Session 7: Tissue Engineering

Transcript of Part 1: Engineering Tissue Replacements

00:00:00.23	Hi, my name is Sangeeta Bhatia. Today I'm going to tell you about tissue engineering.
00:00:05.12	In particular I'll be telling you about engineering tissue replacements for patients.
00:00:09.28	In this seminal paper in 1993 Bob Langer and Dave Vacanti described the concept of tissue engineering.
00:00:19.22	The idea was that one would take a degradable biomaterial, basically a plastic,
00:00:25.25	and mix cells with this degradable biomaterial, culture them together,
00:00:31.00	and create an engineered tissue.
00:00:34.05	These hybrid tissues that are part cell and part material could then be implanted
00:00:40.14	for patients with tissue failure of various kinds.
00:00:43.20	In this same paper they examined the number of procedures per year
00:00:49.20	that were done for patients that had tissue failure of some sort.
00:00:54.24	And you can see that many, many millions of procedures are done
00:00:58.14	for all kinds of medical conditions every year.
00:01:01.07	So, how has this gone?
00:01:04.03	Well, first one of the challenges.
00:01:07.06	If one looks at how tissues are organized in the human body,
00:01:11.04	we see that they're hierarchically organized.
00:01:14.09	So for example, here is an image of the liver and if you zoom in on the liver
00:01:19.14	you can see this fundamental unit of the liver which is known as the liver acinus.
00:01:23.20	This unit is about 1 millimeter in diameter and there are many millions of these unit in the liver.
00:01:30.19	If one zooms in on these further, one can see that within the acinur unit
00:01:36.21	are hepatic cords and these hepatic cords are rows of hepatocytes (liver cells)
00:01:43.05	that are organized in a way to process the blood efficiently as it's flowing through the liver.
00:01:48.25	Finally, if one zooms in all the way to the single cell level,
00:01:53.08	one can see that these hepatocytes, these liver cells, are surrounded by at least 5 other cell types
00:01:59.28	in their home, in their what we call micro-environment.
00:02:03.18	So, these are the challenges of the tissue engineer: to recreate
00:02:08.16	the microenvironment of these cells
00:02:10.24	in a way that there could be many, many of them in healthy microenvironments
00:02:16.07	in tissues that could replace the tissue function of the damaged tissue.
00:02:20.20	So, first you might ask why one would create a bioengineered tissue that's part plastic and part cells?
00:02:30.02	This is a very famous image of one of the first tissue engineered pieces of cartilage
00:02:35.21	in the shape of a human ear in the back of an immune compromised mouse.
00:02:40.19	So why would one do this?
00:02:42.25	Well, tissues engineers would typically do this only if
00:02:46.26	one needed to improve upon one of the following existing medical therapies:
00:02:51.14	if no simple medical device could improve the tissue function.
00:02:57.17	For example, an implantable hip is a piece of metal or a piece of plastic
00:03:02.10	and if that could achieve the tissue function that you were interested in,
00:03:06.08	you wouldn't create a hybrid device.
00:03:08.07	A drug: if one could administer a drug to patient, again, one wouldn't need a hybrid device.
00:03:14.20	A cell that was minimally manipulated: this is the term that
00:03:20.27	has been invented by the United State Federal Drug Administration
00:03:24.05	and it refers to cells that have come out of the body and are re-infused with very minimal manipulation.
00:03:30.14	So if one could minimally manipulate a cell,
00:03:32.28	one wouldn't need to engineer a tissue to improve the tissue function.
00:03:36.27	A surgical reconstruction: if one could simply borrow another part of the patients body
00:03:44.02	for replacement of the tissue that's damaged, surgically, again,
00:03:48.19	one wouldn't need a complex functional replacement.
00:03:52.14	And finally, a transplant: if one could use a cadaveric or donor organ,
00:03:57.09	a whole organ, and replace the tissue function of interest
00:04:00.23	one wouldn't need a bioengineered tissue.
00:04:02.22	But importantly, these criteria are often still not met
00:04:07.17	and one does need to, in fact, turn to tissue engineering.
00:04:10.02	So, there's actually a spectrum of tissues that can be engineered
00:04:15.02	and all of these fall in that category.
00:04:18.19	So engineered tissues include biomaterials and cells in various combinations.
00:04:24.16	And on the first slide I described to you biomaterials that housed cells.
00:04:29.14	So these are plastic, typically, and cells together in a hybrid form.
00:04:34.24	But there are also various incarnations of these engineered tissues.
00:04:40.15	So, for example, there are complex biomaterials that can be implanted and recruit cells.
00:04:46.11	So, upon implantation they don't carry any cells but they can recruit host cells
00:04:51.26	and become hybrid in situ.
00:04:54.11	These are also in the category of what one would call engineered tissues.
00:04:59.04	And finally there are materials that are completely derived from cells.
00:05:04.13	So we know that cells themselves can make scaffolding which is called extracellular matrix.
00:05:09.15	And if one cultures cells in the laboratory, and creates structures that are part cell and part extracellular matrix,
00:05:18.00	those also can count as engineered tissues but they have no synthetic materials within them.
00:05:23.22	So all three of these categories of engineered tissues have been
00:05:29.12	widely experimented on with varying degrees of success
00:05:32.01	and what I'd like to do now is to show you some examples of each of these.
00:05:35.20	So we are going to start with acellular biomaterials.
00:05:38.28	This is an acellular biomaterial which is a very effective engineered tissue.
00:05:46.13	It's called small intestine submucosa.
00:05:50.12	It has no cells whatsoever.
00:05:52.26	So if we look in this bottom image here, what we see if a cross section
00:05:58.04	of the intestine, in this case of a pig.
00:06:01.00	So we see these vila structures of the intestinal lining
00:06:05.28	and that's how the intestine absorbs nutrients,
00:06:08.19	and underneath that structure are these muscular layers.
00:06:12.23	And lying between these two layers is what's called the submucosa.
00:06:17.01	The submucosa looks like this on an electron micrograph.
00:06:22.26	This is a very strong extracellular matrix structure
00:06:26.20	made up primarily of collagen and mixed with lots of other extracellular matrix types of molecules.
00:06:33.17	So what was done originally by Steve Badylak and coworkers and many other groups since,
00:06:38.27	is that one can take porcine small intestine and
00:06:42.25	strip away the cells with various detergent type procedures,
00:06:46.20	and come up with large scaffolds that are acellular, that are very strong mechanically.
00:06:52.25	It turns out that when one implants these at sites where regeneration is required,
00:06:58.02	so for example in tendon and ligament repair,
00:07:01.10	these recruit host cells by inducing proliferation and differentiation of those cells
00:07:07.22	and promote wound healing very effectively.
00:07:10.11	These types of materials have been in millions and millions of patients.
00:07:14.06	Another more complex example is a hybrid example.
00:07:20.13	So I told you before that once can combine biomaterials and cells
00:07:25.11	to make hybrid engineered tissues and this is one of the best successful examples of having done this.
00:07:31.17	So here we see engineered skin.
00:07:34.20	So, on this side we see a cross-sectional micrograph of what human skin actually looks like
00:07:43.02	and we see that there's a dermal layer here and an epidermal layer here.
00:07:47.20	The dermis is composed of connective tissue cells like dermal fibroblasts
00:07:52.20	and the epidermal layer is composed of epithelial cells
00:07:56.18	which undergo what we call stratification as they mature and form this multilayer surface.
00:08:02.12	And way out here is where the skin interfaces with air.
00:08:06.14	So this structure is what's damaged in the case of burns, for example.
00:08:11.07	And tissue engineers sought to create tissue engineered constructs to replace this skin.
00:08:17.20	So, on this side what you see is an engineered version of skin
00:08:21.17	and in this material what's been done is a biomaterial, a collagen gel, an extracellular matrix gel,
00:08:29.25	that has the consistency sort of like jello, has been mixed with dermal fibroblasts.
00:08:35.12	These dermal fibroblasts remodel the collagen and they form very tight mess-work.
00:08:41.19	On top of that mess-work, one can seed those epithelial cells, the epidermal cells
00:08:47.28	and they will stratify and form a multi-layer structure on top of that dermal layer.
00:08:54.25	And this engineered skin can be implanted in the setting of burn or other injured skin
00:09:02.02	and promote wound healing and to great benefit.
00:09:06.11	Another example of a hybrid tissue engineered system is this one.
00:09:11.25	This one was developed originally by Toy Atala and coworkers
00:09:15.00	and in this system instead of using a natural scaffold, like collagen,
00:09:19.19	we're using a synthetic scaffold called polylactic co-glycolic acid.
00:09:25.15	So this is a polymer which I'll describe later in a moment,
00:09:29.03	but for now you can think of it like a plastic
00:09:31.09	and this plastic was cast in the shape of a balloon structure that you see here.
00:09:36.11	On the inside of this balloon, we see that lining cells from the bladder were seeded
00:09:45.03	and on the outside of the balloon muscular cells that are important for bladder function,
00:09:50.04	for the contraction of bladder, were seeded.
00:09:53.03	Together these were cultured in vitro and then implanted in animal experiments and now in humans.
00:09:59.07	And these hybrid tissues that are composed of two cells types and a polymer scaffold
00:10:04.13	together promote better bladder function, better compliance in this setting.
00:10:10.01	Interestingly, in this classic paper, where they first did the experiments in dogs,
00:10:15.23	these hybrid bladders were also re-enervated.
00:10:19.28	So they promoted nerve regeneration in the host
00:10:23.29	and these dogs regained voluntary bladder function.
00:10:27.00	This was unexpected and one of those exciting areas of research that is still unexplained.
00:10:33.16	So I've told you about acellular grafts and hybrid grafts.
00:10:38.25	Now let's talk about completely cellular derived grafts
00:10:42.11	that have no synthetic or natural biomaterials in them.
00:10:45.21	So here's an example of a biomaterial-free engineered tissue.
00:10:50.13	This is  blood vessel that's made by Nicola L'Heureux and colleagues
00:10:55.03	and in this system they do what they call sheet-based tissue engineering.
00:10:59.15	The vision is that one would take a sample from a patient, a little biopsy sample,
00:11:04.23	from which one could harvest endothelial cells which line blood vessels
00:11:09.05	and fibroblasts, those same dermal cells that I described earlier, and culture them in flasks.
00:11:15.16	And if one cultures them in flasks...if one cultures the fibroblasts in flasks,
00:11:20.02	one can see that the fibroblasts deposit their own extracellular matrix.
00:11:25.05	They deposit the same types of collagen that you saw in the small intestinal submucosa earlier,
00:11:31.08	but they deposit it on the bottom layer of the flask.
00:11:34.01	And one can manipulate the culture conditions to promote
00:11:37.00	lots and lots of extracellular matrix deposition.
00:11:40.11	Then one can detach the system and it creates a sheet
00:11:44.14	that can be manipulated and rolled on a mandril.
00:11:47.15	This mandril then, now is a living cinnamon roll of cells and extracellular matrix
00:11:56.13	and it's cultured in this living format so that
00:11:59.19	the layers of the cells and the extracellular matrix will fuse into a very strong wall.
00:12:04.28	One can then seed the inside of these tubes
00:12:08.19	with the endothelial cells that were originally obtained
00:12:11.27	and now you have a construct that has endothelial cells on the inside
00:12:15.26	and this nice, strong surrounding structure made of fibroblasts and their extracellular matrix.
00:12:23.23	And these can be implanted in patients.
00:12:27.01	And these are what these tissue engineered blood vessels look like.
00:12:31.02	These blood vessels have no scaffolding material that was introduced from the outside.
00:12:37.24	
00:12:40.15	OK, so these are the different flavors of engineered tissues that exist.
00:12:44.12	If one were thinking about designing a bio-engineered tissue to replace a particular organ function,
00:12:50.17	one important thing to note is that, as tissue engineers,
00:12:53.23	we can frequently...we are unable to replace all of the functions of interest.
00:13:00.03	So, for example, if one thinks about the functions of skin that are are being replaced,
00:13:04.03	it's simply the barrier function in the example I showed you.
00:13:07.16	Those pieces of skin have no hair follicles, no sweat glands, no immune cells,
00:13:13.19	and that's a deliberate choice on the part of the tissue engineer
00:13:17.13	to go after the function that's most critical for life.
00:13:20.09	Similarly, if one thinks about pancreatic tissue engineering,
00:13:24.10	one often sees that folks are interested in the implantation of beta cells
00:13:28.24	that will produce insulin in a glucose responsive way for patients with diabetes.
00:13:33.10	There are many other functions of the pancreas
00:13:36.02	that are often excluded there from tissue engineering
00:13:38.28	and again this is a deliberate decision on the part of the tissue engineer.
00:13:42.21	So it's important to keep in mind that one can't do everything, one has to have priorities.
00:13:48.00	OK, well then, once you've made your decision about what functions you're most interested in,
00:13:54.02	what would one then to go further and actually engineer a tissue from scratch.
00:13:58.22	So the next thing to do would be picking some raw ingredients.
00:14:02.22	The raw ingredients that one has to work with are cells.
00:14:06.09	There's actually a variety of cell sources that one can consider.
00:14:10.06	So there are somatic cells and stem cells.
00:14:13.24	Somatic cells are cells of the body that are not germ cells, that is, not eggs or sperm.
00:14:21.08	So they are part of the adult organism.
00:14:23.18	And they can come from the person that you plan to treat. Those are called autologous.
00:14:29.22	They could come from another person. That would be allogeneic, of the same species.
00:14:36.10	Or they could come from an organism that is another species.
00:14:40.14	
00:14:40.14	and those would be called xenogeneic.
00:14:42.11	And in tissue engineering one actually sees examples of all of these types of cell sourcing.
00:14:47.18	So, for example, the skin that I showed you earlier was allogeneic.
00:14:52.05	The dermal fibroblasts and the epidermal cells
00:14:55.12	were from a non-self human to create those skin tissues.
00:15:00.21	Another source of cells, which is becoming more and more exciting, is stem cells.
00:15:06.02	One can think about using both adult stem cells or pluripotent stem cells
00:15:10.13	that have been derived from various methods that I'll describe in a moment.
00:15:13.20	So one certainly needs raw ingredients of cells.
00:15:16.29	The other thing one needs is biomaterials.
00:15:20.07	And finally one needs to cultures these together in a way
00:15:23.25	that the cells have sufficient nutrients to remodel those biomaterials and create a tissue.
00:15:30.00	Once one has the right raw ingredients, we have to think about fabrication.
00:15:35.13	How to assemble these structures, how to process these, how to create culture environments
00:15:41.19	how to preserve them so they can get to the clinic
00:15:44.28	and finally how they can then integrate with the host at that site.
00:15:49.03	So in the next few slides I'm going to talk to you about some of the issues
00:15:52.21	that are related to both the raw ingredients and the fabrication of engineered tissues.
00:15:56.27	First, let's talk about cells.
00:15:59.07	So, if one is thinking about somatic cells, culturing primary cells,
00:16:05.12	they can be derived in various ways;
00:16:07.24	from an adult organism, from an embryo, or from an egg.
00:16:11.28	Regardless of the source, what's typically done is these are mechanically dissected
00:16:16.14	in the laboratory and then they can be further processed in one of three ways.
00:16:20.14	If they are just slightly processed and they retain their three-dimensional structure
00:16:27.20	from the in vivo environment, we call that organ culture.
00:16:31.18	These organ cultures are often done at air-water interfaces
00:16:35.04	that mimic the environment of particular tissues in vivo.
00:16:39.17	For example, the skin that I showed you earlier is actually cultured at the air-liquid interface,
00:16:45.03	just like the skin on your body is at an air-liquid interface.
00:16:48.05	One could further dissect these tissues, finely chop them,
00:16:51.28	and culture them in a flask and allow cells to grow out of these structures.
00:16:57.19	That's called explant culture.
00:16:59.11	And that's actually the way that the bladder cells were first derived that I showed you earlier.
00:17:03.26	Finally, and more commonly now, these tissues can be enzymatically digested
00:17:09.22	to create a single cell suspension, a suspension of cells in a liquid.
00:17:14.18	And those can be cultured in flasks and if they are a proliferative cell, if they can grow,
00:17:19.20	once they grow to what we call confluence, once they fill up that flask,
00:17:24.13	one can remove them, put them in a new flask and passage them.
00:17:28.16	That's called passaging--grow them, expand them, over and over again.
00:17:32.00	And that's how you would create the cells for your engineered tissues.
00:17:35.26	One limitation of using somatic cells was first described by a scientist named Hayflick in 1965.
00:17:45.26	And that's known as the Hayflick limit.
00:17:48.02	So, what he observed in his experiments was the following.
00:17:52.04	If one takes fibroblasts, those same fibroblasts that I was describing to you earlier,
00:17:56.11	from patients of different ages (so, fetal fibroblasts all the way up into your 80s)
00:18:03.24	and cultures them and passages them, in the flasks as I showed you and counts doubling time,
00:18:10.04	one sees that the number of cell doublings that one can achieve is less as the patient source is older.
00:18:19.21	So as patients age, their cells are able to double a fewer number of times.
00:18:25.23	And what that implies is that cells have a finite lifetime in culture,
00:18:31.05	and, in fact, in vivo.
00:18:33.00	And we know now that the molecular basis of this is that
00:18:36.00	the ends of chromosomes have something called telomeres, the so called molecular clock,
00:18:40.29	and every time the cell divides, those telomeres get shorter until the cell reach senescence.
00:18:46.18	So, this observation has led to much of the excitement in the field of tissue engineering
00:18:53.03	about stem cells because in fact stem cells don't have this property.
00:18:57.11	They are what we call immortal.
00:18:59.06	They elongate their telomeres every time they divide
00:19:02.21	by expressing an enzyme known as telomerase.
00:19:05.06	They actually are not susceptible to the Hayflick limit
00:19:08.26	and they can grow in perpetuity in culture.
00:19:12.06	So as a cell source they are very exciting resource.
00:19:15.18	Furthermore, stem cells have been identified that are pluripotent.
00:19:23.27	So here's a diagram describing a number of ways that one can make
00:19:27.29	stem cells that can differentiate, that can be encouraged
00:19:33.23	to grow into the various cell types of interest for any tissue of interest.
00:19:38.15	The idea here is that you could have a single cell source.
00:19:41.20	It could be immortal. It could be expanded.
00:19:44.14	And then, it could be used to engineer any tissue of interest.
00:19:47.10	A further idea that's been very exciting in the field
00:19:50.19	is the idea that one could make individualized pluripotent stem cells.
00:19:55.18	This is the idea of patient specific, or personalized medicine.
00:19:59.14	So let me walk you through how this idea goes.
00:20:02.05	There are a variety of ways to make these cells.
00:20:05.03	So, in this system what we see is that you take, again, a fibroblast, a skin fibroblast
00:20:09.19	just like the ones I showed you earlier
00:20:11.17	and they have two copies of DNA. So these are somatic cells.
00:20:18.00	One can take out the nucleus of that cell and put it in an enucleated egg.
00:20:23.21	Those cells then will start to grow into a blastocyst structure shown here.
00:20:29.29	And the blastocyst, which is the early embryo, has a pocket of cells known as the inner cell mass.
00:20:36.14	If one cultures cells from the inner cell mass,
00:20:39.22	one can create so called pluripotent stem cells or embryonic stem cells.
00:20:44.25	Pluripotent stem cells, the embryonic stem cells, will have the same genetic identity
00:20:49.28	as the original patient.
00:20:51.15	Another way to create pluripotent stem cells from that patient
00:20:55.15	would be to take the same somatic cell that we saw earlier
00:20:58.15	and fuse it with an embryonic stem cell.
00:21:01.13	And that process of fusion also generates patient specific pluripotent stem cells.
00:21:07.12	Finally, recently, in a very exciting set of discoveries,
00:21:11.03	Yamanaka and coworkers have defined a way to reprogram somatic cells
00:21:16.26	without going through an ES cell intermediate or an egg intermediate.
00:21:21.29	What they did, in their classic experiments, was show that
00:21:25.18	a certain number of molecules known as transcription factors
00:21:29.06	could be expressed in a somatic cell and that transduced cell would be reprogrammed
00:21:36.09	into a pluripotent stem cell.
00:21:38.02	So, this now offers the potential to create differentiated cells
00:21:43.19	that are genetically matched to patients.
00:21:46.27	It offers the further possibility to make pluripotent stem cells
00:21:50.27	from patients to fix a genetic defect in the laboratory
00:21:55.14	and then to create a tissue out of those genetically altered cells
00:22:00.07	to give back to the patient.
00:22:01.29	So, given the Hayflick limit,
00:22:05.20	the promise of stem cells has been very exciting in the field of tissue engineering.
00:22:10.21	OK, so we've talked about cellular ingredients. Now let's turn to biomaterials.
00:22:17.00	So, one classical tenant of biomaterials in the tissue engineering field
00:22:23.13	is that one wants to match the degradation of the biomaterial
00:22:29.17	with the synthesis of extracellular matrix scaffolding in the host.
00:22:34.12	So, one would not want the biomaterial to degrade too quickly.
00:22:39.01	Otherwise, it wouldn't perform its scaffolding function.
00:22:42.15	On the other hand, one typically would not want the biomaterial to be persistent indefinitely.
00:22:48.21	Because one might incur what's called to foreign body response.
00:22:52.02	So, where does this concept come from?
00:22:54.26	This concept was originally defined by Yannas and coworkers
00:22:58.27	in these classic experiments that were done in the skin.
00:23:03.01	In these experiments, what they did was they made tissue engineered skin
00:23:07.27	that had a variety of degradation rates.
00:23:10.25	And what they looked at was the wound healing time of a full thickness wound,
00:23:16.07	a cut through both the epidermal and dermal layers of the skin in an animal model.
00:23:22.09	And what they found was that if one didn't put any scaffolding in that wound,
00:23:29.19	it would contract and close very quickly and form scar.
00:23:35.02	That was what we were hoping to prevent with the use of the tissue engineered scaffold.
00:23:40.18	If one then implanted tissue engineered scaffold that has a very rapid degradation time,
00:23:46.09	those wound proceed to contract, just as in the control conditions,
00:23:53.02	as if there was no scaffold in the system at all.
00:23:55.04	However, if one further cross-links the material,
00:23:58.24	one can see that one can delay that contraction time as seen here.
00:24:05.18	And then after that, if one makes the material even less degradable,
00:24:09.11	there's really no benefit to that and the wound actually just persists the same amount of time.
00:24:13.25	So these experiments led to the idea that there's an optimal degradation rate
00:24:19.07	for tissue engineered scaffolds
00:24:22.06	and one can actually see that in the SYS experiments, in this bottom graph here,
00:24:27.13	they conceptualized this as follows.
00:24:29.21	So, the degradation time of the material is decreasing
00:24:33.06	and the production of extracellular matrix is increasing.
00:24:38.26	And the SYS actually bridges the gap in those two time scales.
00:24:43.20	So, it's important to match the degradation with the host synthesis.
00:24:47.25	So that's a material property that's important. What are the materials actually?
00:24:53.18	So there are a variety of synthetic scaffolds that one has to choose from
00:24:58.06	and if one looks in the community we see new chemistries coming out on a daily basis.
00:25:03.17	I've just listed for you here some of the classical chemistries
00:25:07.22	that are used for tissue engineering.
00:25:10.07	Many of them are what we call polymers. So, for example, PLGA is polylactic co-glycolic acid.
00:25:17.26	These are macromolecules that have repeating structures.
00:25:22.03	In this case, a lactic acid and glycolic acid.
00:25:25.16	And when they're implanted and they degrade in the body by hydrolysis,
00:25:30.13	by the interaction with water, they degrade into units of lactic acid and glycolic acid,
00:25:36.02	which already occur in your body.
00:25:38.10	So these are actually originally what sutures were made of, biodegradeable stitches.
00:25:45.01	So there are many, many novel, polymeric materials that have been developed.
00:25:50.09	Another very popular one which has the property of being a hydrogel...
00:25:54.14	Again, a hydrogel is a material that absorbs a great amount of water and
00:26:02.02	has a sort of intuitive feel of jello, gelatin.
00:26:04.27	Here we have a polyethylene glycol which is a very popular biomaterial these days
00:26:11.15	and you can see that it looks a lot like a soft contact lens.
00:26:14.05	What's interesting about some of these hydrogel materials
00:26:17.08	is that rather than being pieces of plastic, where cells can be attached to the plastic and they can remodel the plastic,
00:26:26.07	these are materials where cells are imbedded within the plastic.
00:26:29.28	And this material in particular is very interesting because it can be crosslinked with light.
00:26:35.04	So the way the experiment works is
00:26:37.10	you take polyethylene glycol macromers (so long chains of polyethylene glycol).
00:26:43.19	You mix in cells and a chemical that's sensitive to light
00:26:47.22	and when you shine light you get a network of polymer
00:26:51.17	that looks like this contact lens structure with cells imbedded within it.
00:26:55.09	So these are examples of synthetic scaffolds
00:26:58.27	and one can tune these to have different material properties.
00:27:02.08	They can be inert, like polyethylene glycol.
00:27:05.05	You can modify their chemistry to interact specifically with cells.
00:27:09.17	For example, one can put in peptides that can bind to cellular receptors like integrins.
00:27:15.08	They can have different mechanical properties
00:27:17.08	which we know is important for some cells types.
00:27:19.19	And then they can have different mechanisms of degradation.
00:27:22.04	So they don't all degrade just by interactions with water.
00:27:26.00	Some of them can degrade in very specific ways;
00:27:28.07	for example, by interacting with enzymes that the cells are making.
00:27:31.18	Another example of a kind of scaffold that one would use are the so called natural scaffolds.
00:27:38.11	So I already mentioned collagen earlier.
00:27:40.26	Collagen is one of the most popular natural scaffolds.
00:27:43.29	Hyaluronic acid is another natural scaffold.
00:27:46.13	The SYS material that I showed you is a more complex natural scaffold
00:27:51.00	because it contains all the extracellular matrix molecules that were in the submucosa
00:27:55.16	in addition to the growth factors
00:27:59.03	and other bioactive molecules that would be in that natural material.
00:28:03.25	An example of such complex natural scaffolds that is becoming quite popular these days
00:28:11.23	is an idea that was first described by Doris Taylor's group a couple of years ago
00:28:15.27	and these are whole organs that have been de-cellularized
00:28:19.23	in much the same way that the small intestinal submucosa was de-cellularized.
00:28:23.26	So here what you're looking at is a picture of a heart that's being perfused with the detergent.
00:28:30.12	And you can literally see the cells melting away
00:28:33.13	so that you have what they call a ghost heart at the end.
00:28:37.01	This preserves all of the microarchitecture of the organ that I described to you earlier.
00:28:43.10	You can see here under the microscope that it has a fibrolar collagen organization
00:28:49.14	and what she did in these experiments was re-cellularized this de-cellularized heart
00:28:55.29	using that as a scaffold for tissue engineering.
00:28:59.23	So various groups have now gone on to do this in other organ systems including the liver.
00:29:06.07	And what's exciting about this potentially is one can think about reusing organs.
00:29:12.03	Instead of having to just transplant a living organ from a donor into a patient,
00:29:17.11	one can think about using cadaveric organs as complex scaffolding
00:29:22.03	for cells from some of the cell sources that I described earlier.
00:29:26.00	OK, so I've described the raw ingredients; cells and biomaterials.
00:29:31.01	I won't say much about nutrients except for to say that
00:29:33.24	there is a whole science about how to feed cells in culture.
00:29:39.10	And that's pretty well understood at this point.
00:29:42.19	The next thing I'd like to just touch on briefly is how to fabricate these organs;
00:29:47.15	how to assemble them, how to process them before implantation and
00:29:51.17	importantly, how to preserve them so that one can get them to the clinic at the time of need.
00:29:57.11	OK, so I mentioned earlier that tissues are organized hierarchically.
00:30:03.07	One can see here in this diagram that when one starts at the level of the liver,
00:30:07.23	we have this multi-scale organization where the liver acini are 1 millimeter in dimension,
00:30:14.17	the liver cords here are 500 microns long,
00:30:17.22	and the cells here are about 20 microns in diameter.
00:30:21.07	So it's this hierarchy which tissues engineers have to try and grapple with
00:30:26.07	in the sense that cells respond, actually, only locally to their microenvironment.
00:30:31.07	So even though you're trying to build a large structure,
00:30:34.12	if you care about cells performing the functions of interest
00:30:38.22	and here are some functions that one might care about,
00:30:41.04	proliferation, differentiation, apoptosis, migration, or metabolism,
00:30:46.23	If these are the functions of interest for having a healthy tissue,
00:30:49.29	that we know that the cells are actually responding to their local microenvironment
00:30:54.23	to these kinds of cues; soluble factors, interactions with other cells,
00:30:59.17	extracellular matrix interactions, physical forces, and cell shape.
00:31:04.24	So the challenge for the tissue engineer is to maintain the cellular microenvironment
00:31:09.25	that will impact the cell behavior on the sort of 10 to 100 micron length scale
00:31:15.12	but simultaneously be scaling up the structure
00:31:18.06	to be a large enough body of cells to implant to achieve a therapeutic outcome.
00:31:23.17	So a good example of how cells have local microenvironmental interactions
00:31:30.12	that one calls 3-dimensional in the field
00:31:33.09	is cartilage.
00:31:34.09	We see this is cross section of articular cartilage
00:31:37.17	and if one looks at cartilage one sees that the chondrocytes
00:31:40.23	that make the extracellular matrix that makes up cartilage,
00:31:43.26	have different organizational structures.
00:31:46.09	So here we can see that they are in these columnar structures.
00:31:50.06	Whereas, in the superficial layer of cartilage that sits right next to the joint space
00:31:54.25	they actually are flat, elongated and in clusters.
00:31:58.24	So if you were to look down at the cartilage, you would see that they were in clusters.
00:32:02.18	So, it's been known for quite some time now that chondrocytes in culture,
00:32:09.07	when one puts them on plastic of any type for tissue engineering purposes
00:32:12.28	they will actually spread out and lose many of these functions
00:32:17.14	They will start proliferating and stop producing the extracellular matrix
00:32:20.25	that one is trying to recapitulate in a tissue engineered construct.
00:32:24.23	And so, in fact, if one embeds them in  a 3-dimensional microenvrionment,
00:32:28.24	where they have this round shape and more favorable mechanical properties,
00:32:32.23	one stimulates them as they are stimulated mechanically in vivo,
00:32:36.18	one can actually improve the quality of the extracellular matrix that they produce
00:32:41.23	and therefore the mechanical strength of the graft.
00:32:44.26	So this is just one example of a tissue where
00:32:47.11	the 3-dimensional organization of the microenvironment,
00:32:50.14	in addition to the chemical composition of the microenvironment is very important.
00:32:54.19	And one will hear a lot about 3D tissue engineering in years to come.
00:32:58.09	One can then think about how to assemble these microenvironments
00:33:03.18	where cells have very precise, local chemical and mechanical cues into larger structures.
00:33:09.12	There's a set of technologies emerging as 3D fabrication technologies
00:33:14.05	that have come out of the rapid prototyping field
00:33:17.23	that are starting to be borrowed in tissue engineering.
00:33:20.10	Some people call this organ printing.
00:33:22.12	This is an example of a couple of parts that were made in a layer by layer fashion
00:33:27.17	known as stereolithography
00:33:29.28	and stereolithography is one example of one of these rapid prototyping technologies.
00:33:34.23	So, in this technology, what's done is
00:33:37.28	a 3-dimensional drawing is drawn on the computer and sent to this robot.
00:33:43.14	And the robot has a movable stage that sits in a vat of light sensitive material.
00:33:50.16	And in every layer of the material, light is shone in a pattern.
00:33:55.21	It polymerizes and the stage drops and the next layer is built and so on and so forth.
00:34:00.28	And using this, one can create complex structures; some that are actually biomaterial structures.
00:34:06.06	Some folks envision that one could make personalized tissue engineered parts
00:34:10.26	using a technology like this because one can envision
00:34:13.09	going from a 3-dimensional medical imaging data like CAT scan data, to tissue engineered parts.
00:34:19.13	
00:34:24.02	Another thing that's required is to culture these tissues before they're implanted
00:34:28.18	and these are a couple of examples of the kinds of bioreactors that are designed and used in the field.
00:34:34.06	At the top what you see is a quite well known bioreactor developed by NASA.
00:34:39.06	This is a circulating bioreactor and it never allows the cells to touch the walls of the bioreactor
00:34:47.02	so they're forced to aggregate in 3-dimensional clusters.
00:34:49.29	And this has been shown to promote tissue formation in both cartilage and heart cells.
00:34:55.01	Furthermore, techniques from the computer chip industry
00:35:00.13	are now being borrowed to create micro-reactors.
00:35:03.22	So here what you're looking at is a coverslip and some inlet ports from a microfluidic device.
00:35:09.18	And if one looks in the microscope, inside that coverslip,
00:35:12.19	one can see these little tissue units that have microstructure,
00:35:16.24	that have been fabricated in the computer lab at 100 micron length scale.
00:35:22.06	So they have 3-dimensional microenvironments
00:35:25.01	that have been specified in a scalable fashion for building larger pieces of tissue.
00:35:30.17	So, one can assemble tissues, one can culture these to mature them in the laboratory,
00:35:37.08	and then finally one needs to think about how to preserve them the deliver them to the patients.
00:35:41.16	So, there's a whole set of technologies that have grown up in the field
00:35:47.06	about cryopreservation. I should say that not all tissues are frozen.
00:35:51.18	Many are actually just refrigerated to 4 degrees C
00:35:55.06	and in fact, the skin tissue engineered product that I showed you earlier
00:35:58.02	is sent that way to the clinic.
00:36:00.13	However, in the field of cryopreservation,
00:36:03.15	which most of us agree will be important to really make this technology have it's highest impact.
00:36:11.03	There has been a lot of research on how best to make cells, especially in 3-dimensional structures,
00:36:17.10	survive the insults of cryopreservation.
00:36:19.18	So, the insults of cryopreservation have now been categorized
00:36:23.21	into two classes of insults and those are depicted here.
00:36:27.03	So I need to walk you through what a cell feels as it gets frozen
00:36:31.14	so you can see what the two classes of insults are.
00:36:34.25	So if you have a cell here and it's been cooled just under the seeding point of ice,
00:36:40.03	and one artificially seeds external ice in the environment,  two things can happen.
00:36:45.15	If one slowly cools the solution further, you can see that water has time
00:36:52.03	to cross the cell membrane and leave the cell and form ice in the extracellular space.
00:36:57.25	And this causes what people call dehydration injury to that cell.
00:37:03.07	You can see that its shape has shrunken. There is a concentration of the intracellular solutes.
00:37:09.19	And those cells when they are thawed again, exhibit damage from this dehydration process.
00:37:15.13	If instead one takes this solution and rapidly cools it,
00:37:20.03	one actually finds that you can have intracellular ice crystal formation.
00:37:25.02	The formation of intracellular ice crystals, in and of itself, can cause damage to cells.
00:37:30.10	So the process by which cells are susceptible to slow cooling or fast cooling
00:37:35.11	turns out to be cell type dependent and dependent on various additives in the system
00:37:40.21	as well as the 3-dimensional architecture of the tissue.
00:37:44.01	And so this is being worked out now for many, many cell types in the field.
00:37:47.18	OK, so to conclude, I've told you that engineered tissue replacements can
00:37:52.15	be made from combinations of cells and biomaterials to replace a subset of tissue functions.
00:37:58.17	I've told you that the cells can be derived from somatic cells or stem cells.
00:38:03.06	I've told you that the biomaterials in these tissue replacements can be natural or synthetic.
00:38:09.02	I've also told you that tissue structure in the body is hierarchical
00:38:13.18	and therefore the tissue engineer has the challenge of maintaining the cellular microenvironment
00:38:18.27	in a way that can be scaled up to build a tissue that's large enough for therapeutic impact.
00:38:25.03	And finally, I hope you've noticed that the convergence of
00:38:28.20	cell biology, medicine and engineering is really advancing this field.
00:38:33.11	In part two of my seminar, I"ll be telling you about our work on liver tissue engineering
00:38:39.05	both in the progress we're making towards therapeutic goals like the ones I've described,
00:38:44.18	as well as progress that we're making in using these engineered liver tissues
00:38:48.20	to advance scientific discovery.
00:38:51.02