. So in this regard, Dino also participates in the surgeries that we perform here at the Millennium Science Complex. So we have other surgeons too, who participates in surgeries and [inaudible 00:13:39], like Elias Rizk from neurosurgery department. So sometimes, the surgical room in the in-animal facility can be quite crowded. Right, Dino? Two surgeons, these are really big surgeons, right. And then I have two, three students, and technician, and postdocs, all together. Five, six people are working on intraoperative bioprinting.

Cole: And this will be with rats, mice?

Ibrahim: Yeah. We perform mainly on rats. We have other bioprinting work with Dan's group, on mice, nude mice. So now, we actually received a project from Osteology Foundation from Switzerland, where we will intraoperative bioprint a bone on sheep. But that will be performed in Hershey, not in the University campus.

Cole: Wow. Well, what would you say is right kind of at the forefront of what's possible with this technology right now? Do you think it's happening here at Penn State? Are there others internationally, that are sort of in the ballpark with you?

Daniel: In the area specifically, intraoperative bioprinting?

Cole: Yes.

Daniel: Ibrahim would probably know better, but I think we're definitely at the forefront of that field. I mean, it was largely created by Ibrahim and Dino. And I'm just kind of along for the ride.

Dino: Adding that crucial middle step, you got to differentiate those cells, man.

Ibrahim: Well, we're one of the institutions that is at the forefront of this technology as we're doing both on large animal, we're doing more a composite-take tissues that's not really single-tissue type, but it's quite challenging for me to stratify layers of multiple tissues and interfaces. And now, the other goal is to induce some vascularization in them in order to keep those tissues viable. But I'm not a surgeon. Of course, I mean we discuss a lot with Dino and other surgeons, and my students what we should do in order to make this process successful. It's not just the technology itself, but there is also a significant biological component of it, right. I mean, printing on the animal doesn't mean that you'll really make it a functional tissue. So you got to make sure what you print is going to turn into a functional tissue that's going to repair the defect inside. If it's a bone, it will be a solid, strong bone. If it's for craniofacial defects, right, it's going to keep the skull really intact and strong, so that the brain won't really get any damage if there is any sort of loading on the skull.

So we discuss a lot about the biology, about the process itself, about the technology. Should we do just a 3D printing or should we do a [inaudible 00:16:46] printing? Or we can directly print on very complicated surfaces, but we don't really have to print vertically. But can be approaching from different angles, or can we use surgical robots that we can put a tip on a surgical robot, that the surgical robot can print? Because surgical robots have been used in clinics. I mean, Dino can tell more about those, right, for a decade or more. So these are my, of course, my contributions, right. But we also get a lot of contributions from the surgeons because we don't really know if what we propose is going to work or not. But these are the guys that really know if our proposed technology's going to work on patients.

Cole: They can take that in the practical reality, right, which is what the collaboration is all about.

Dino: Yup, exactly.

Cole: Fantastic. So a couple things come to mind listening to you speak. You're talking about vascularization of tissues. I want to make sure we touch on that, and why that's important, and why that's tricky. And also, the different layers of the different kinds of tissues. Can you speak to me how many different kinds of tissues have you printed? I think I heard something about bone being printed and the different kinds of cells. Could I hear a little bit about that? What's been achieved so far that we can share with the world.

Dino: I think what we've worked primarily, with Ibrahim, I think, is bone. I think blood cells have been printed, I think skin, we've done some work on. Ibrahim has also done stuff with pancreatic cells, beta cells. What else, primarily, Ibrahim? The main ones.

Ibrahim: Well, these are the main tissue types that we've been working together. But I mean, in terms of vascularization, one of his clinical practice is microsurgery, right? He deals with a lot of anastomosis related to it, where he connects little blood vessels to each other so he can tell more about why the tissue needs a vascularization.

Cole: Yes. And for our listeners, do sort of defining what vascularization mean. We're talking about veins, we're talking about arteries. Is that right? And I understanding?

Dino: Yeah, veins and arteries, and then primarily embedded kind of at the end level of the vascular tree that's supplying the nutrients to the tissue or the capillaries. And so a lot of the stems cells or progenitor cells that we're able to harvest from human patients, especially under the correct circumstances, we're able, actually, to form capillary sprouts. The only thing is that we need to, basically, engineer these capillary sprouts so they're part of a contiguous vascular tree, that when you implant each structure, they're able to be rapidly profused by the recipient site. And so I think that's one of the major limitations, still, of tissue engineering, is how do you perform this anastomotic connection? Following implantation, the body will grow blood vessels into the tissue you implant. And it will probably grow a little bit quicker if the implanted tissue already has an intertwined vascular tree but it's still a little bit too slow. So we're trying to figure out ways of how to speed up those vascular connections.

The nice thing as far as with microsurgery and super microsurgical techniques now, we're actually able to hook up vessels that are half a millimeter in size. And so I think, we don't need to basically integrate the capillary tree with a large vessel. It can be just you going from 10 micrometers in size to 500 micrometers in size. And then we actually already have the surgical capability to integrate that.

Cole: That's fantastic. One more question, and part one here, is how new is this whole field of bioprinting? I mean, how far back to we go? I mean, I hear about bioprinting a lot, but I don't have a real strong sense of the timeline. When did people start messing with printing biotissue? Printing tissues, et cetera?

Ibrahim: I think the foundation of this technology was basically demonstrated by a scientist called Klebe back in '89, where he modified an HP inkjet printer into a bioprinter format. At the time, the technology was not really called as bioprinting so he called the technology as cytoscribing. And he actually printed some proteins, not really cells. So early 2000, Thomas Boland, now he's currently in Clemson University... Sorry, he was in Clemson University at the time, but now, he's at the University of Texas at El Paso. And he printed living cells for the first time using inksheet printing technology, same thing that Klebe did, modifying a printer, then he print his living cells. And it was a time where people started to print cells, only a few groups. So at the time, it was just cell printing.

Nowadays, we are doing tissue printing. In the future, we'll call this technology as organ printing because we expect that in the next decade or so, we could be able to create larger organs like solid organs that are vascularized and that are scalable in size, and then they're surgically relevant in terms of dimensions and function.

We expect that it may not really be a fully functional organ but it will at least meet the requirement that the body needs. Like if it's a pancreas for example, it's one of the tissues that we've been bioprinting, so I will go suggest to make an organ, bioprint an organ that's [inaudible 00:23:02] and excrete insulin. But as you know, pancreas has quite a bit different functions like endocrine pancreas, exocrine pancreas, secreting different hormones. But we're just interested in the insulin secretion function of that. And there are other organs like lung, heart, kidney. These are some complicated organs. Complicated in terms of the structure, complicated in terms of the biology, as well as the mechanical function, like the heart. But with the advances in bioprinting, as I mentioned, the next couple of decades, we'll be able to see some tissues and organs translated into clinics.

Cole: Fantastic.

Dino: And I think the one interesting thing maybe, especially if you have the endocrine or the exocrine organs, the things that ultimately may be printed may not look exactly like the organs they're intended to replace. So I think the goals is probably to restore the function, but I think for a lot of organs, it doesn't necessarily need to be the exact form. And so then, that really broadens how you can tackle these problems of vascularization, implantation sites, and things like that.

Cole: So it makes you think sci-fi. It makes you think of almost, cyborg people with whole new organs that didn't even exist before. We're getting to that level. Fantastic. I'm Cole Hons, this is The Symbiotic Podcast. We'll be back in just a minute with part two of our conversation.

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Cole: Welcome back to The Symbiotic Podcast. I'm Cole Hons, you're speaking with three of my colleagues from Penn State about bioprinting. So for part two, I'd like to launch into a topic that we always cover on this podcast, which is the interdisciplinary nature of what you're doing and the transdisciplinary nature of people getting out of their normal box, working together to have a bigger vision to do something they couldn't otherwise accomplish. And we've already touched on that a little bit with the three of you folks in the room today. Who else have you been working with and what are they bringing to [inaudible 00:26:02] that has an impact on what you're doing here?

Ibrahim: Sure. So I've been working with various different scientists, both at the University Park campus, Hershey campus at Penn State, as well as other collaborators out of Penn State. Like Jackson Lab for example, that we've been working on 3D bioprinting of lung tissue that they are interested in creating a platform technology as a tissue model where we can use that to understand interactions of the immune cells with the bacterial or viral infections in the lung. So unfortunately, we can't really have access to the human lung that we can use as a model platform. And animal models, they don't really represent the human physiology closely, so in this regard, we need to create lung models.

And I'm here to make lung model, but of course we need a lot of expertise from different areas, like the lung biologists is one of them. We have a collaborator from Ohio State University Children's Hospital, that he brings a lot of expertise with the lung biology. We have quite a bit collaborators at Jackson Lab, their immunologist and their T-cell expert that really understand what the immune cells do, how they behave. And we have also people that are expert in microbiome at Jackson Lab. The microbiome is the bacteria culture in the body. So other than that, I have collaborators on other projects, like in this new project, hopefully, we'll receive it in the next couple of weeks, funded by NIH. So then, it will be funded that time. So as we mentioned, we intraoperative bioprint composite tissues like multiple layers, as Dan suggested. Sot hat has soft tissue, that has hard tissue. And that's being done on the, currently, maxillofacial region on the body.

So we need a plastic surgeon that's really expert in soft tissue. We have a neurosurgeon Elias Rizk, he's at the neurosurgery department. His expertise is in the bone regeneration and bone surgery. And then we have Tom Sampson, he's an expert in kind of the maxillofacial surgery and he's also a plastic surgeon, right. So he'll bring a lot of expertise, not just on the cranial side, but if he move into other directions where we want to repair other tissues on the face. I think tom Sampson's going to contribute significantly. So these are some people. Of course, we have other people that Dan and Dino, they've [inaudible 00:29:10]. They can tell more about those people.

Daniel: Our group focuses a lot on, obviously, the nanoparticle-based drug delivery, for delivering the tools to regulate gene expression post translationally. And that is a very materials-focused problem. And so we've got collaborators Jim Adair, Josh Stapleton here at Penn State, who helped us a lot with both the design and characterization of those particles, making sure that we're making what we're trying to make and that they behave the way that they're supposed to so that we can deliver the microRNA mimics to their appropriate tissue types. Beyond that, we have some collaborators here who help us with the stem cell differentiation work. So Lance Lian, who's in the biomedical engineering program, helps us a lot with the stem cell differentiation. Additionally, we have [inaudible 00:29:55] Penn State, so Jeff Kimball has been a great collaborator for us at Tulane University and helped us a lot with mesenchymal stem cell differentiation and really has helped us refine our process for controlling them.

Cole: Perfect. Dino, how about your collaborators?

Dino: Yeah, I think that's really the gist of them. I think we've touched on on most of them. We have some additional folks at Hershey, where we've collaborated with the OB-GYN department to allow us to get umbilical stem cells. So most of the tissue that we retrieve is from adipose tissue. Those are from our group's practice, but we've also been able to use both the umbilical cord and umbilical cord blood to [inaudible 00:30:39] those stem cells as well, so that might be a potential outer area of starting cellular material. And we've worked with some of the other folks now we've got interested in that, the manufacturing even to do 3D printing of not necessarily for biologic processes, but for surgical appliances and models and things like that.

Cole: Interesting. Fantastic. And this very building that we're sitting in right now doing this podcast was designed to kind of bring all these different folks together and give them cutting edge technology that they can share for the materials or life sciences, et cetera, and be able to facilitate these kinds of collaborations. Would you guys care to talk about what it's like to work in these kinds of facilities? Is it doing what it's supposed to do?

Daniel: I could speak for our work. I mean, it's absolutely enabling. I mean, having access to both the expertise in terms of Josh Stapleton and his team here at MCL, Material Research Institute. But also then, the direct collaborators, having us all in the same space, in the same building, having our students be able to cross-pollinate, it's been great. And I don't know that it would have happened had we not actually had this convergent science building to really drive it.

Ibrahim: And we pretty much don't really go outside of this building except the micro-CT scanning work that's being done in Hershey. A collaborator of ours, Greg Lewis, in orthopedics department. So we send the surgical [inaudible 00:32:16] like when we utilize animals. We take the regenerative tissue out, and then, we ship that to Hershey, and then he does micro-CT scanning. And he send the results back, and that's how we collaborate with some other Hershey folks. But I can say nine percent of the work that's being done in our papers are all being done in this building. So we're not really utilizing just more than a couple of things outside this building.

Cole: Good to hear it's doing what it was built to do. So I was curious about what Penn State's contributing to this field that's unique that other institutions aren't really touching in a way that we are? What contribution are we making here?

Daniel: I can't say that anything we're doing is absolutely unique, but I can say that the focus of this collective group on complex tissue regeneration and creating functional tissues, and when I say complex tissue regeneration, I mean the tissues that are composed of different cell types, but they are all playing their appropriate functional role. And that we're creating complex tissues through 3D printing and gene regulation that actually function appropriately, that have the appropriate structure.

Ibrahim: So in terms of other strengths that Penn State provides, is a material science that sometimes, when we double up any materials, we're not really super sure but the quality of what we produce or the novelty of what we produce because we're not really material scientists. So we have quite a bit contributions from material scientists of Penn State. And particularly, for example, we have a project on 3D printing of biodegradable metals. That's something new that we're interested in. 3D printing biodegradables. So the printed metal compared to the other metals like titanium or other, steel-based implants, these biodegradable devices, they don't really stay in the long term. So they dissolve, and they also support the regeneration of the bond. Segmental, large scale [inaudible 00:34:43] bonds. So in this regard, we work with material scientists, that you can't really find such quality material scientists in other institutions. As Penn State is known with its strength in materials engineering, so now, we're developing new materials, biodegradable metals that can be 3D printed for fabrication of biodegradable implants.

Cole: Oh, fantastic. What do you see Penn State doing that you don't see elsewhere?

Dino: Like Dan says, I don't think it's completely unique, but it certainly helps. I mean, there's only 140 medical schools in the United States. And so the ability to have a large scale university with an academic medical center, it really allows that clinical translation to be easily developed in both directions, both bench to bedside and back, and bedside to bench. And I think that's something that really helps evolve in the entire field, both from a clinical discipline and from a research discipline forward.

Cole: Right on. Thank you very much. Well, this is The Symbiotic Podcast. I'm Cole Hons, we're going to take a little break and we'll be back with a third and final part of our conversation. Stick around.

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Cole: Welcome back to The Symbiotic Podcast. I'm Cole Hons, here with my colleagues from Penn State talking about bioprinting. So I'm really curious to know, how far off do you think we are from being able to apply these technologies and these techniques to human beings? What would it really take to get there?

Daniel: That's a pretty complex question and a lot of it depends upon how the FDA would respond to the use of stem cells and genetic manipulation of stem cells. And so the short answer is, we don't really know because they're not approved yet. But I'm guessing anywhere from 10 to 15 years, we're going to start to see this type of technology, stem cell based technology, manipulated stem cells, finding their way into the clinic.

Cole: Do you guys agree with that assessment?

Dino: Yeah. I think the biggest drawback is now, cells really can't be manipulated if they're going to be used for other applications. I think for engineered tissues though, clinically, once kind of the regulatory hurdles are cleared, I think the probably thinner tissue is probably going to be able to be most easily clinically translatable. And again, just because of the more volume of tissue that you have, the more vast your blood flow you require to profuse it, it's harder to integrate it. And so a good example would be skin grafts.

Skin grafts, when we implant skin grafts, we don't have to basically hook up their blood vessels. We just laid the skin graft down onto a recipient site. And as long as the patient is healthy and the site is healthy, new blood vessels will grow into it. And because it's just not a very large cell mass, most of the skin graft survives. But if you would try to do that with tissue that's much thicker, then obviously most of the cells will not survive. So I think from a clinical perspective, that will probably be the forefront, as far as in the initial steps of these emerging technologies.

Ibrahim: Yeah. I actually explained that to my students yesterday, in my bioprinting class. So we went through the bioprinting tissue types, from skin to cartilage, to pancreas, to liver tissues. And then we discussed the possibilities of clinical translation of these tissues. Of course, as Dino mentioned, the tissues that are thin, that are avascular, that is, without blood vessels are easy to translate. And particularly, connective tissues are the very first tissues that we'll see in the clinics. As Dino mentioned, skin is one of the first one because it's very thin, it doesn't really have blood vessels that it needs to be printed inside. Cartilage is the other one, because the cartilage doesn't have blood vessels. I can see bone is very well studied and then we have a very good understanding of bone regeneration with these bioprinting technology. And then I can see that the, not really super large segmental defects, but smaller defects could be easily repaired with the 3D bioprinting technology.

Of course, there is also the FDA-related issues that needs to be overcome, right. But except the FDA clearance, the major challenge with overall organ printing is vascularization. Creating multi-scale blood vessels, from arteries and veins, down to capillaries, that you can profuse them and when you implant them, get long term patency. I mean, these are some important challenges. At this point, we haven't really seen any large scale, bioprinted organ that has multiple scale of blood vessels in it that is transplantable. So as soon as we overcome that hurdle, I think we'll see more tissues types in the clinics. But [inaudible 00:40:37] organs like kidney, say, heart, lungs are very complicated in structure and in function, so these are going to take long time to be seen in the clinics.

Cole: What advice would you give to other researchers who are thinking about breaking out of their discipline to work either on bioprinting or other transdisciplinary projects in terms of the special things that you have to do as a researcher to get out of your box and kind of play in this transdisciplinary arena?

Daniel: I guess I would say, you have to be patient and you have to be comfortable with the fact that you may not be the expert. We've all been trained to become experts in our field. And when you start working in these interdisciplinary areas, you find out that often, you don't know a whole lot about that interdisciplinary space. I realized that I know very little about surgical interventions. I know very little about 3D printing. And it's humbling at times, to discover how little you actually know. But that, to me, it's making yourself comfortable. Or at least getting over being uncomfortable with hat lack of knowledge.

Cole: And I imagine too, you get the play the other role as well. With these brilliant people coming to you, going, "Oh, I had no idea how that worked," right? So that's got to be kind of a cool feeling too, to be able to educate one another.

Daniel: Yeah. That is definitely nice. I mean, sometimes, when your colleagues come to you with questions that you take for granted, you're like, "Oh my gosh, other people don't know this."

Cole: Right, right. Exactly. Dino, what do you think about all of this?

Dino: I think part of it is, at least learning a little bit of the other disciplines' lingo. And I think, at least being able to have some common overlap. I think the best way is to kind of collaborate together. Because when you toss around ideas, there's things that you would never think of on your own. And they kind of develop spontaneously out from kind of crosstalk, I guess, of different disciplines. The ability to be innovative is made much easier when you have other minds kind of chiming in.

Ibrahim: I think most of the bioprinting research nowadays focuses on just printing cells and then doing some very short term testing. It just shows the cell survival, and then they can be functional a bit. But in terms of the tissue function, the tissue maturation, there's very, very small focus. In addition, we want to see these tissues are really working in the animals. So transplantation of this into animals. Small animals, and then large animals, and then showing that these tissues are really functional is a key factor. So that is why I'm a mechanical engineer by training, but nowadays, I do just tissue engineering work. I don't really do a typical mechanical engineering does, so it's a very different field, but I learn. And in my opinion, in order to be successful in this field, just printing cells requires a lot of biological expertise and facilities too. But even just printing cells is not really satisfactory at this point. So we want more successful outcomes, and then therefore, people should train their selves in tissue engineering, and sometimes, in clinical practices too.

Like a student of mine who is working on intraoperative bioprinting for about five, six years, he actually did a little interning in Hershey, in the hospital. So he got a certificate, so that means he's now learning surgical practices. Not really as a surgeon, but at least he has some sort of an understanding. He's an electrical engineer by training, but he maintains the animals, he participates in the surgeries, he does the medication to animals. So these are very critical for the success of our work. So we definitely need people from different expertises, but sometimes, we need to educate ourselves towards the transdisciplinary directions.

Cole: Got it. Yeah, makes a lot of sense. What's next for you guys? With the work that you're doing and everything, all these papers coming out, and grant proposals being written and everything, what do you see is kind of next on your agenda?

Ibrahim: So the next things are more larger projects that is basically done with the contributions of a quite a bit number of people, more than 20 investigators from various different disciplines. And particularly, NIH has U19 type of grant. NSF has a [inaudible 00:45:42] center grant. So here, the goal is, can we bring those center grants at Penn State? And particularly, can we create a center here on tissue engineering, [inaudible 00:45:53] medicine, and bioprinting, biofabrication, biomaterials, and in the meantime, bring such larger grants to create an atmosphere that we can exchange the ideas and make all these, not just bioprinting efforts, but gene therapy, surgical efforts are all successful.

Cole: Thank you.

Dino: Yeah. I think I would definitely agree with that. And I think the nice thing about kind of the approach or the understanding, is that ultimately, you'll be able to get more and more tissues involved, and more and more applications. And the interesting thing is, especially with stem cells, and you're able to really differentiate into multiple cell lines, the combinations that you can make is really limitless. You just have to really see what are the best ways to integrate it. Hopefully, in the future, we'll figure out ways to basically integrate these printed constructs with the recipient vasculature quicker. So I think, hopefully, that will continue to develop.

Cole: Thank you. Dan?

Daniel: I have very little to add, other than say, I think that definitely, what I see is the future where my interest is that integration of surgical technique and engineering along with the biology to target some low-lying fruit and try and get tissues developed, make a clinical impact. And so as I just said moments ago, we're probably 10 to 15 years out, probably, from getting some of these things approved. But can we position ourselves so that as this move towards approval, that we're ready to actually take them to the clinic and impact people's lives directly.

Ibrahim: And then the other good thing is Penn State is now hiring more faculty. I think some of the directions are there are new centers in biomedical devices, and some departments are hiring faculty that lines up with our interest, like Amir Sheikhi is just showing in chemical engineering department. His expertise is in hydrogels, mainly cellulose-derived materials for tissue engineering purposes. And I hope we'll hire more people, more faculty members that will actually fill the gaps that we need now.

Cole: So what about immunology? I mean, we hear about bodies rejecting organs when they're transplanted sometimes. What does that look like for engineered tissues? How are we addressing that angle?

Dino: I think it needs to be a little bit regarding this initial cell sourcing. And so there's some thoughts that some stem cells are immune-privileged, some aren't. And so a better understand of that will hopefully allow the correct tissues to be developed. For example, is it possible to take a skin cell from somebody and reprogram it to an induced pluripotent stem cell where then, you can differentiate it into any cell you want, and then that cell would have the same genetic background as the person you're ultimately implanting into. And so then, you would think, well, hopefully, that would be immune-privileged. Hopefully, it would be rejected. Now, you don't know how it would be transformed if it was developed in vitro for a portion, or how it's transacted to be an IPSC cell. But the idea is that if you're able to use the stem cells from the patient that you're ultimately implanting, hopefully, that would be minimized.

Cole: I see, yeah. And do we have anybody here at Penn State addressing that specifically from an immunological perspective?

Daniel: So Lance Lian, and he's a Huck member in biomedical engineering. He's working on creating universal IPSCs. So manipulating the cells genetically to make them as tolerable to largest number of potential patients.

Cole: Oh, fantastic. And I want to just salute you guys for all the amazing work you're doing, and thank you again for being on the podcast. Best of luck with everything you're doing. Thanks.

Daniel: Thank you.

Ibrahim: Thank you.