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WSCS 2015- Bio-Materials & Regenerative Engineering

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WSCS 2015- Bio-Materials & Regenerative Engineering

Bio-Materials & Regenerative Engineering

Video Transcript

MODERATOR

Saverio La Francesca, Executive Vice President And Chief Medical Officer



SPEAKERS

Samuel P. Franklin, Ms, Dvm, Phd, University Of Georgia




Timothy Ganey, Phd, Atlanta Medical Center



I'm a bit hurry orthopedic surgeon primarily doing surgery on small animals meaning dogs and cats but my areas of interest also include regenerative medicine treatments for cartilage defects bone healing and osteoarthritis and so I was asked to participate in this because I had some experience looking at use biodegradable implants and particularly moving toward treatments above way and/or joint replacement.

I'm Donna go for a little bit of data that we generated a few years ago this was done back at the University of Missouri when, I was doing my doctorate degree in residency there at the time and so on of the laboratories major goals was to develop a methodology for Biological joint replacements or something they could overcome the limitations of current joint replacements using artificial materials metals plastics and ceramics in which we could ultimately end up with functional bone cartilage and so on of the initial studies that was done through the laboratory in conjunction with the laboratory at Columbia University NewYork was replacement of the humeral head of a rabbit using Pole its own caprolactam which is a biodegradable polyester material should also know that the disclaimer there right now.

I am neither a chemical nor mechanical engineer and so my expertise is limited accordingly but they perform the study in which they used CD I'm sorry I'm computed tomography reconstructions of the native proximal humerus to create computer-assisted design three individual specific implants which they then ultimately infiltrated with a TGF beta delivered within a bovine collagen gel or scaffolds without such supplementation places in the rabbits and then ultimately sacrifice them three months later performed immune histochemistry as well as histology and what they found was that particular with the TGF beta 3 supplement and scaffolds.

They had very good integration into the bone in into the implant itself so in terms of gaseous regeneration and also very promising Lee had high levels of type 2 collagen staining as well as tragic and which can be seen in the two images there so it appeared they got both gaseous in cartilaginous regeneration within these results are encouraging but the rabbit is a very forgiving model in terms of its regenerative capacity and so when the next steps was trying to identify a situation which this might be applicable to a person and then also a large animal model so this is just one example in which capacity is performed people that's individuals with deterioration of the humeral head and shoulders so very similar to that rabbit model that we just showed you but then there is also osteonecrosis or a basket necrosis of the femoral head in both dogs and in people and so in looking to move this toward a large animal model.

We looked at the canine in the canine hip specifically because dogs do get a vascular necrosis of the femoral head in which case the acetabulum can be normal in the canine hip is the one situation or one joint that we very commonly do joint replacement so there's a great deal of similarity between the dog in between the person and so on trying to translate that studying what was achieved in the rabbit to the dog we had several areas that we needed to look at the first was designed a mechanical testing of ephemeral resurfacing scaffold because of course the rabbit is much smaller and some mechanically speaking alone again are more forgiving model than a dog which is Donna play more force and so on of the big questions that remain as well this compunction in Iraq that but will these constructs mechanically hold up to the lives that we would expect me their dog in the normally human being a secondary.

I won't really go into details development of certain surgical technique of protocol ultimately be able to test us and then the third would be assessment of the bio compatibility of the constructing conjunction with canine either chondrocytes arms income will stem cells and so for that first part is canned we make a scaffold is sufficiently strong to withstand loading in the canine hip and this was also a personal question because a lot of the work that's been done up until that point in time looking at gaseous regeneration including in things like mandibular defects had been had generated promising data but again was not a load bearing joint or bone and so engaged the first thing that we did was created some models for replacement of the femoral head and then ultimately place them into cadaveric canine femurs and this situation.

We actually cemented them in place with bone cement potted the femurs and then articulated them with the commercially available ace tabular prosthesis and then loaded them to failure and interestingly enough what we stand was that the alleged that failure was as high or higher with the implant that it was with the lateral native canine femoral heads and where we are seeing failure around 700 news was actually through the stress riser hole created with part in the femurs so those data were really encouraging that at least from a mechanical stand point.

We could reach created construct that was sufficiently robust moving on to the separate area and looking at the bio compatibility we had greater problems and one of the things they first tried to do was identify ways in which we can either get the canine again chondrocytes worse or ultimately stem cells to actually adhere proliferate within the constructs coming back to one of the major questions if you have a biodegradable material how much extra Bebo conditioning are you going to do with the cell source of interest in order to optimism the mechanical biological performance at the time of implantation and what we think found was that we're really getting poor adhesion an attachment and there was also very much limited to the surface so the image on the left hand side of the screen shows some fluorescence suggestive are indicative of live cells in here but then when we looked or section these constructs.

There was really poor infiltration into the interior of the constructs and so then tried to move toward aphasic constructs which would involve culturing of cells within our gross later that's essentially bonded to the PC also hoping that the PCL would serve as the gaseous regeneration whereas the sales within the Argos would serve as the contrast layer but in performing this experiment they stumbled upon a couple things and that is that the manner in which we prepared in sterilized the PCL had significant ramifications for the cellular viability and so this picture is of PCL constructs after they have been bonded to the Argos with cells as well as some are gross constructs containing.

The cell's which are actually sitting in the wells that appear pink and so what we had actually done was arrest sure in a state with US Cellular viability and what we happen to find was that there is a very obvious and gross distinction that any of the constructs or any of the well sorry that actually had the policy at Old Capitol lactone within them had no viable cells and so we followed that up with a more controlled study where we had several groups using some sterilization methods such as isopropylalcohol and UV light which are you sometimes prior to doing live animal studies but which ultimately would not be adequate before putting anything into a person and then compare them to hydrogen peroxide gas plasma which we had used plus that and in a subsequent washing of the constructs and then followed that with again the same register and subjective cellular viability and what we found is one of those four groups this image alone very much demonstrates.

We found that is very poor cellular viability I'm for the group shown in the upper left-hand corner which is the group that was still as with the hydrogen peroxide gas plasma there was moderate cellular viability for the group on the way in which hydrogen peroxide gas plasma been used followed by a wash and then good cellular viability went ahead not just you be lighter isotope alcohol disinfection to perform then performed and those results were statistically significant and so that's kind of who really a very small snapshot and brings me to just touch upon you mentioned some things they think they're person regarding the combination of the biodegradable materials and stem cells and that is that number one.

I think gaseous regeneration using poets on capitol actress easel ball and certainly other investigators labs have demonstrated this to an even greater extent than we have that ultimately looking for osteochondral resurfacing the bigger challenge is going to be there generation of the cartilage not surprisingly and we probably need to be capable of developing constructs that are either by stays occur try stays so that they facilitate the regeneration of the distinct layers of the joint and then a so-so and then Associated questions are what then would be the ideal cell source to use in conjunction with such materials and what is the ideal XP bill preconditioning protocol to maximize the mechanical well as biological performance prior to implantation leave it with that and I'll take any questions at this point or we can wait till the end, Thank You same questions what do you guys think about it actually.

I do have a couple was a very interesting but away and I'm sure he's a huge market and need for dead so the question as a whole design interesting because we kind of use polymers as well so I guess I'm trying to learn PCL we'll see I swear to absorption time cause I will tend to you would choose a drone absorb all material for hips Julie it's very slow so PCL is one of the longer-lasting polyester so it would take at least six months before deterioration amoeba so facts are a lot of the group's my understanding is that have looked at using PCLN be believed that you wanted to hasten the re absorption or degradation of material over time and then have done other modifications such as incubating with things like sodium hydroxide prior to implantation.

So that they have actually initiated the degradation in that case they break down more rapidly didn't need him to you have all this regeneration of deal also its component that's going on in their Christian was your association met you had a run-in with plasma and the Broadside and plasma it didn't work and is the same maybe they got anything to do with hydrophilicity of the PCs that are you a chance to present you think what do you think is happening there so what is happening is that he followed this up with a couple more experiments and so what we think is happening in and found was that number one we think that there was a residual hydrogen peroxide within species of the constructs in that initial or in those initial experiments in which we would perform the sterilization the day before we actually moved them to in Vito culturing and then what we did is we followed up looking at what would happen.

If we did is we use hydrogen peroxide then followed with either washing aeration over a period essentially a four to six weeks in which case there were still greater toxicity to the cells in comparison to for example the UK later alcohol controls but the washing or the aeration did mitigate that to a degree ultimately I think that if one is looking to use these are Donna get Tote point in the future of their potentially usable in the future for people gamma irradiation probably provides a less toxic methodology but the disadvantages of gamma radiation are its expensive availability so an initial studies in animal models that would be more cost feasible if we can use gas associated sterilization protocol.

Thank You Me Rachel using guns and you see how could it be expensive but you know I see no it's painful it's so good right yes so I can't remember exactly the Young's modulus that we quantified but we did that both will be quantified it using cylindrical desks the constructs that we were making were similar to the cancerous bone so not as stiff as mature cortical bone but certainly on the order of what one might expect in the region of the femoral head in which the majority of the bone really is a relatively dense and cell phone as opposed to cortical right and my limited understanding is that the molecular weight of the PCL that one uses influences it as well and so we were using the needs.

We were using the 15,000 molecular weight which was actually the issue that we are running into is it seemed to be more brittle and so we are really concerned that it was going to fracture and so when we were doing the quantification of the modulus of them using just the discs and typical load so we were essentially getting fracture at the point of failure and so we were concerned that everything later was Donna be pointless but what we wanted to do a part of the motivation freshly creating the femoral head and articulate what the acetabulum was seen whether it that distribution of load was more representative what we might expect in the joint and spread out over larger surface area with the performance be adequate in that scenario we found that it appeared at least feasibly adequate the gamma radiation it changed some qualities the physical qualities of the carriers for the songs.

I've tried and it made them more brittle right and so when you try again irradiation I would suggest maybe redoing right that Young's modulus to see if that is not in effect absolutely and I think I've read that somewhere else as well where they've quantified that so I mean ultimately I think the point is wherever we move in terms of prior to actually putting it in the animal models any subsequent steps of testing we really need to validate with the exact protocol we're going to use because everything that we do is going to influence either the biological and mechanical characteristics of the combination we did and XPS up to her damn mind its own controls chemistry.

I change it sounds like it was another front so I could determine your company thank you was just wondering if you had any proteins or did you code the PC all before you actually put the files on or anything like that media initiated we did have FBS but we didn't do anything special in terms of treatment of the surface in order to train a chance that the only other thing that I tried to do is develop a methodology for actually delivering the cells to the interior of the con- struck but that failed which is when I then moved to the arose I guess I should follow up to mention that bonding to the PCL did not work and I tried repeatedly didn't work and then we had collaborators who tried and actually wanted to go to in vivo study which we did and it didn't work and then I spoke with a chemical engineer who now work with the new mercy of Georgia who told me you know up front that there's no way they are gross will ever buy into the PC also even though I'm it tends to work with lax vivo in terms of culture in the cells.

I don't think that ultimately in the long run that's going to be the material of choice I think you just have to be another combination to serve as the congress later and I think that other groups have already been able to combine the macro porous constructs for the gaseous portion some work we describe in conjunction with microfiber electron spun woven for the cartilage layers tremendous amount or so yeah I don't know how he died on this project but he was using IP o's with titanium and use racial antenna on the edge of a nice to kind of have a contract and you get a chance to evaluate any of that type working your yes but we talked about here so they were they use that and ultimately made it as far as an enviable resurfacing of the patella and dogs and they used different combinations to use the tantalum medal in conjunction with the arose.

They used so I can sell us allograft bone for the bone layer bonded with in all of them in terms that patella resurfacing I would say that the results were they are at best and so it was part of the motivation for move in this direction toward the PCL was based on the success of the rabbit study I will go so far as to say that that laboratory has moved'm very much in the direction of osteochondral allograft doing because they have had more rapid in greater success in improving allograft preservation such that use of allographs now is feasible for them to do not only focal defects and people but they're even doing large and almost total joint replacement with osteochondral allograft and now he's doing that large joint osteochondral allograft replacement the University of Missouri's yeah.

I would suspect that Dr. bug be another at UCSD and SandyDiego no bodies have been doing a lot of osteochondral allograft in a long time and they will they're probably doing a large regional replacement to at this point it's not really a question it's more an answer to your last two questions what'st he deals cell type and the ideal pre-treatment and if I could depend what you want to do because if the target is doing in engineered constructs well I the assault-type its embassies and pretreatment as mechanical loading and I were they then cycle than her hurts but if you want to go clinical the answer is no pretreatment and no cells and that's more to challenge in designing the scaffold that could working those conditions right balance right if we could do clinical repairs that are one step and don't involve multiple surgeries and ex vivo preconditioning that would be easier and faster but there are few of those that meet our goals at this point in time from a Bailey you know with the biological approach to it but have you are you doing work in the acetabulum.

I mean can you talk to that a little bit of what if anything you think has the best shot of working in sort of reshaping if you will the assets having a portion of the acetabulumI don't because most of the time when my understanding is most of time when HenryArthur plastic is done we're looking for the femoral head replacement rather than ace tabular while maintaining the femoral head and so the femoral head osteochondral allograft replacements that they've done at Missouri in the last year had been that they've been femoral head replacements rather thanacetabular but theoretically is there any reason why this match these materials in your mind like if they didn't work they did work on the female had side could not work on the ace tabular said mr. League reverse morning now.

I don't know of anybody who's used these materials in vivo for the femoral head replacement and a large animal model yet okay thank you very much when his team game and I'm chief scientific officer for my picsbiomedical also the director of Orthopedic Research Atlanta MedicalCenter and speak today on a couple of different topics a look at the peak application but I want to show you a couple different ideas will be looking good composition I think Sam is a great job and give you some idea of composition and change in immigration how that might affect you speak about something a little bit different kind of going through some big city and packing as I'm back and forth between Atlanta and Tampa Jacob pronounced we talked about the new magic and science it's the magic of technology.

div>Let me show you two different technologies today that you may not have seen and to me these are unique evolution material certainly know both in form and function in composition examines book to elect own ability to there to guide to build as you might suspect I think at one point we tried to imitate but then its materials that change and certainly can change materials with coding we can take peak can take hydroxyapatite but putting these on the materials Labor to effect a change we change the binding machines the opposition would change the mechanics of the cells themselves will also change the chemistry of materials as well you can go from my peek to a peck and all of a sudden going took tones now that you have a different complete set of molecules for long time we get to these ball and stick models.
I think as we develop these compositions were able to look at something completely different you may look for serotonin for feeling good having his insights on preventing your gaining you also now able to change the surface chemistry armies materials and create Electric she's still actually function as receptors are reporters for ways to actually a billion and interviewed the materials themselves so maybe also working with his isomorphic chemical topography will you be good to use electric fields of molecular surfaces and in doing so you actually create the surface of the binding and you guide the direction of the cells and their activity known for a long time that these functions will change selectivity is coming to work that was done no 10 to 12 years ago but what you find is and if you look carefully don't know that I have a there's a bed it's ok so if you look at this here to find his money reducer you construct the flow to his own cars and you don't get it behaves like anything else anymore you remove this pressure they go out and flatten out so you're able to restore it just by changing some function dynamic get rid of some event Castro the turbulence and go back to a laminar flow back to normal when you just a little model by the way.

I'm thinking about this and I call it kind of linear development so if you look at each of these brackets and you consider each of these circumstances could mean temperature it can be flavor can be color candy size restrictions in every cell has its way in the dynamic effect time the teachings of the move through these become something different and it shows you how they develop if you look at it like this and you don't change any of the parameters of the cultures, I continued culture and they just go right to their cycle so those orange balls representing a cell early later and then in its terminus and if you've been changed one variable have the ability of them to also destroyed that cell layer so that tissue pathology you're moving development you can do the same thing with this suggestion that many other ways to do this by combining a totally different variables can actually extend an interface would you want to see how you're Donna see if you wanted to that this value.

What we're going to see in a lot of the regenerative medicine so you can change this point with the largest size but you can take it to a couple of different functions to get it there so to speak to particular types of material modifications and talk about the kinetic response in a magnetic response to the kinetic first then reason I have friend ed named Jesse hunting he came to us we should resist issuing says what do you think of this item on this fantastic it's making their timetable look at this cage he says what doesn't work as well you know that's a good question.

Let's figure this out so you look at the amount of building you see the fact you get really nice reversal lines but it's a trust building is attributed to the mechanical engineer just work with compression and tension the same time on these materials and in doing so as we know it is very much response to mechanical forces should be able to predict and making changed the bone you want we're not the first people who recognizes high-frequency merchant transaction suspension for a long time I can agree upon a time when Webb diva still at Utah and care what it was a unity party that's cool and so it works with him he says you know if you just need a little bit you realism thatmicrosoft itself as a way to make it happen and prospectus McKenna step function when you look at the amount of culture Stadium look at the microphone over time you're able to predict whether you have a bit rest of the building gain so we looked at these trusts' system here.

We could have a piece of the trust and would you find that if you take this out and you think about the osteoblasts on here that's about 20 to 30 microns and you think the length of the trust maybe ten millimeters that you only need to move 37 manometers in strain to effect change the process predicting so we did is we look at this way of maybe becoming a mechanical generator so we find the Jetty calcium influx of BMP illusion able to make bone and it should be appropriate to the radius of the strike so good even to demonstrate this that you can give either have a stunning titanium where you can have an active mechanical structure and stretch it and in doing that change you able to transducer section the genome of the ice cube last name or bone so this service matter.

Here Georgia Tech get a lot of papers on the Titans spine and able to show you if you haven't been discovered if you have an edge service you actually able to change the link to the full web trust it wasn't the kinetics and just to be able to move it would you find that references to be characterized it's rough all the way along but if you think about it in terms of the variations between the roughness you realism that the reference altogether the higher the ramp the more the amplitude so you get more transaction of the material itself with the model to look at this and how it's going to change and you look at the disparity ratio of the height difference in the reference this is a crevice in what you find is that the clothes need to come together if he keeps the Russians the same the more you amplify the response you could pick forces so you may only get a hundred and eighty or 200 Mike restrained.

You're able to get twelve times in the crevices get close also be changed the height get a similar type of application for this content to this one more time for us to make and instead look at bone loss of bone game you put this in a bio chamber you actually measure CT in real time you should make a measurement of the movement doesn't trust itself did by knowing how far the bone moved as well did you measure this thing that stain is proportional to load and inversely proportional to the strike diameter in so it upset the magnitude struck by a matter and these are all 3d printed you can actually tailor your trust to the patient knowing its weight size weight and length so this is the piece of translation that I'm Donna carry into the next piece oh now did you have mechanical what is going to be the next step in this case we know roughness.

We know amplification we also know that the cells to test himself to the point of strain boom models to reduce its train we've been able to demonstrate cultural expression this morning two percent over normal BNP to expression which is considerably above normal and bill without the carnage intermediate I want you to carry these pieces into the next part of this talk this is the medics base this is something I'm doing a lot of this is actually my images right now my emphasis probably what I'm doing to buoyancy in micro gravity and how to do is to compare you think the buoyancy you think of the ability for our terrestrial mammals you look at Wales and I spent four years at the College of Medicine dois to Florida and then we did a lot of Marines Mammal Stranding soldiers defecting dolphins and whales and seals and other things as well you find when you look at that but if you look at the left.

You see the human cancellous bone in his an isotropic type of the signature tunes is learning to history we see it why it's learning that the same each time is responsive to gravity when you look at the way you can sell us bill have greater surface are about looks more like the micro gravity better astronauts experience when they leave the planet and one of the things this very different between astronauts and Wales is well worth it reaches a veterinarian with tell us as well and then do so they might about tension so what we're trying to do is to establish the distribution where you can look at this field anyway so if you look at it this way you look at this way are you look at it out of field you're able to find you get the same service during the same dynamics the same loading some remodeling history so Donna go up in a blue collar well-made always.

Gonna talk about laying bricks you know when you get this thing started don't let you get that first course down giving it happens so this was a resulted in a corporation that was developed with their space cooperation in bio medical here in Atlanta Georgia we've been able to work with Dr Frank living stone rank is probably the ultimate laser specialist in the world therefore these lads throughout the planet and he has built the mall he is able to actually materially change composition of plastics metals and bones without actually affecting the chemical structure so the nuclear speaking with Frank what we want to do is want to create like he and I spoke to Paula Jones get back to that brick pattern would be able to kind of laid down but in geometry and deaths and right now.

We're kind of limit is something like a NATO and War saw a router and so frank says what I can do this is to bring together Micro CT data I give you with depth geometry and again due to my own personal laser so this is what it looks like when Frank is cutting laser with us he's getting Simpson 72 second resolution on laser and fingers clear to send two lasers behind each other the first one he's doing putting up fluency and not really going into the physics P critic lose you were to near melt almost like you have some is almost a soft it follows with the second laser and you know how you can hamper trump the same thing is takes out a phase-in freezes it would bring bin Forrest.

He was throws this material includes a nuclear by resident implant so not only do we get the geometry and the death and the topology yes indeed electrically conductive so over seven so having the truth it's an insulator material that's an active conductor as many people known this room when you look at stem cells you look at the ability to differentiate the bar you're able to have a polarized Donna goes to the bone phenotype to less than 50 so that they differentiate we have something we thought was pretty interesting when you look at the electron microscopy unless we also try to get the same order and structure here as you would get in the letter type of bone in effect at the same magnification you get the same college and birefringence and same damn rebuild it to have that same resolution as an as a contract also serve as an is very critical to bone formation.

When you look at the size of these cuts you look at the top left at your standard that's coming out of the milling machine and people like Tracy Anderson to build the experts at that machine a big piece of machinery they know how to represent that what frank was able to do is created this kind of orange peel looking material which unique about that.

If you think your basic geometry squared being a sphere for this service under steer you know these conspiracies to par squared so in an effort to create is electrically conductive Beijing on geometry he also created twice a service area and every sleep so we doubled the surface area modeled and isotropic distribution made electrically conducting in one pass some Wendy Wilson has been able to actually look at these cells behave.

I mean it's great to have a beautiful piece and it's a nice hard piece where does it work so we tested these on normal stem cells we look for the ability to differentiate towards bone we challenge that ten days with announcing glued to look at the alkaline phosphates you may also follow them outwith alizarin staining to look at the calcium standing at twenty days as you can see on the left this is our control this is a standard peak material untouched unblemished on the red panel you begin to see what happens to that material when you offer an electric conductivity and 10 days we begin to see complete crossing complete closure across the surface like 20 days in stem cell culture will be in to demonstrate that matrix with money in it and by thirty days we had a very collected matrix.

We don't have you considered this but these medications begin to see the monocle phenotype star pravah nuclei we looked at it with the electron microscope as well and what you find is that you find that the bone test to them electrically conductive surface preferentially over the pic material in fact we had five times in matrix within the Jeanette geometric phase as we did on outside on the peak advice no intention is to certainly brings into the clinical applications as quickly as possible the FDA requires you to a particle analysis to be sure that you don't share materials are quitting is very brittle or something is going to be compromised the patient with a lot of accelerated inflammatory responses yet it requires that you do for me I'm in rabbits 56 milligrams you be under this before you go into a clinical trial as you can see in this slide at 600 new 250 pounds maybe loading 800 at a hundred and seventy pounds what you find is that we're under a million in total and five million cycles of testing on this material so we have something against me just true Donna bring me so much together or something its geometrically isotropic conductive and at the same time.

It looks good you know in the lab what is he doing proof of concept we want to be sure that we have a stable fixation restriction and were able to get an efficient fusion we did a large animal model of the interstate 40 pic Callahan was our surgeon down there and we did that term in the bed resident implants and messing with age but the standard peak we look for them either alone or with graft and the contrast of them out at six months and even though this one more time you think of you in this room we all have a little bit different size we have a little bit different gate and the idea is to use its peak implant to create this area the surface here that allows the only been cells to settle on those then have a team of bodies from each animus toward individuals will then create this compliance in the idea is to accentuate that model explains own right down to the implant itself so at three months we're already astounded and member Dennis crazy deals holy cow that is great but you find the complete dismissal bone that forms between the two vertebral bodies in this is with no grafting material when you think of the standard of care particularly the United States you have an allograft plus a spacer of Pete.

We're doing this with nothing will be compared to sit three months with their standard graft and a pic implant that we had two typical analysis we didn't get the tapes it so one of the things that really wanted to look at his table 3 getting information you know it may be great just quiet the fibrosis but does he actually getting his bone so we look at the projected surface of the wire frame this is what the laser so I was going down this is actually the candidate geometry the cancellous bone who wanted to cut you superimpose us over the sheep spine I think it's pretty clear posterior column you look at the trabecular structure is very wide open very lazy this was the web based in size it was placed into the vertebral body was found here was that the bone that formed immediately adjacent to democracy TV serial section is exactly.

The same as only two were actually able to resonate the design with the bones that form behind we did histology in this of course maybe get a better look at that the medical no graft on your left my medic with the Grafton the right seem to be a bit of an impediment just by having the thing full actually slowed in three months which resolved itself but when you look at the histology and on the left you're going to find the plane not attached material on the right you're seeing our magnetic material you can even see the bone for me because the fibers sheath around the material on the left and that's standard of care with graft with peak at three months by six months to a complete consolidation of our graphs that had the peak on them the standard with the graft we had good a position.

He was a tight fusion but you still have a lot of fibrosis around the graph you'd gone around it just took it and penetrate kind of that inner space around the graft itself and you look at the standard medic at the six months with mild nicely home again you cannot see the pic implant within this frame relative the same size same magnification compared to the medical the people so in summary we have some six-month histology with extensive billing contact we have very little evidence of fibrous tissue interface with a very healthy bone that was formed adjacent to the implant we've taken this technology and also had entered on bone we were able to do this on a limb and actually machine building with lasers to create new types of materials.

I think in terms of your interested in looking at allographs from being able to do maybe uses into population but we need to do the same way Frank did with fluency the plastic materials he actually took this and also he can do the same fluency within bone so you're able to acknowledge not only the inorganic appetite but you're also able to shrink that college in around it and so would you look at this he said he's almost like a little color flower heads a very tight inorganic type of the calcium hydroxyapatite the things we gained on this too.

I think is really important and I heard a couple questions let me go back to something to be a good asset minute ago we did it reduced spectroscopy we looked at right now as we did com positional analysis like a dispersive spectroscopy as well and we found there was no change in composition is exactly the same what frank it actually does just football carbon so they all looked up to you see leaves on a rainy day waiting for trains you seem older and services are all up and turned all the carbon sides of the materials so going back to kind of this principle of tears and coordination we'd ask frank and as I think any of this rule no you need about a three-to-one orientation of the flat place over the group to actually make bone growth the best type of surface it likes and so frank was able to achieve this same 321 so if you go back to that magnified piece you realise you created this service within the context of the 321 so in any of the slowdown in bone.

We have met with material changes in your micro material changes well so again this material taken advancement extension in translation go back to may be closing piece here for services making which has so many years ago took about knowledge being our but I think maybe it's more important that we can recommend nature set by a banger and I think the same thing when asked he was talking about was being totally different team black magic and white magic you don't wanna swim upstream the whole life you want to go with the flow you want accentuate you want to integrate those things as best you can so in this context.

I think of cell-based therapies in the world summit that we've been allowed to participate in here they'll be able to change those materials be able to change the interface and change the reaction of things that are still in our hands think you should mention today from Tracy Anderson by the CEO and founder when you listen to the seller David King used in the IP drink living stone TimTaylor who set up the airspace collaboration Jason Kenney bumps up in just a couple of me some slights thank you very much so we thank you for making it highly quickly we were differentially asked to present some time but at least.

We could announce some locally believe the sort of ground breaking down from our large animal study and you only and I guess it's ok because we only have a few minutes leftmost likely have been given to answer all the questions if there are any because we still haven't analysed the data and now I work for a company and being a publicly traded company unless we tell the entire world I won't be able to get into details unless we ready somebody's medical officer of health problems with technology we are saying attack you know depends how you look at it and it all depends on what you think is if that's a true believer in the genetic medicine.

I get that we do sowe volunteered in plans that we surgically implant and it's called police to restore the structure and function will vote we are focused harlequins namely yes although this may mean a way which included a track and demand rom-com I guess now would be a good game going to Lincoln trademark technology little bit so we're dressing a whole spectrum of diseases that affect guess hospitals and the airwaves gloating cancer and cancer he pusher majority will suggest trauma contractions congenital abnormalities being called up until it reaches affects one in Port TownsendUnited States which means daddy away when you hear that means that he shoot hadn't begun look forward to our next decade McLeish yes off against which is a team as and develop fully into locked it can be like the hourglass shape.

I can be completely corrupted and this can be found out and Wales right now to try to keep your cool and esophagus and tried to make it grows going concern to solve ago consider all this article's cancer of the lung when you think about a cancer of the lung we all think about the long but eventually came to reflect give bronchial tree and a good portion extra40,000 cases of lung cancer are located really nothing to prove we forget the long as I gained probably sometimes we think all the games that is located in the main blown so you would think ok though this kind of that and that's what we do with just you know we're only five butcher will we do with cut up the bill on problems and then if you probably when he was sent to any cancer you don't just accept the cancer you go through the world a little live there will be there to make sure there is no one single cells that he couldn't seethe single cells.

He will stay up because that sounds about wake up at some point and headed home to you but you can't get online to cut a larger piece and when you can unlock your PC world sometimes you have to let the novel and he gotta get along and everybody give me a hard time that is healthy lines in apocket because we can't touch it is not in with some not Donna elaborate on what your life is Donna be without one long because we almost become almost commonsense I don't know I'm not going to waste any time we also address attract a cancer Israel trauma no cost too much and the track is really something that when you respect that decision can be as creative and has ended but we just can't put it back together imagine the pipe and he gave me not even if you can deliver peace who can probably put it back together if you like is you have a large cheese you just can't put it back together somehow he is like you need another piece of pipe and bad I guess we do even though.

We don't have to do it and we have a pipe if you will dad will transform itself into an organ or will guide the growth of an organ how do we do that so we don't have any emergency and it ended we're Donna be working on it will be Sunday you can schedule is going to be a six months but a patient can wait for three weeks so we get a pensions and with surgery and surgery scheduled for a few weeks later we get the cells from the patient's I can do more matter derived or adipose derived mesenchymal stomach population we're still in and of course of identifying collectors.

I am recognized the Appalachian but that's what it is that we get the sales they go straight to the lab and wet spot them for around two weeks which is a class you heard of that is always some magic number their cells don't really differentiate in anything they're just expanded number then we can we go this way we do we have a scaffold of a scuffle it'sit's an electric pump skillful and as Apple you a thing and has a very thin scuffle and disease and SEM picture discovered it looks like but this is where we spent a few million dollars because defied their size the full size and this is what makes it different and the only way to actually know that is to do a large animal implant otherwise he'd do small animals.

It all looks good and then you do a large animal implant and all of a sudden it doesn't work so you have that you have a conduit there by you don't have something that actually drew generates at all we are so once we cede discomfort with a lot of effort led by Rabat and prove true tight days we test the appropriate he knew better as the purpose of evenly distribute the and solid this come from access perfect home in Dubai's for the stem cells again this Sunday we worked because this makes it different a stem cells.

I don't know you like that kinda like it or really like the micro structure and that's the secret if they're not that happy is the Renault going to be able to start of regeneration process at that point this is ready so this goes to the operating room and the surgeon Dr.the surgery so I'm not Donna but that's besides the esophagus the only reason I guess I'm going to show it is good that this is about 25 centimeters long sole t's say 10 inches so when I want to stress this because this is an Oregon is not as much fun without talking about it as moll 42 could be a college in the polymer and you put the cells and the cells go where no return to really I guess we're very ambitious we're trying to really regret in Oregon the portion to sell their goods are sold to go portion at Palazzo portion and a small fortune to belly that connects the stomach what does it do we do now well the surgeon gets his stomach cancer gets us tarmac you actually come this time.

I eat because the stomach looks like a bag pattern looks like a pipe and you put it into chest and that catch up to the remaining oesophagus well that doesn't sound too appealing to me that's it they are better options than a physical on and lead to start with theirs but get a piece of Cologne and bring it in the chest I don't think I need to expand on medical 12 used to calling will not have the stomach no vehicle available only to chest today the problem is dad if you do that you have what is cold and dumping syndrome and all you had to be on dietary restriction have no said that I guess of the Geography lots and I can keep going what are the issues the complications and the co morbidities when you do that kind of operation itself further large amount of patience and it is very conservative as we decided to sort of we don't want us with a tracking number day but I said thousands and thousands of patients and in Europe I'm not even going to Japan a date with the overall Asia would incidence is higher.

I get our tank this is one of our guys we've been having is out of decades looking for food obviously there's no food around here but anyhow so we use a lot you get a minute being somewhat large animal model and that's what that's what you need to do that's what the two to go down in two patients you just can do in a small animal on so on and so forth so this is where we follow this debate but you said before that's so this is a native oesophagus a dead pig and this is the one that we grew now again I'm not talking about a little thing I'm talking about a piece of the oesophagus up these of all again this is something that tomorrow a patient who use is not we're not thinking about a hard-to-reach consulate this is a good beginning this is now this is the real deal due to the sub mucous to the older later represented this is her and we have chemistry.

I can show otherwise have give some get fired only get to see a sec it's sort of growing up to us you notice other bills gotta muscle tightness would you can get before well the muscles all the layers are represented and it is something bad I'm not all I can say now expected either buck plexus de Nova intervention as part of sympathetic and sympathetic whether we get represented it that's where gives the most in a disciple good that's why this offer discounts for a living it contracts any condo pushes the food down load into this time so it would be good to have something that is not a stomach or the column but now we have actually and we well oesophagus just we just do a piece of this all goes that is covered bowl and we're done with limited test which is the bag and swallow.

Dr DeBary usually patients do they went down link the biggest life is short so they did you did abundance well they just kind of showed us into the trial that baseball is it and we have you do you have a CRM so under x-ray you do you see the bag and a film and we have no other commodities it seemed absolutely normal to me so that's what we all and I just going to elaborate on ad decisive game 5 will readily account and Daniel hour and 46 a time.

I'm Donna stop here but we also work like I alluded before the main bronchus and on the track you the principle is the same the platform is the same only when it comes to the airwaves through add on to the electric spot scaffold something else that I can tell but it just would not elaborate on what I give you depend highest-ranked because when custody a way you need to be able to stay open ride the oesophagus actually usually closed it just opens to let the food cool it down to work the stomach but instead the raised me to be open air is to pursue go out that doesn't go to work obviously the patient dies.

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