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Trinity Bivalacqua: Good morning. I'd like to thank Josh and the Engineering Society for giving me a chance to talk today about, number one, a clinical trial that we actually presented last year in the late-breaking abstract, session and also is currently under review.

I'm going to ask everyone in this room to keep your mind and sort of scientific rigor open for this first discussion because I'm going to tell you sort of the lessons learned and how we hope to fix it. By way of disclosure, the data that I'm going to present today was a sponsored by a company called Tengion and I'll go over that more later and some of the work that we're presenting today will involve a provisional patent.

We heard a little bit about sort of the principles of tissue engineering and how this relates to the production of a new urinary like tissue. It involves biological components in particular cells. I'll touch base on some of the cell types that we're currently utilizing as well as what we utilize in our trial. It involves the actual local microenvironment.

So, in the case of urinary tissue, the urine itself, as well as the vasculature and the urothelium that we're working with is actually providing that niche to stimulate regeneration. And then there's a nonbiological component. This is what we just heard about, scaffolds, biomaterials and this is actually and sort of what we found is probably more important than some of the other things that I just discussed.

So tissue engineering and bladder cancer is a little bit different than what you've heard with the pediatric condition because we're dealing with patients that have, that have cancer, bladder cancer, we're removing their bladders and we can't take an actual biopsy of their bladder to isolate autologous cells.

The reason why is because clearly they have cancer, so we have to come up with a different approach. So we have to be able to utilize a different population of cells to seed the bladder. So when you think about bladder cancer, obviously, the urinary diversion or reconstruction is where all the complications that all the late effects occur. As we just heard from Josh in the pediatric population as it relates to augmentation and the majority of patients getting a cystectomy will ultimately have an ileal conduit or an incontinent diversion.

So, the hypothesis here was is that if we could figure out a way to use a tissue engineering or regenerative approach to make a tube or a neo-urinary conduit for which we can facilitate passage of urine to the outside without using the GI tract, we may be able to mitigate some of the complications.

So, first thing we had to do was, is identify a way to isolate cells. We ultimately used autologous cells isolated from the fat or abdominal wall. Here's just an example of how we can isolate smooth muscle cells from, obviously, the bladder itself. But once again, we can't do this in bladder cancer patients or we can take it from fat or actually from peripheral blood, so PBMCs.

So, we ultimately chose to use the fat because it was a rich source. So, the concept here was that we could isolate adipose-derived smooth muscle cells, implant them, and allow them to grow on a material. We heard about the materials that were, that have been ... heard about a little bit about the material science from Josh and actually the reason why we use PLGA in this trial is because it was FDA approved and Talla had used this in his first trial. And then we removed the bladder and then we use the omentum to wrap the PLGA material that seeded with us, with muscle cells and then we bring that to the outside.

The sort of translational challenges that we encountered were mostly related to the actual material that would, that we could potentially use. I will acknowledge right now that the material that was utilized in this trial was the wrong material and I'll show you why. The autologous cell source, which was smooth muscle cells, it actually was able to make urinary tissue, which I'll show you. However, it would probably wasn't adequate enough to completely proliferate urothelium.

The surgical approach, we had to use blood supply. Omentum was initially chosen, and I'll show you the results of the trial. But first, we had to use a preclinical model. So preclinical models as Josh just discussed are really important when we want to recapitulate the human condition. So we used a swine model where the bladder was removed, a cystectomy was removed and the ureters were actually sewn to the biomaterial, it was wrapped with peritoneum, not omentum because the peritoneum was utilized on the abdominal wall.

Now, the difference between men or women and swine or pigs is that they are on all fours, men stand up, women stand up. So when this neo-urinary conduit was used in the porcine model, it was not brought through the abdominal wall like we do in humans. So this was actually a mistake because ultimately we didn't predict or understand the importance of that, and I'll show you why.

But, in the porcine model, three months later, there was good tissue regeneration. Here are examples of the urothelium with Cytokeratin staining. We see early calponin and alpha-smooth muscle Actin staining, suggesting that we were able to regenerate urinary tissue. Now this is the result of what happened to the kidneys in these porcine or swine and what happened with the kidneys on the left and right side was that we saw an increase in size, so we all probably can recognize that increase in sizes are probably related to hydronephrosis.

And, you can see here that actually they developed diverticulums in some of these animals and this is because of resistance and ultimately contracture. It's easy to say this now because we're looking back, but the reality is is that they had upper tract early deterioration, but, statistically, there was no difference in creatinine or size of these pigs.

This went to the FDA as an IND. They approved a phase one trial to do this in 10 patients. The end result of the 10 patients was actually to have safety. So was this procedure safe? Where are we able to show patency and the PIs for the trial was myself, Mark Schoenberg. At that time, he was at Hopkins, ultimately left and the PIs in Chicago were Gary Steinberg and Norm Smith.

This is the cohort of cystectomy patients as their normal cohort of muscle-invasive disease. They receive neoadjuvant chemotherapy as well as non-muscle invasive disease refractory to BCG. Here's the length of stay was five days. What I will tell you, it was remarkable. It was like we did an open prostatectomy, patients were up and walking around on a regular diet the next day ready to go home, but by protocol, we had to obviously watch them in the hospital.

This was the protocol. Once again, biopsy the abdominal wall, removed smooth muscle cells, seeding them on the PLGA and then placed them into the patient. Once again, rapid omentum, provide the blood supply, bring it through the abdominal wall, just like an ileal loop, and here are the results. Here are eight patients who are actually enrolled. What I will tell you is all eight patients needed explantation of their neo-urinary conduit due to upper tract deterioration.

In the early stages, we had some problems with the surgical approach. We made modifications in the surgical approach that allowed us to start seeing patients that were going out to eight and ultimately one patient out to 16 months. But, all patients developed contracture of the biomaterial and stricture at the stoma and upper tract deterioration.

Here's an example of a patient, six, who was actually, unfortunately, died from an unrelated to the trial, actually had a myocardial infarction and his family allowed us to explant this and he was seven weeks out and we could see it. Or as early as seven weeks, we're seeing smooth muscle growth and we're seeing some urothelial or cytokeratin positive cells. At 10 months what we noticed when we explanted this neo-urinary conduit, is that our conduit had actually contracted to nine centimeters. It initially was 15 to 16 centimeters, and actually, the conduit was only about five centimeters.

And if I showed you this picture without telling you what it was, you'd probably say, well, that kind of looks like a ureter. So what we ultimately made, or from this early trial was actually a ureter. It had, it was cytokeratin. It had urothelium from where the ureters connected to the skin. The skin was mostly made from squamous and we had beautiful calponin and alpha-smooth muscle contractile protein expression.

We actually had growth of nerves, as well. We actually saw an ingrowth or a neuronal innervation, this was mostly around that where the ureters had sewn in. So there's clearly proliferation of nerve fibers. But, the problem was, it wasn't functional. We saw no function, none of these patients had a good functional result. They all contracted, they all developed upper tract deterioration and needed explantation.

So, the ability for us to regenerate and make urinary like tissue was present. However, it was not functional. So we did not meet our endpoint. So why was this? Well, we hypothesized that it was probably the biomaterial that was really was not adequate to allow there to be a full compliance or a tensile strength. So we sought to fix this. And the other problem was is that the neo-urinary conduit, when it's brought through the abdominal wall was kinking because when the patients move there was actually kinking and this prohibited or caused problems with blood supply.

So, our issues were scaffold, cell type, nutrient supply. I'll show you about work that we've focused on in the lab to improve upon the scaffold. The first thing that we did to do this was we hired a material scientist, Ani Singh, who is a champion in this work. He is a biomaterial scientist who has special interests in collagen-based scaffolds.

So, the question is, what is unique to a material that would be important for a bladder, to make a good compliant bladder and how do we prevent urine extravasation? Well, what I will tell you is in order to prevent urine extravasation, you have to have a co-culture of cells. And I'm not going to show you this data, but what we've now done is we've used the epithelial cells from a buccal mucosa to actually, to seed the inside of our material. And then we use smooth muscle cells to provide the stimulus for vascular regeneration.

But, if you see a synthetic material, you have stiffness. So as you have the increased strain, you have stress or increase in stiffness. With a urinary bladder, you get a J curve, which actually is indicative of compliance. And this is ... the reason why this occurs is because of the interplay between collagen and elastin. So, when the bladder fills, you get this, you get this nice crosslinking of collagen and elastin and you have the compliance that is necessary.

What we see here is, is the PLGA, the first series of experiments we did was look at the degradation of PLGA and within 14 days we're seeing significant degradation and we're seeing a significant strain or a lack of compliance. So this would tell us automatically that this was the wrong material to utilize. The collagen-based scaffolds for which Ani's using now have a completely different characteristic. So if we use collagen-based scaffolds, the benefits, at least to regenerate the urinary system is, is that it's physiological relevant, it's consistent with native urinary tissue, it's FDA approved and it's also available to sort of the off the shelf.

The challenge is making shape, mechanical properties that are similar to the bladder, and can we suture on it, is it durable? So, I'm not going to sit up here and pretend like I know about the production of a material, but this was Ani's ability to make the collagen formulation for which he did the material. And unfortunately, I can't speak to this part. But, what he showed was, and what we've been able to demonstrate is this collagen-based scaffold is more compliant and actually we can suture on it and if you look at it and actually were able to make it into a tube as you saw previously with the other fibers utilized and we're actually able to make bladders, we can make actually ilium, which we've already done and we can actually make viscous organs.

What we found was is when we compare it to ureters or pericardium, SIS is another material we've utilized, is that our material actually has wonderful compliance and it doesn't, and when you cycle it through different cycles or heat, you continue to have the same ability to be compliant with low pressure.

When you look at different cycles of heating and with the pressure we see an improvement and when we look at suture strength, we're able to suture on this and we're able to keep its properties in vitro. So, as you just heard about animal modeling, we've now moved this into an animal model of augmentation. As we just heard Josh talk about, this is actually a modified pericardium we've done this with our modified collagen and what we see with our modified collagen and pericardium, we're actually able to get good ingrafting of neoangiogenesis. We have a lack of immunogenicity with less inflammation and we see good improvement in compliance as we looked at it in Maya graph. We have not done urodynamics.

So, in conclusion, the translation of technology in our preclinical models demonstrated a native-like tissue in the phase one trial. However, this was completely not functional and durable. So we've chosen to use newly designed collagen-based scaffolds to improve upon our ability to have compliance with dual seeding of cells to ultimately make a neo-urinary conduit. I want to thank my collaborators and the team and the lab, as well as my clinical collaborators who helped with this surgery. Thank you very much.