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>> For a cardiac surgeon to come to Georgia Tech to talk about tissue in cell engineering is a little bit like taking coal to Newcastle. But I do appreciate the opportunity understand I'm going to bring to you. The perspective of a clinical surgeon. My area of surgery is reconstructive cardiac surgery rebuilding the heart replacing the heart closing holes in the septum repairing valves.

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So we use a wide variety of materials. And in fact with any given situation. We may use day KRON we may use P.T.S.D.. We may use patient's own tissues. We may use crop preserve home a graph tissue tax you name it we have a whole panoply of materials just like an artist would have their materials and we matched the performance characteristics of those materials to what we wanted to do in the reconstruction and none of these materials are perfect.

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So I'm going to tell you about today is a little bit of my odyssey in the area of valve replacement and valve reconstruction and starting off a little bit with with the real clinical challenges that we face as surgeons. This is the so-called learning tree outside the. Medical school of Brown University the seed for this tree came from the hill on which the tree stood under which a packer tease taught his pupils and by various works of art.

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They've determined that this is in fact the species so supposedly this is a direct descendant of apocrypha he's plants us organ Tallis which is a relative to the second war now when we talk about replacing the valves in a heart whether it's a child or an adult. We have a number of options today.

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We can always repair the valve or we can always have. The option of repairing the valve and when we can repair the valve and leave the patient with a well functioning valve with their own material that is always our first option but in most cases in fact repair is not an option because either the valves not there to start with as in congenital heart disease or the valve is so badly damaged as an acquired adult cardiac disease that some replacement is necessary.

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In today's world. These are the replacements that we currently have available. We have mechanical valves of which I'll show you one in a minute which has a number of advantages. It's manufactured it is manufactured to specific tolerances every time you open the package it's exactly the same if it's a twenty three valve It's always a twenty three valve it has excellent gerbil you today.

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Most of them are now made out of carbon are relatively non from the genic all the thermal images and Bill ism and the need for any quagga lation remains a problem. It's a very reproducible valve. But it does have some problems I'll talk about in a minute. Zina graph manufactured valves are primarily poor seen the tissue is treated with glued or aldehyde which cross-link.

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All of the college and makes it a very stiff very firm easy to handle valve easy to suture valve they can be on stance or they can be so-called on standard valves instant valves is basically the order to root of a pig chopped out given to us after being good or aldehyde treated.

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And then we saw it and place of the patient's root. Good aldehyde also has the other advantage of being extraordinarily toxic and kills all viruses bacteria etc. There are manufactured valves out of cow para Cardium once again treated with blood or aldehyde And once again they are on stents they have the advantage of the human to Nam makes can be optimized by the way you sew the valve leaflets on to the stent.

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And so in fact bovine paraquat. Valves are actually a little bit better in terms of their hydraulic performers their mechanical engineering characteristics than the poor scene valve which is intact from the pig or a group. Then we have a whole group of homo grass the most commonly available home a graft modest transplant from one human to another is a so-called crop reserve Tama graft.

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These are obtained from multiple organ donor harvest in which the heart is not useful for a whole heart transplant and so the valves are chopped out carefully then prepared with antibiotics preserved and after sizing and then are kept in liquid nitrogen until we use them. Now in the history of homo Gras this actually goes back to one thousand nine hundred sixty two when the first day or two valve was home a graph was sown N. and that was a fresh one.

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As the story goes. Donald Ross was operating on a woman in London. Young woman who had rheumatic heart disease and he was D. calcifying or valve at that time. Remember that the star Edwards valve had just been invented in one nine hundred sixty was not yet available in England was not had not been manufactured in England was not available.

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This young woman's aortic valve disappeared up into the sucker and he was looking into her or Dick root with no valve in sight. And of course she was essentially dead at that point he sent his resident in England called a registrar down to the morgue where they cut a valve out of the only available cadaver there he sewed it into the patient and when I was in London in one thousand nine hundred four some twenty two years later she was still alive she had three kids and he took her to many of the lectures that he gave around then the problem of course with fresh Homa grass is that you don't always have the right stuff hanging around that you need.

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And. The harvesting and the quality control and the bacteriology and all of those things are would be overwhelming. So what Donald Ross did was every Christmas. He went all over London to every autopsy room in London and gave a case of scotch to the dinner. And then he would send his technicians around the rest of the year and they would harvest aortic valve.

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From all of the cadavers that came through those autopsy suites. They put them on antibiotics then they put them in tissue culture medium and they bring them to die down. Ross's Laboratory at the at the cardiac hospital where they would stay in the refrigerator intel they became contaminated it which time they'd be thrown out or if they were needed.

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They'd be sewn in a child or an adult and that's called the so-called wet stored valves and once again very cheap costs a case of Scotch a year or two to get them. But once again quality control little difficult. Decided I was on the grass we're going to talk a lot about it and they decided I'd seen a graft and some of you in the field may be aware of the center graph story pig valves have been tried and the experiment has been tried in humans and experiment failed.

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This is the most commonly used mechanical valve today it's a by leaflet designed Parlett carbon This is a St Jude. There are two or three other companies that make similar valves. This is a standard first generation course scenes in a graph. You can see the three leaflets and you can see that they're so into the sewing ring this area where the pliable leaflets are sewn to the rigid sewing ring is the area of difficulty for this valve.

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This is where all the stress of flexion stress occurs. This is where the calcification occurs. This is where the ruptures and the tears occur and this is where this valve fails the other way the tissue got. Fails is with gradual calcification fibrosis and stiffening as the body responds to this foreign object with inflammation here you can see holes calcium very rigid valve.

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These are valves we've taken out of patients and had to replace the life of a pig valve a standard pig valve in a child because of the revved up calcium metabolism can be as short as two years. In a D. Young Adult it can be as short as eight to ten years in somebody over the age of sixty five.

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It may be as long as fifteen years or so as surgeons we replace one disease with another disease. We replace value to heart disease with prosthetic valve disease process develop disease is a consequence and has varying components as a consequence of which prosthesis we choose they can be more from a genic and therefore we have to antiquity late the patient not a good thing for a six year old kid anybody years a parent and has seen six to ten year old kids run around and play on the playground know that being an to coagulate it is not a good thing for a child that age all valves manufactured by anybody else other than God has hydraulic dysfunction inherent the valves do not work as well as the original equipment there are always gradients there's always areas of turbulence.

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There's always resistance and that can change over time. He may Dynamics' performance is what we refer to in terms of how well does it adapt to the workload of the heart and this is it the right size for the patient is it obstructive does it prevent or does it cause so much obstruction of the heart has to increase its muscle in other words high perch your feet.

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What is its actual human damage a performance size mismatch is a big deal in my area. If you're operating on a two year old child. You obviously cannot put an adult. Valve and with these manufactured valves into cordite this since turbulence is always a problem and these are unnatural materials manufacture materials the risk of seeding the prosthesis with bacteria or fungi is reasonable and in fact there's a probably about a fifteen percent chance over a twenty five year life of valve that it will get involved with into cordite as.

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The valve scan catastrophic Lee fail. For example a leaflet could flip off or tear or it can fail over time with the theory ation and so we talk a lot clinically about the dura bill of the valve put yourself in the position of the patient the patient comes in and says gosh I've got to undergo open heart surgery.

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How long is this going to last. That's our. Definition and Erbil ity and engineering definition Derby L.T. would in fact be a whole lot more precise. Now I could spend an hour giving you. Capital Myer curves of Dura billet he and patient outcomes with the various prostheses and that's a little bit of a different talk but this is the way surgeons think we think and terms of these Kaplan Meier performance curves and here for example is a patient survival curve over six to eight years for a freestyle or that's a step less valve This is the root of an aorta from a pig sewn in to replace that your dick root of the patient versus that standard pig valve that I showed you a picture of an interest only enough in this group of patients and this is fairly representative of many trials there begins to become a separation after about five to seven years where the UN standard valve seems to confer a certain survival advantage to the patient this catches our attention as a clinician.

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The. Derbez of the of this valve may be very good but for some reason the patients are doing a bit better with this valve over time and we'll talk a little bit later in the talk about why we think that may be true. Now Homa graphs are the sort of the ultimate understanded valve.

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This is the Arctic root of a human cutout as I indicated there's a so-called fresh fresh like Diana Ross's first one. There are the so-called fresh white stored where they're simply rest often antibiotics and stored in tissue culture medium in the refrigerator. There is what Dr Yacoob calls homo vital and these are valves that are harvested with the intent of maximizing viability.

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There was a experiment with the radiated valves at the Mayo Clinic in the seventy's in which they put in about six hundred ready to valves the advantage of the radiation was that the valves were sterile. There's no risk of infection whereas in all of these you can potentially transmit disease from the donor to the recipient.

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Unfortunately they put in six hundred in about three or four years later they took out six hundred valves as they all calcified. Today we have two versions of the crop preserved valve I'll show you a little bit about car preservation minute. One is crop preserved by a commercial company for maximum viability the other is cross preserved by a not for profit organization for some value building.

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And we're entering the era of the diesel your eyes in the tissue engineer bouse this is a heart. C. It's just delivered to the tissue laboratory where deception is begun it's harvested as a whole heart put on ice and like any other organ transplant is sent. Here is the or tick route from the patient from the donor that has been the sect and you can see a little bit of remaining muscle here which is important it is impossible to prepare.

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Air of whole erotic root without leaving some of the donor muscle attached. No more about that later. This is the end surely for the mitral valve which shares the fiber skeleton along one third of the or tick valve which you see right here this is the aorta. And this is the first branch vessel to the head and right arm and this is normally cleaned off a little bit but what we get when we thought the home a graft is prepared with D.M.S.O. ten percent step down replace the water to make sure the cells don't license and then as crop preserve to minus one hundred eighty degrees and stored in liquid nitrogen until we thought out in the operating room.

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Now here is a five year study in which homo grass were compared to the stent was Zina Gras which I showed you compared to the to the tissue standard valve and you can see here that in least in this series and this had some younger patients ninety percent of the patients are alive and doing well.

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Similarly between the two and then he Few looking at centers that have gone back and forth between via so-called viable allograft or homo grass. I'll use those terms interchangeably or with anybody sterilized. You can see that sort of the less you do to the homo graph. It seems the better the homo graphs survive and there's some reason for that when you treat the home a graph with antibiotics there's a certain amount of cell death cell necrosis so necrosis is by definition pro-inflammatory so that if you're leaving behind a cell remnants in the chronic wall.

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You're going to generate inflammatory response in the human body the inflammatory response is always followed by scar formation and calcification. From a certain standpoint there's also some interesting. Lessons that we've learned over the last twenty years with some grass and here's one of them a root replacement seems to have a longer durable and we were now out now fifteen twenty years in this particular series has a do this is from England from Dr Yacoob a group the root replacement seems to do better than when you cut the valve out and implant the valve by itself.

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Well that's kind of interesting because if you think about it. Now I'm sort of putting myself in your place as an engineer when you put the root in you're leaving the entire erotic root complex the function as it was originally intended. In other words the annulus can dilate with each systolic expansion.

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The root can dilate as matter of fact and the valve leaflets in the Holy Order group complex functions as an entity. Whereas when you saw the leaflets in percent. You get fibrosis around the sewing points and the whole complex is not working as a unified valve structure and therefore that leads to ultimately tissue failure here you can also see that unlike manufactured valves both the home of graft in the freestyle actually drop their pressure gradient over time after implantation.

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And then it Hold Steady there after I didn't put the the slide in but in fact over time all manufactured standard valves actually increase their pressure gradients over time increase in the load. And then in a real macro type of view this is a series that I put together all about fifteen years about ten years ago in which I took four or five kind of classic home a graph series out of the literature from the seventies and eighties and compare.

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Or them to mechanical and tissue valves and this is just an children. And just grouping throwing everything in together. This used to be college review and it is now called a medicine Alice's you can see that there is a significant separation as you come out ten years in terms of patient survival and freedom from replacement of the valves.

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So for whatever reasons. These are working better than these over a longer period of time. This is the semi lunar valve leaflet of an aortic valve This is one third of an aortic valve the next cost. Approximates here and the other cost approximates here to create the tri leaflet design.

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Unlike the mitral in tricuspid valve is a similar valve is relatively passive it does not have muscular attachments underneath the leaflet there is some active changes in the diameter of the Arctic root complex itself. But this is primarily a disco elastic entity and responds passively to currents and changing pressure gradients.

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Now this is what I do for a living I so these things then and I make them fit into various size patients and here is one way of doing this. Here's the already group from another human. Here we've opened the already root of a patient have cut out all of the obstructing tissue and are simply sewing this down into the ventricular outflow tract of the patient.

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We then can do a number of things with everything above the valve we can throw it all away and replace it as a root taking the patients corner arteries and implanting them into the sinuses of the replacement valve or we can keep part of the home graft and enlarge we often do this in children who have hypo plastic a orders enlarge the order group with a piece of the home.

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Graph. But so around the corner areas with this piece leaving the patients native tissue in over here and taking care to research spend the semi lunar valves that I showed that picture to you. And here's one the so-called sub corner am plan in which we've opened the door to a group and so on the patients are already valve or the home of graft in place of the leaflets all around on the tri leaflets one two three all around replacing the functioning part of the valve the similar valve as I told you this probably isn't quite as good over a long term as replacing the entire area group.

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Why do we do this. This is LARRY do poorly who came to me when I was a Georgetown he had valve or heart disease at age thirty. He had not graduated from high school he was the winningest jockey on the West Virginia Virginia circuit. He had a family and he needed his valve replaced and obviously the textbook answer for a thirty year old would be a mechanical valve it'll last longer than anything else but he'll be taking it a quagga lation human and he could never ride a horse again because these guys hit the dirt all the time so we put a homer graft into him and he sent me this picture a few years ago when he was winning this is the finish line of his three thousand thrice So not only are we saving lives with valve replacement but we also have to be a tentative to quality of life and matching the prosthetic valve qualities and in fact their poor qualities to the patient so we often pick the valve that is the least intrusive into the patient's lifestyle.

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Terms a home of grass What are some of the advantages even as we use them today one the engineering design is clearly been proven. To the size matching with the patient because this is soft tissue and not real. Aged I can usually put an adult sized home a graft in any kid that's larger than fifteen kilograms.

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So by doing some tailoring and some nipping in tucking we can usually get in an adult sized or large valve to improve the human Amec so. It is a vis go elastic replacement it is not rigid in anything rigid in the body has a problem as a problem with fatigue where it has a problem with causing fibrosis and sterile inflammatory reactions.

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And as surgeons we much prefer to work with tissue quality material we like sewing tissue. I'm not I do that I dislike sewing day crime to be honest but it does make because it's a very flexible material. It makes many of our very complex multilevel surgeries much much easier.

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Because it is Biscoe elastic the after load to the heart is actually reduced because during systolic ejection of the heart the Homer graft conduit can actually dilate functioning as a hydraulic capacitor the valve closes and then it contracts giving back some of the hydraulic energy you have none of that none of that in a fixed stented prosthesis.

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It has the best hydraulic and human and it function and for a number of reasons. Probably more so this than the fact that it's human tissue and others that there's less problems with turbulence. There seems to be a markedly reduced in the cordite it's risk. After homo graft implant.

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I mentioned to you how these are are prepared. There are donor harvest D.M.'s. They're stored in the quick nitrogen the way I got into this field was I was asking myself in the eighty's what is the best way to crop reserve homo grass at that time we kind of had the idea that maybe.

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We should improve the viability of the mile Fraggle blast the interstitial cells deep within the leaflets we never wanted the into feeling them to hang around because in a feeling was very energetic. So we did a series of experiments looking at A.T.P. retention throughout all the steps across the preservation and to make a long story short cropped reservations pretty tough on cells.

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They're pretty much depleted of intermediate metabolism metabolites and they're they're pretty pretty hungry by the time you put them in. In addition to that and this is some work that Steve Hilbert did with us in the laboratory Georgetown most of the cells are essentially doomed and they've already initiated a popped USCIS so that in fact by nine months after implantation of a holographic virtually none of the cells that were living at the time of implantation are in fact alive but what we did find was that you the cells were pretty resilient they definitely were alive when you put them in maybe not very happy but they were definitely alive.

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We defined the fact that these were mild fiberglass and not just fiber blasts. And we also felt that we've refuted the immune privilege these cells are not immune privilege they generate the same rejection phenomena that any other cell does. And I'll show you why we think despite all of that that these Homa graphs work so well and this is actually a slide that Steve prepared with us from a sheep but we have many human X. plants that look very similarly this is a valve leaflet and you can see there's just the occasional cell and then you see the host she thing this fiber She thing or Neo into the formation that in effect splints the valve leaflet.

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So what's happened with the holograph is it's become a mandrel it's become a basis on which the pay of the patient has. Created a fibrous superstructure which now functions for a long period of time and in fact prevents these few remaining donor cells from really coming in contact with MacHall with white cells.

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So if you look at the here's the tris classic tri leaflet structure of the valve leaflet and here you look at the allograft valve thirty days later you're beginning to lose that tri laminar look and you're seeing very few cells remaining one of the disadvantages of homo grass Well they are energetic.

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This is been clearly showing they generate panel reactive antibody responses and the more mismatch they are the the worse they do and in fact a very interesting study was done. Comparing Homa graft implants which we do not give anti-rejection drugs with whole heart transplants the vals from a whole heart transplant in which the patient we sees any rejection therapy look normal.

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Whereas all the Houma graph transplants look like those pictures I showed you. Why they do so well. So let's learn what do we as we enter the tissue engineering era. What do we need to not lose from the home of grass will we still use them every week.

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Well first of all the processing does strip into Thalia the matrix is left variably intact depending upon the processing but even some of the sayable proteins remain the mild fiber blast if you strip the into Theall IYAM and you strip out by Little simply leeching some of the more superficial mild fiber blast most are buried within the collagen So except for that piece of muscle tissue that I showed you these cells to a certain extent are in fact there's a physical barrier between it's being exposed to the to the recipient.

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This is I think and this once again comes from Steve helpers work with us this is critical when you get rid of process in such a way that the cells are removed as opposed to being destroyed then the cells leave by a pop Cosas and not cell necrosis and if the ptosis by definition is not inflammatory they just disappear.

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Whereas if you destroy the cells break them up and leave them behind. It's very very inflammatory the fiber She thing we talked about the wall of the home of grass almost always does calcified make sense it revalued arises. There's more exposure of the donor cells to rejection phenomena.

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So the bioengineer person about how do we make the step from home a grass to engineering out the defects of the home of graft without losing the advantages of the home the graph L. transplants. Well what do we want. What do I want. Most of your bio engineers all of you graduate students.

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How many graduates in any undergraduate insurance. OK So as a graduate bioengineers you're going to come to me or I'm going to come to you more likely I'm going to say I want an ideal valve make me one. And I'm going to tell you in surgeon speak what I want.

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And then you're going to translate that into engineering and mathematical language and six weeks later. Give me what I want. So what I want I want a valve that resists infection. I want to valve that can change its matrix to adapt to changing growth of the patient to hypertension and to other stresses a valve does not state the valve you've got at age twelve is not the valve you got when you're forty.

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And in fact you take a pulmonary valve which is a thin diaphanous very delicate looking valve if you put it in the order position we actually do that in patients six months later looks like an aortic valve it's to conduct it's got more college and it's got more last and it's got more control and sulphate So I want something that dynamically remodels in both repair and growth modes.

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I want something that looks like a valve and here's valve leaflets with the three layers of fibrosis fun geocentric you Laris upon which there's into feeling I'm on each side. Each layer is a little bit different. It's not constructed this way by accident. This gives the best bending stress parameters the lift allows the stress of the movement of the leaflet to be dissipated across the entire leaflet avoiding those flexion creases of the manufactured biological Val's there's a reason for all of this.

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So as much as I'd like to know those reasons and we know some of them more importantly I say let's replicate it and then figure out why it's so good. A living valve replacement involves not just matrix but also cells. We've done some work in our laboratory looking at various cell sources and I know that all of those of you who are working with tissue engineering know that there's a lot of different ways of doing this anything from stem cells circulating stem cells embryonic stem cells.

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There are fiberglass everywhere is a dermal fiber blast as good as a fiberglass that comes or a mild fiberglass it comes from the heart itself and I have some biases about that based upon our work. But we've taken fiberglass from many of them. We have done bone marrow yet but we've done all these others and even have developed a way of biopsy the tricuspid valve that would be clinically useful and can then grow up the cells as you see here in tissue culture so that.

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Potentially if we're going to bioengineer your valid fell for you in vitro we could biopsy your tricuspid valve then proliferate your cells and then come back and see them back into a kind structure. We've looked at ways of separating the Maya five blessed and cells we can do that pretty easily just by changing the media and then we've tried to characterize the cells other words well how do I know that this is a valve leaflet mild fiber blast as opposed to a skin fiber blast.

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And we do it with a number of you mean a set of chemical stains we use and I've met an alpha smooth muscle actin fiber necked and can drop in sulphate and then of course they can they should not stain for Factor eight which would make it into fuel cells.

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Let's talk about matrix. This is a valve leaflet in which all of the cells have been removed and you see that try laminar structure and interesting enough you see all the spaces left behind this is not the same valve it started out to be it looks like the same valve minus the cells but I can tell you it doesn't feel like the same valve it does.

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And so like the same valve and I'm going to show you that it doesn't exactly act like the same valve. But it's pretty neat that we can take out all the cells and leaving very little cell debris and still have an intact aortic valve in our hand. And this took about five years of work to get to this point there are a number of ways of the said arising.

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There are non ionic detergents there and ionic detergents there's E.D.T.A. and then there's some pull just fluid just water wash out with sterile water. What we ultimately found this is a whole nother talk but we found that what worked best for us which is little bit different than what's in the literature right now.

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Now is using in an ironic detergent with enzymes Arnesen D.N.A. in zines and then using a prolonged war shot phase of about twenty four hours and then using an ion exchange column to remove the detergent left us with the best valve in terms of having essentially no debris.

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I say essentially not show you why I say essentially And yet you retaining the structure. We've tried the other methods in the literature and they've always left out a step and you know a lot of do that your patents but you can do that the peer reviewed literature because we've never been able to repeat the steps of some of these other groups around the country and get the same valve.

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Here's one of valve looks like that's been the cellular arises upon our evolving see that the leaflets look very nice and intact and then we said OK how do we know that cells like this kind of tissue. So in parallel with the work on the centers ation we started looking at a qualifying as say that we could do in vitro we call it our static seeding qualifications should say qualification be qualification and say.

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Well this is sort of a combination assaye it's a little bit of a toxicity essay. It's an ass a that ass will cells in static tissue culture and I talk about a bio reactor now will the cells and here. Will they migrate and will they proliferate. In some of the materials that we have tried to scaffolding is the coastal smooth muscle tissue from sheep so called as I.A.S. the diesel your eyes valve tissue crop reserve valve tissue fresh tissue good aldehyde fixed para Cardium and then photo oxidize which is another way of cross-linking the pair of the college and Procardia are assay is a million tricuspid valve cells from passage three they've been.

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Apple fied. In culture and we apply it every other day for three applications and then carry the the tissue culture out about another week. And the idea here is that we can test materials and then say we find a nice material then we can work through a series of pretty treatments etc and it would be a very much quicker than sawing this material into sheep.

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So this is what we call our patch studies for scaffold performance now as a surgeon. What do I offer that's a little different in our laboratory than what you can replicate or what's easy for other laboratories to replicate and one is that we can actually saw these in living animals and see how the animals do over time.

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That's a very important step. SOKOL chronic large animal implant studies. There's a reason why in the regulatory world that that is a regulatory requirement before you go with devices into humans. However it's it's inherently inefficient. It's expensive got to keep the animal live you have to put them on heart lung machine.

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You have to take care of them afterwards sheep like many of the animals require anabolic for a long period of time daily veterinary care. So anything that we can go back to the lab and do in vitro is very very important in terms of qualifying and SES before we go back to the sheep.

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So the process goes back and forth and you'll see some examples of that. This is S.Y.S. material just put this up. This is a four ply S.I.D.S. and you can see that the cells are still there and I'll show you some S.A.'s lives and he's got a little bit out of order.

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This is photo oxidized para Cardium which is the main seated with pride custom fiberglass in our assaye you can see that there is essential in this particular photo limited in migration the cells do it here. Or anytime there's a de facto find their way in and they seem reasonably happy.

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On the SCI ask material they don't seem quite so happy. They're rounded up. There's no in migration at all so S.Y.S. is sort of a less happy substrate and then is the photo oxidized pericardial Now if you take the S.A.'s and and treat it with trips and we've done this with trips and Clagett days you can open the structure up and you can get the cells to migrate in and there was the poor sizes larger but the problem with that is it is now just like wet Kleenex and you could never saw it.

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So it's clinically irrelevant. Here's Boban para Cardium And here's what happens when cells get layered on to something that has been there all died and it will go anywhere and they look very rounded very unhappy very mild fiberglass like no warm side of plasma processes looking like they're trying to crawl into the leaflet Now if you take a poll or artery and here's one we call this the vitalized with the cellular eyes and then it's just freezing vitrified.

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And then rethought. This is a low bang for cation view of all of the tissue with mild fiberglass and you can see here that in the same conditions in vitro you see my father was on the surface but you see my fiberglass beginning to crawl all through the matrix of the of the scaffold material and then here's devitalized that's not been that's not been vitrified and you can see that there are cells beginning to come all the way through and once again this is in the one week in vitro preparation.

[00:39:45]
So we can now take this to the surgical model and put this in a functioning position in sheep. And my going back I'm going backwards and this is why. A pulmonary valve that's been divided lies looks like but notice that there's still some muscle at the bottom. This is not the same as a piece of tissue like the patch in which we've cut it out here and which there are no residual cells there is residual material here we know that because we've developed a panel reactive in a body as say a flow cytometry base for us an antibody test that replicates the P.R.A. assaye that we use in humans and we know that when we put these in sheep we get a P.R.A. antibody response.

[00:40:36]
This is one of our fellows in planning the valve. This is what a pulmonary valve looks like in the pulmonary outflow track position the head of the sheep is up here and the heart is down here pulmonary arteries are up here. Had a sheep is actually. This way.

[00:40:57]
Or had a little bit differently to sheep than human. And this is what we wake the sheep up immediately after the surgery it's one of the tricks to getting them to survive we're up to about ninety five ninety percent survival now. And then they grown boy they grow like crazy over the twenty weeks they'll grow from twenty kilograms to forty to sixty kilograms.

[00:41:18]
If the valve is functioning well are right for to go off a track model or pulmonary valve model we sample at ten weeks and at twenty weeks we've done this with all of these kinds of material so that we can compare more are going to be showing you bits and pieces we just don't have enough time.

[00:41:34]
I'm going to come back to the slide a minute but this is a valve leaflet and think about that valve leaflet that I showed you before of a hologram found in which all the cells died and you got fiber she think this is a decent arise valve this valve looked like that valve that had all those spaces and all the cells were gone and you see from the luminal side from the wall.

[00:41:57]
This is the hinge point of the valve leaflet. That you're seeing these valve leaflet cells. Streaming and these mild fiberglass streaming in. But this is still relatively a cellular you're seeing some attempts at into the legalization here but you see our old friend here the fiber She thing just like the home of grass.

[00:42:16]
I think Steve you did this slide and one of your beautiful slides. This is an S. I asked material that we've been planted as a model cusp in the right track was ition Here's our old friend the sheath Here's the S. I asked material without much happening. And yet where there is a large space that the fiberglass will find their way in and begin to repopulate.

[00:42:43]
And that's just a high power view of that. But as I asked material in general in vivo now that we've gone now we're in the sheep implant X. plant studies in general respond with inflammation and with fiberglass She thing on the album will side away from the lumen of the pulmonary and on the luminal side so you see this more organized Neo animal formation and then a sort of fiber optic response and not much happening in the material and I would be pretty I would not be very optimistic that this material is going to replace with donor cells and just another slide to show.

[00:43:24]
Not much happening in the material itself and just the spaces remaining and not filling up as this is an area of Sawyer's ation and not filling up and here's the area of sheath formation looks very much like the scar response that we see almost anything that we put in the human body.

[00:43:43]
So the S.A.'s material is not resell you arising it's not tissue engineering itself. It's being lined over by the body's own cells as almost a foreign body material would. And here's kind of a frightening slide. In which you see the material. Beginning to deal laminate and actually break apart.

[00:44:03]
No cells and they're supplying structural proteins and all you see is this little she thing along the leaflet edge on each side and I'd be very worried that this thing is going to fall apart. Pretty soon. Just more and more slides of the same. Now back to R R D said her eyes to show here's what it looks like with cells.

[00:44:28]
Here's what a leaflet looks like without cells. And here's what some of our first X. plants looked like at the leaflet margin we sees some hemorrhage. And we see some fiber she think but we also see some cellular zation occurring. More about that. But again with some of our early days Sellars ation methodology the tissue is was causing some clot formation something some in migration of blood some breaking apart and some fiber she formation and a little bit of cells coming in but not exactly what you'd really want to see.

[00:45:10]
Go back now with your mind. We're now looking at a different material going to come back to a new generation of material in a minute this is a crime preserve she pulmonary artery. This is not the leaflet Now this is the wall and this is just what it looks like when it's thawed you can see the cells all through this.

[00:45:26]
So these are donor cells. This is what a typical crop reserved piece of cardiovascular to shew looks like. You put it in to a sheep as a patch in this case into the pulmonary artery. We put this one the descending order. You see a big thick pseudo enema formed this is the lumen of the aorta.

[00:45:48]
This is the outside here you see an inflammatory spot response in fibrosis occurring here. And here's the original crop preserve leaflet it is the void is so. Ells is gradually disappearing is is not tissue engineering. This is scar formation. This is not resell your eyes. It is a high powered view of the same thing works.

[00:46:11]
It doesn't blow out and the valve leaflets work and you do see a little bit of research of of in growth of the fiberglass here but I wouldn't call that. An engineered hard film. Here it is at twenty weeks and this is the lumen side and this is now in a pulmonary artery just notice that the neo in him as a little bit less thick.

[00:46:36]
Everybody remembers the pressures fifteen and they already mean pressure is eighty or ninety and therefore the response to the human Amec stress is less than a pulmonary so you get less thick Neo enema. But look what's happening out here were all that inflammation was occurring on the abnormal side now are starting to see counts for cation inflammation and really sort of bad things happening.

[00:47:04]
Now we come to a pulmonary artery which has been the cellular ised and this is what it looks like here is the luminal side if you will in the adventitious side of a patch that has been divided lies and then freeze dried and then we rehydrated it before sewing it in and you see lots of voids where the cells were and you see a lot of this kind of a Morpheus material remaining behind and this is what it looks like after ten weeks in the the sending aorta fibers She thing material is going away fiber She thing out here but a lot less so that inflammation and no cost cation and here when you get on high power you see a lot more cells are already beginning to distribute themselves into this crop preserved matrix.

[00:47:56]
I'm sorry matrix where there were no donor cells. And here and here again at ten weeks a little higher power a little different view. And here you can see that the material particularly from the evolutional side is beginning to repopulate was cells that look all the world like mild fiberglass.

[00:48:16]
And here's one of the descending aorta. And again you see thicker fiber She thing you see the material beginning to disappear and you see cells begin to distribute through the same thing again. At twenty weeks of material you begin to see the cells distributed all the way through one's skin lumen side.

[00:48:40]
This is one that was twenty weeks in the pulmonary artery. This is probably a more true this is a little bit on angle but the patch is beginning to disappear and is beginning to become resell your eyes to the higher view to show the distribution of cells and we stain these for smooth muscle act environment and fiber Nekton them all those marker proteins for what we think is the phenotype of a mild farmer blast it lights up so these are mild fiberglass and not macrophages and not lymphocytes.

[00:49:14]
And here's one in which was crop observe first and then divine allies and you say well why would you ever do that. Well if you think about it. Sitting around in liquid nitrogen freezers all over this country are about twenty thousand Crile preserve hard fouls that are getting ready to go out of date and be pitched into the garbage dump.

[00:49:34]
If we could take those human valves have been crop preserved and take them through the processing of diesel or ization and resellers ation we could in fact probably recycle all of those valves. So one of my pleases so far not been heard very well but one of my pleas to the companies that control these valves is do not throw them out yet because some combination of these.

[00:50:00]
Techniques may in fact be able to salvage literally thousands of else. What's one of the major problems with human tissue transplants or human organ transplants. It's availability. So anything we can do to maximize availability. Would be important. And here's one of these this is an artifact of cutting here.

[00:50:20]
But here is a crop observed the cellular eyes. Patch that was placed in the order for ten weeks and you see the cells beginning to distribute through minimally but still there ten weeks and then twenty weeks you see more and more of the cells coming through. None of these patches became aneurism or any dimensional changes so they at least in the chronic implant model were safe.

[00:50:48]
This is a may see one stains on fresh now frozen tissue. This is normal ph and you can see positive staining indicating that these are H.L.A. positive as one would expect we did this because there's still a few people out there who believe that for some secret reason homo Graf owls are not and Janick they certainly are.

[00:51:12]
And here is a diesel your eyes valve leaflet and actually these do. There are a few remnants that are not visible to light my craw Skippy but on on your monochrome staining diode it does show up as positive for M. A C. of one and two. So there are still even in the disorders ation techniques so we're working with.

[00:51:38]
Still a few cell remnants behind that might cause mischief. We're starting run out of time but I do want to point out some interesting features and why use you may have been asking why you put them in the order in the pulmonary artery and patches and leaflets Why do you so many different models were asking slightly different questions.

[00:51:56]
And the descending order model is interesting because it's. You recall right above the heart. There is systolic closure there's no diastolic flow in the aura. The in fact the flow in the ace in the aorta actually reverses at the end of system. And falls back towards the heart.

[00:52:12]
So the valves snap shut but when you put a similar valve in the descending aorta. There's continuous diastolic flow. So we have what we call the fluttering leaflet model. Now these leaflets aren't slamming opening shot they're sitting in the bloodstream fluttering our question as well gee if we're not stressing the valve what happens to the cells in the researchers ation is there something different going on in this.

[00:52:37]
And interestingly enough they re cellular eyes. Perhaps even better in the fluttering position than in the functioning position. Now why would that be. We're not entirely sure but I'm going to show you some slides. Remember this one I showed you before see where the cells begin to peter out this is the wall.

[00:53:03]
This is the hinge of the leaflet typically for the somewhere out here and these cells are having a hard time getting past this hinge point. So maybe it's just a physical block at the maximum point of bending and here's a little bit further out in the right for profit track model which would be working leaflet model and you see that there's minimal research is asian at twenty weeks.

[00:53:29]
And here's the fluttering valve and this is just past the hinge point and you see these cells beginning to really flow in to the leaflet. So that maybe is just a physical obstruction problem. Here's one in the descending aorta for seventy five days. You see our old friend the fiber sheath.

[00:53:52]
But you see some cells beginning to get out into the mid portion of the cusp that you never see in the crop reserve hard felf and you don't. See in the right Fredricka Alpha track working position. So there is a race going on here between recyclers ation and fiber She thing.

[00:54:12]
Now this is the issue for you. Engineers here is the root of and the order sewn in to the pulmonary artery position. OK And aortic wrote an aortic root in the low pressure side of the heart. The only thing is it's been the cellular eyes and what do we see we see an aneurism beginning to form.

[00:54:41]
So the material properties while we have biologically improved the home a graph by decided arising it and we're now getting researchers A should begin to occur. There's a little bit of a race between the was of the material properties an aneurism formation and wall thinning and reset or ization by the host.

[00:55:04]
So right now this is not something that would meet safety criteria for sawing into a human are via our valve model is a functioning value we do echocardiogram like to be a fellow in my lab doing echoes and sheep every week but they do and they do excellent echoes to document that these valve leaflets are moving in functioning we do dimensions of them plan a next plan we do the exploit morphology and we do weekly pen reactive antibodies as long as there's no muscle attached to the valve leaflet to the valves and we don't see a blip in paying reactive antibodies.

[00:55:42]
If there's tissue left behind. We always see it which validates it. One quick side we do have a mitral valve model which we have tested the S.A.'s material where you see an X. plant morphology and here is the bow. That is. And constructed. This is the name behind this is the constructed papillary muscle.

[00:56:05]
This is a native haploid muscle behind this also is a twenty week model. You can see where we've sewn in the pathway muscle here to the wall of the ventricle the pledge it's are outside the suture kind of curls its way down from the leaflets of been constructed up here down this neo haploid muscle and then out the ventricle as you can imagine in flat inflammation and companies all sutures and so I switch or is heel.

[00:56:35]
So this is calcification along that reconstructed papillary muscle. You can see here. You can see a little bit of calcification occurring up there. So what do we know. Well first of all we know that it's feasible to the cell to a biological in point we know that we can get to a point there essentially no cells in a in an intact similar valve it still looks like a similar Val's it's still functions.

[00:57:04]
There are no remnants by a chimney. There are some minimal cell debris by electron microscopy and by the image she wanted to stains. We know that this material functions relatively well in our seeding qualification assaye not qualification I say qualification I say in comparison to other materials that we've tested that either perform horribly or perform exceedingly well we've done a nude mice implant model that shows no calcification when the materials the cellular eyes.

[00:57:33]
We see no calcification in D.C. truly do sell your eyes material in the chronic implant studies and we know that the D.C. material has minimal to absent P.R.A. responses we know that it resell your arises and this is a very unscientific term this is a surgeon's term it recently arises sort of OK right now with it's sort of cellular arises.

[00:57:57]
One of the challenges is how do we quantify. That and how do we time it. We do feel however that it reset it arises in Vivah with my fiberglass which is what we want as opposed to fiberglass and we've also documented that there's minimal inflammatory responses we do know that fiber She thing does occur around this relatively inert material we do know I didn't show you this but reenter feel is a should does occur it can be relatively confluent it can also be patchy have we totally restored the trial laminar valve cell tissue structure.

[00:58:33]
Not yet in some of the areas in short segments. Yes. However the adequate biological D. cell that we currently have come to clearly results in material strength safety margins being very very narrow or even exceeded we see thinning we see fraying we see aneurism from ation we see increased dimensions.

[00:58:56]
I didn't show you this but we do see Frank and risible dilatation in the descending order. What we do not and what we need to know is how do we accelerate resellers ation in vitro and or in vivo do we use preimplantation mechanical preconditioning or chemical or protein pretreatment do we need to optimize the cells sitting in other words are we taking the cells from the wrong place or are we treating them improperly can we cycle them with protein shock or whatever to make them better at hearing and migrating all these experiments need to be done.

[00:59:34]
Can this be done better in a pot reactor a lot of the information that's come from the Georgia tech laboratories here from Dr Nerem Dr yoga Nathan. Would suggest that a pulse told by a reactor would be very useful for accelerating in vitro reseller ization. How do we enhance reseller's ation in vivo well as my bias that if we understand how to do it in vitro we'll be able to direct it and improve it in vivo.

[00:59:58]
Can we pre-treat. Diesel your eyes to Eric valve with attractants are signaling proteins or do we seed it. Do we seed it there is one paper from a German laboratory that in a bio reactor seeded some valve material. I don't think it was intact valid it was valid material for six weeks.

[01:00:16]
It took to get the cells. Now that's clinically very impractical guarantee that nine out of ten of those valves get infected in the hospital environment if you actually tried to do that. So if we're going to see do we do it the day of surgery do we do it a week at a time do we do it and cetera.

[01:00:33]
We have found that we can take the leaflet cells up to two three weeks before like you would say a bone marrow just biopsy the patient's only fluid and we can grow those cells up we have plenty of cells in two three weeks but how do we actually do this in the what's the timing and how do you prevent scar from ation as I said it's a race between reseller's ation and scar formation.

[01:00:59]
This is this is a huge How do we define the mechanical engineering performance parameter so that we can predict the material properties I can tell you as a surgeon when I saw something in that it's a good feel or a bad feel I can tell you when the aneurism forms ten weeks later.

[01:01:16]
This was not a good material. But how do we actually define from an engineering standpoint so that we can predict that behavior after we've used the material in the patient. What are the safety criteria as I said there's a there was a European study with by the said arise in a graph material.

[01:01:36]
That either blew out as aneurisms with rupture formation or became calcifications to not a very very rapidly obviously safety market safety was not achieved. Prior to implanting into humans. I do believe that we should be able to rebuild a valve in vitro so that we can understand how to rebuild it in in vivo.

[01:02:03]
We need to prove that research arise valve cells express the appropriate phenotype in response to ongoing human Amex that's a proxy for telling you this valves going to be still functioning twenty years from now that's when you look at those Homer graphs and say why did that one last twenty years twenty five years or even thirty years and some patients and other patients they calcifying are rejected in two to three years.

[01:02:32]
I think that the answer is what happened to the cell biology of that particular valve. But how do we prove that the reset or ization of the valve once it's in vivo once it's in the patient whether that patients are sheep or human is actually resold arising with the proper kind of cells in the proper way.

[01:02:51]
So the we can predict the performance of that valve in the patient. It's not his future six you might think to be able to actually do that with with the kinds of imaging equipment that we have now with PET scans etc We made fact be able to follow the resellers ation So in a tissue in your valve we have various options we have scaffold options we have cell options.

[01:03:14]
We have in vitro options. We have in vivo options we do like the semi lunar valve design potentially if we solve all these problems in our grasp potentially that could be extended to Zina graphs. If we can get rid of the energetic protein problem. And the whole question of what minimal processing is prior to transplanting this tissue is an interesting issue one or two more slides.

[01:03:38]
What are the practical issues from a surgeon standpoint. I'm going to do this to a patient. How do I get the cells. What's the duration of pre-employment pre-implantation preparation is this a one day thing. Or do I have to have the patient sit around for three weeks. Is it a breadcrumb approach where we're treating the valve leaflet or is it a hybrid approach where we're treating the valves and see.

[01:04:00]
Adding the valves. This is huge. Whatever it is that we put in has got to be functional and durable with an appropriate safety margin at the time we implant it not six months later that the progressive host interaction with the valve must be gradually improving the valve as opposed to degrading the valve as in our current generation of zener grass and in the valve performance and the corollary to that is the valve performance over time must be no worse and performance characteristics than the current clinical options.

[01:04:41]
The regulatory issues we could leave to another discussion. Thank you very much. Bant glad to answer any questions and we'll say that we have about forty of these customers sitting around right now with very separations of valves put in I do want to mention one thing. This is why we're doing this is a six year old boy that came to us with a valve that needed to be replaced.

[01:05:07]
He loved hockey. We were able to put a homer graft and him he's now playing hockey again this is a picture he sent me of him playing hockey. He's growing the valve is doing well but it will need to be replaced. Probably for if I replaced him about four years ago probably four years from now he's going to go back surgery for another valve.

[01:05:25]
I'm hoping that the valve we can put in four years from now will be a permanent reset your eyes fell. Let's take a few questions. Well we have this guy here in the terminal like anybody want to come. We're going to have Yeah I think it's. That's a rather.

[01:06:05]
That's a real good question right now we're doing hyper plastic left Hearts for example as newborns first operations of phase one operation they get a phase two operation and phase three operation they've had three operations by the age of one they have a holograph Alvin that's functioning and now McGrath valve will have to be replaced two years later five years after that ten years after that.

[01:06:29]
So the time they're twenty five or thirty. You're looking at maybe five to seven different operations. So the issue of growth the ability to put a valve in that will grow the patient right now we use tricks to put in an oversize about it sort of like those old Hot Rods used to see with the motor was so big it stuck out the top of the hood.

[01:06:46]
We can we can do some of that but if we can actually put a valve in that will grow with the patient and maybe we'll have to do it once or twice. The question that's best bad no questions. Holes. Yeah that's a real good question. We've really focused on it was what I think happened with that lady is I think that the cadaver was dead just long enough that all the cells are sort of gone they probably put the the valve in Lactaid ringers which we know strips off in the theory I'm probably a lot of good things happen for nobody knew why.

[01:07:43]
But probably when curing it up from the morgue to the fifth floor operating room. The end of feeling them stripped off most of the fiber blasts were stunned or dead and she got basically in a more fist material the did very very well but you're absolutely right. Can we alter.

[01:08:00]
The host response. There are a number of centers that have talked about even with the crop observed from a graph using any rejection medications cycle sporran in particular steroids. The problem with that is we're now taking a valve that we've all the sudden decided is less than perfect.

[01:08:18]
We're instituting a clinical treatment to protect this valve from rejection but the treatment we're giving the patient carries its own risk of infection and problems with cycle sporran and Prince prednisone and if in fact you lose a child to bacterial or Curtis or meningitis because their own any rejection is going to go on and I put a pig valve in so you keep you know two wrongs don't necessarily make a right in clinical medicine.

[01:08:47]
There are having said that there are current trials I think there's one in Chicago and there's one in Germany in which patients children and as young as an innings are receiving up to three months of any rejection. Therapy following implantation of as of a of a crowd preserve home grafting that was one that contains donor cells with the thought that they're going to prevent this rejection inflammatory response during that period of time that the cells are actually dying.

[01:09:17]
It's possible I don't know the answer that but you're certainly putting the patient at risk and if we could just get rid of the engine. And and implant a valve that's not energetic that is more attractive to me that's a great question and the other great stuff.