9UNP0172SD_William_Murphy_WordPress

- Thanks everybody for coming today and thanks to the organizers of DART as well. I think this is a wonderful idea for a program and I hope it spreads across the country and ultimately across the world. This concept of giving talks that are streamed virtually around the world. What I'd like to talk about today is some of the approaches that are being developed with the ultimate goal of engineering tissues using stem cells. And I know that in this series in the past you've had some biologists speak, and you've had a few different perspectives. I'll try and take a different angle today and tell you a bit about some of the challenges associated with tissue engineering using stem cells and try and describe a bit about how we're trying to address those in my lab as well. So I'll start out just by describing the need for tissues for transplantation, and the need in short is tremendous. If we look for example at the number of transplants that were performed in 2006, in these areas in particular approaching 30,000 transplants. And the number of patients that remain on the waiting list for organs, about 95,000 patients. 

There is this large disparity in the numbers. This has been widening over time, so this separation between the number of organs available and the number of people on the waiting list continues to diverge, so there is a real necessity to create new strategies to replace tissues and organs. This is a graphic that actually goes back to 1998, and this was maybe the peak of the excitement that surrounded the early stages of tissue engineering where the concept was that essentially any tissue or organ could be re-created using combinations of cells, materials and molecules. I think that hope still exists today, and I don't want to go in too much detail into what the potential of the field is because I don't think it takes very much imagination to come up with what this field can do. In almost every disease state in the body and almost every tissue type and organ type in the body there is tremendous potential for tissue engineering to solve problems that haven't yet been solved. So the organ transplant diversion that I just described, or the general concept that there's this widening gap is just one of several examples where tissue engineering can be a very important field. But there are a series of very significant challenges that limit the potential of the field as well. So what do we mean when we say tissue engineering? As a broad definition, it has been defined as an inter-disciplinary field that applies the principles of engineering and life sciences toward development of biological substitutes that can restore, maintain or improve tissue structure. So this is a field that is interdisciplinary. 

It involves a lot of different concepts coming together to try and solve some of these problems. And if we think about it in a rather cartoonish way -- so if we start with a patient and the concept is we would isolate a biopsy from that patient, isolate and expand cells from that biopsy, seed those cells onto a scaffolding material that ultimately degrades over time resulting in formation of a new tissue that can then be transplanted into a patient. What we have here is sort of the pie in the sky goal of tissue engineering, the concept that a patient's own cells can be used to create transplants. There are challenges associated with essentially every arrow in this cartoon, and every arrow has got multiple investigators and multiple fields really working on trying to solve problems. So, for example, the source cells -- expansion of source cells, procurement of source cells for tissue engineering purposes is a significant issue. Designing scaffolds that can promote new tissue formation is a tremendous issue and one that we work on in my lab in particular. Identifying factors that can induce stem cells to do something specific, to make a bone tissue, a skeletal muscle tissue, etc, is an important area as well. And then just the pragmatic issues -- being able to biopsy tissues, being able to preserve those biopsies if needed, being able to effectively isolate stem cells in a pure form so that they can then be used for tissue engineering purposes -- is a tremendous issue. So all of these arrows have got challenges associated with them. And if we think about what's inherent in each one of these steps in the process, what we have to think about in each step in the process, we can come up with a pretty significant list of things to consider. 

So if we start on the left side of the graph here with source tissue we have to think about procurement, we have to think of storage. If we then go to the idea of being able to culture and expand the cells, we have to think about cell culture conditions that will maintain stem cell character or provide environments that promote differentiation in some cases. We have to think about generating bio-reactors that are scaleable so we can create large amounts of either stem cells or differentiated cell types. We have to control the environments for those purposes as well. We have to think about selecting and purifying stem cells from a source and then we also have to think about cell delivery and homing. So if those cells are going to go directly back into the patient, we have to think about actually how we deliver them, how we get them to their target area. The next step in the process, the concept of being able to create materials that promote stem cell function, we have to think about bio-compatible materials, materials that degrade over time, materials that interact with stem cells and then materials that have mechanical properties that are conducive to the environment where they're going. You wouldn't want to put a material with the consistency of jelly into a bone in most cases because it wouldn't be appropriate. If we think about then inductive molecules that can affect cell behavior on the upper right, then we have to think about stability of those molecules, the fusion of those molecules through a construct or through a developing tissue and then we have to think about ligand receptor binding so some of the kinetics that have been well studied in typical cell culture conditions but not so much in the context of tissue engineering constructs. And then during formation of the 3D tissue, we have to think about 3D cell culture, cell-cell interactions and tissue development phenomena that are fundamentally different than typical cell culture conditions on polystyrene. 

Once we have formed a tissue that we might want to then implant into a patient, we have to think about survival of that tissue, the surgical methods that are appropriate -- so we need clinicians' input to figure out what's practical. And then we have to think about issues like immune rejection down the line. So there are daunting challenges but a great deal of potential and a tremendous importance to the field. So where are we in tissue engineering and where have we been? So if we think about sort of the history of successes in tissue engineering, I think they can be represented fairly well by these three examples. So in each case the concept involves combining a mature cell type with a material carrier and then placing that cell material construct in vivo to promote new tissue formation. So skin regeneration is a good example. The company OrganoGenesis had success in seeding cells onto a three-dimensional scaffold and promoting new tissue formation. Cartilage regeneration as well, injection of chondrocytes into cartilage defects to try to promote new cartilage formation has been an important area. And then one of the more high profile successes from Tony Atala's group involved urinary bladder regeneration in this case seeding two different cell types onto a material construct and allowing an organ to form over time in vivo and actually a functional organ so in this case a dog bladder that ended up being functional in vivo. 

Now the common thread, one could argue in these success stories is that these are relatively simple tissues, at least in comparison to some of the more complex tissues that haven't yet been successfully engineered. These tissues tend to be thin, so they don't require a functional vasculature in the early stages of development. Cartilage is poorly vascular anyway. And these cell types don't include a very large number of different cell types, they're not broadly heterogeneous, so they're the sorts of tissues that one could imagine would be the first successes in tissue engineering. And it doesn't belittle the successes that we've seen, it just indicates that there is a long way to go in some of the more complex tissues. I want to point out here that these success stories really contrast with natural tissue development. So in the case of combining mature cell types and material carriers, the general concept is that you're taking cells that are capable of doing something like forming skin, and you're placing them in an environment and allowing them to essentially do what they do. In tissue development it's quite a different phenomenon. So typically in tissue development, you start with precursor cells that haven't yet been specified as to what they're going to do. They're exposed to environments that then promote their differentiation down a particular lineage or promote particular functions, so it's a really complex process that's highly controlled in time and space where the extra cellular matrix and other cell types that surround a stem cell type are instructing its behavior over time and in specific locations. 

So there is a real contrast between these two concepts. And if we are to extend beyond into some of the more complex tissue and organ types there is a likelihood that there are lessons we can learn from tissue development. So what are the critical questions in trying to develop emerging tissue engineering approaches? Maybe the first question is what is it that drives tissue development and regeneration in natural systems? This is a nice template, so obviously natural systems know how to make tissues very effectively. We can ask the question, can we mimic what they do? And the answer in many cases is stem cells. So, stem cells in vivo during early development as well as regenerative processes are really the workforce in creating new tissue types. This is an example of human mesenchymal stem cell that are cultured on tissue culture polystyrene, but there are several other cell types that are present in vivo that promote development and regeneration. The second question -- given that stem cells are quite powerful, are capable of forming new tissues, the question becomes what makes a group of precursor cells or stem cells generate a functional new tissue? 

And this is where we have yet quite a lot of questions to answer, not just in terms of tissue engineering but also in terms of just fundamental tissue development. We do know though what some of the major players are. So what are some of the major factors that promote particular stem cells functions like differentiation or self-renewal? One class is inductive soluble signals like growth factors. This is an example of fibroblast growth factor-2 which promotes proliferation and differentiation of a variety of stem cell types. Another example is cell adhesion signals, so signals that cells see as components of their extra-cellular matrix. So, as cells bind to their ECM, they interact with these signals and it effects their internal structure and ultimately their behavior. And then cell-cell signaling. This is an example of an eight cell stage embryo, and you can see it's difficult to distinguish individual cells. At the eight cell stage the embryo compacts and you get upregulation in cadherens junctions for example and you get enhanced cell-cell signaling. 

And that has important functional consequences during even the eight cell stage of embryonic development. So there is a likelihood that cell-cell signaling is going to be really an important set of signals to try and control stem cell behavior. So, we know what some of the major players are and what we're trying now in the field to figure out is what are the more specific parameters from tissue development that we can use. So we know that soluble signals are important. We have to know what is the identity of the soluble signal that is important. What are the appropriate timing regimens and spatial regimens to expose stem cells to these signals? And how is it done in natural development? Okay, so I mentioned sort of the history of the field and some of the successes that we've seen. Where we are now largely involves sort of a logical extension on what's been done. So what's been done combining mature cell types and material carriers, what's being done now involves combining stem cells types and material carriers. 

And for lack of a better word, seeing what happens. Essentially, seeing how cells behave when they're exposed to a three-dimensional environment. A couple of key examples here, one from Bob Langer's group at MIT involve seeding human embryonic stem cells into plastic scaffolds that degrade over time. What they found was these cells are capable of differentiating down all three germ layers, all three germ lineages and they found that growth factors that these cells are exposed to actually influence their differentiation. So this was an encouraging step toward embryonic stem cell based tissue engineering. A second example comes from Kristy Anseth's group at Colorado involves seeding human mesenchymal stem cells into hydrogel matrices and maintaining viability and starting to look at differentiation down mesenchymal lineages, skeletal lineages like cartilage and bone lineages. There are also tissue specific scaffold materials that are oftentimes used with stem cells. Hydroxyapatite based materials in bone are a good example. Hydroxyapatite is the major mineral constituent of bone, so it's a logical material to use as a scaffold for regenerative medicine purposes. And stem cells have been seeded into these kinds of materials. 

The example shown here is from Scott Hollister's group at the University of Michigan. So this is sort of where we are, and where we're headed really involves trying to as I mentioned determine what are the signals that stem cells need to see, and how are they most effectively presented to stem cells to recreate the processes of natural development and regeneration. So I mentioned before the definition of tissue engineering and some of the things we need to think about. If we then go a step further and think about stem cell based tissue engineering, there are some additional things that we need to think about or some things that we need to emphasize. And so the things that are boxed here are sort of additional issues or particular issues that we don't necessarily think about in standard tissue engineering practice but that really become quite important when we talk about stem cell based tissue engineering. I'm not going to go through all of these but if we just look for example on the upper right, initially the concept behind inductive soluble signals, proteins like growth factors, involves understanding their stability, their diffusion and ligand receptor binding. With stem cells we also have to think about mitogenic effects, so are the stem cells proliferating, if so are they maintaining their stem cell character as they proliferate. Inductive effects, what lineages are these factors potentially sending the cells toward? Controlled timing, so in early development the timing of growth factor exposure becomes very important, spatial control, so in different parts of a developing organism or regenerating tissue, you typically have up regulation of very different sets of signals, so trying to re-create that process. And then crosstalk, so there is the possibility that a cell can be exposed to a factor that would promote its differentiation except that there's another factor being secreted by an adjacent cell that either inhibits that factor or somehow otherwise affects that factor. 

So, the general theme here is enhanced complexity, and the need is for enhanced control, the ability to control engineered systems to promote tissue regeneration. So if we re-state these challenges in the context of a classic paper on the subject, Bob Langer and Jay Vacanti published a paper in Science in 1993 called "Tissue Engineering" that reviewed sort of what had been done up to that point and what the potential of the field was, what the challenges were. And so they came up with a list of challenges, so this is adapted from their paper but there are a series of challenges, they listed about 13 challenges in their paper. So what controls new tissue growth? How do ECM components affect cells? How can we address immune rejection? How can the cells and tissues be preserved? How do we solve nutrient and transport issues? How do we model tissue regeneration, or do we need to model tissue regeneration? How do we predict events that are going to happen in vivo after we take a cell material construct and put it in. 

How do we design large scale cell cultures to expand? Natural materials -- are there batch to batch variations in these materials? Is is required that we use synthetic materials, and if we do use synthetic materials, are there biological interactions we have to think about? Can we process porous support scaffolds that allow for new tissue growth? Can we develop sustained release systems and where do we get the cells that will be used for tissue regeneration? And so if we then extend out -- so these are sort of the old challenges from about 15 years ago, if we extend out to the current challenges in the field, many of these challenges remain, but they maybe are recast a bit. So what controls new tissue growth remains a significant challenge. How do ECM components affect cells remains a significant challenge. A variety of them actually maintain -- remain significant challenges. So these challenges are all still quite significant. 

But one of the challenges that is potentially addressed by stem cell engineering is this challenge, so where do we get the cells that will be used? That answer is coming from the stem cell area, so there are enough now stem cells available that one could envision that we have the cells that we need. If only we know how to manipulate them. And so a new set of challenges has sort of popped up based on the use of stem cells, how do we combine signals in bioreactors, how can we mimic tissue and organ development processes, how do we combine multiple cells and tissue types and then how do we manipulate stem cells in vitro and in vivo. So if we can solve some of these challenges then I think the potential of the field is almost unlimited to treat the maladies that affect people today. So one way of viewing the field in its current state is that we can get stem cells into materials and we can create materials in which there is a homogeneous population of stem cells initially, and what we'd like to figure out is how over some time frame do we promote formation of a desired tissue structure and function where the ultimate goal is to create a tissue that isn't homogenous but is heterogeneous in its function, in its structure, in its cell types, in the signals that are present, etc. So, the signals that are cartoonishly represented on the right here, these green and red signals that cells are exposed to are what we need to figure out. So the question is how can one use materials to promote stem cell based tissue regeneration. This is the question that we address in my lab. So instead of describing to you what the potential of the field is, what I'd really like to do is describe what the major challenges are in the field because I think it's an important time to be talking about some of these challenges and some of you may be the people that ultimately do solve these problems. 

So I'll describe three different really major challenges in the field. I'll try and describe a bit about why these are challenges, how they're being addressed and specifically how my lab is starting to address them. And I'll describe a little bit of our data for your interest. So the first challenge involves throughput. What I mean by this is that in tissue engineering experiments in academics and industry, the typical concept is to run one experiment and get one result. So if the question is how does a particular extracellular matrix molecule influence differentiation of a particular stem cell type, then that's the hypothesis. How does that one factor affect stem cell behavior? And that's -- answering just that question, that very simple question is time intensive, it's labor intensive. The hypothesis that's being addressed is very limited. The complex hypotheses are very difficult to address using that kind of a mechanism, and it's very different than a -- it would take a very long time to get to mimicking tissue development this way because during tissue development there are tens or hundreds of different really important signals that cells are being exposed to. 

If we start to address one signal at a time it's going to take us a very long time to figure out what the really important environments are. So there's a real need for higher throughput so that we can develop systems in which we can answer several questions at once, address multiple hypotheses simultaneously and start to accelerate toward finding environments that promote tissue regeneration. So standard cell culture has figured this out. People can do high throughput experiments in standard cell culture and these are a couple of examples from the relatively recent literature. One is from Bob Langer's group and this is a paper that was published in Nature Biotechnology in which they basically spotted out 1,700 different materials onto a surface. They seeded stem cells onto that surface and characterized essentially what influence that material substrate had on the stem cells. So this is a two-dimensional substrate coated with a material, so it isn't three dimensional in the sense that we've described thus far, but it's a lot of interactions to screen at once, so they're answering a lot of questions. Similarly with soluble molecules one can seed stem cells into high throughput culture conditions and expose cells to a wide variety of molecules. So this is Peter Shultz and others at Scripps screening 50,000 soluble molecules in a single set of experiments to identify a small molecule that promotes self renewal of the cells. So this is terrific and there's a lot of questions that can be answered this way in two-dimensional culture. 

One of the questions that we and others are trying to address is how do we then scale this to a three-dimensional system that might be more relevant for engineering a tissue? So this is one concept of trying to define the stem cell microenvironment using materials and trying to create materials in an array-based format. So one can think about the concept of having spots in a hydrogel or some other three dimensional matrix in which each spot contains a different complement of signals. So these spots that are being represented in red here would contain a particular type of soluble factor like a growth factor and a particular type of adhesion ligand that might be derived from an ECM protein that cells can bind to. It might also contain a particular type of cell and a particular density of cells. It may also degrade in the controllable time frame, so there are all kinds of variables that can be affected in particular spots. And then a second complement of spots can contain a different type of set of signals. So a different soluble factor, a different cell adhesion ligand, a different degradation time frame, a different cell density, a different cell type, and we can then start to ask a wide variety of questions. So what one might consider is what do we try to control in systems like this? Well the first is cell adhesion. 

What cells are bound to is very important in affecting their fate. The second is degradation, so the ability of cells to remodel their extracellular matrix dynamically over time is really vital to regenerating new tissues. And then the third is soluble signals, and it's very clear based on a huge body of literature that soluble signals are important in promoting both self renewal and differentiation of every stem cell type that we know of. So the way that we've been addressing this in our lab involves creating hydrogel arrays in the format sort of similar to what I described cartoonishly on the previous slide. So this is an example of a hydrogel array on the upper left here. This is a three-dimensional gel. It contains 16 spots. Each spot is filled with an automated liquid handler, so this is a high throughput system. We can address a lot of conditions in a single experiment, and on the right what you see is a 24 wall plate that's completely loaded with these hydrogel arrays. So this is a single portion of a single experiment. 

It's not even an entire single experiment. So you can see how quickly you can scale up to ask thousands of questions in an array format, yeah. 
- Tell us a little bit about what a hydro -- a hydro matrix. What is that? 
- What the material is? Sure. Yeah, sure. So these are hydrogels which means that it's a three- dimensional network of polymer molecules, and it's highly hydrated, so one way of thinking of it is as analogous to a natural protein matrix in vivo. So most or many protein matrices in vivo can be characterized as hydrogels. They're hydrated and they form sort of a network structure. 

In this case, the material that we start with is a polyethylene glycol based polymer. We've worked with a variety of polymers, but this particular one is polyethylene glycol. The advantage of that is maybe not an obvious one. We choose this polymer based on its lack of interaction with proteins and cells. They just don't interact with it very strongly at all. The reason we're interested in that is because from an engineering perspective this is as reductionist as you can get. We're starting with nothing and anything that we present on this backbone is something that we can address very specifically. So if we want to look at the effects of an adhesion molecule, we're only looking at those effects. We're not looking at a thousand other things going on. That's a very good question. 

Other questions at this point? Before I move on? Okay. So we can get up to hundreds or thousands of conditions using this kind of an approach and a few of the things that we've been able to vary without too much trouble. One is the cell density. So this is what you're seeing is a live dead stain. Green fluorescent staining indicates cells that are alive and red fluorescent staining would indicate cells that are dead. And you can see that if we change the seeding density in cells per mil included in these wells, as you increase the seeding density, you increase the number of cells that are present in the well, so it's a predictable relationship, and it's a pretty linear relationship which is what we would expect. So we can control cell density. We can also control the presence of soluble signaling. 

So one way we've been doing this is by presenting molecules in this gel construct that bind to proteins. Since they bind to the proteins they effectively sequester those proteins in the local environment, so they do what the ECM often does. The ECM can sequester growth factors in local environments and affect signaling, so we're trying to do that as well and what you're seeing here is fluorescently tagged ligends on the top, the blue and the green and fluorescently tagged proteins on the bottom that are binding to the same places where we include the ligands, so we can colocalize the ligand and the protein as we'd expect for a sequestering interaction. So what we can do with systems like this is not just look at single -- the effects of single factors on cell behavior but we can also look at the effects of multiple factors combinatorily, seeing how they effect cell behavior, and we can start to identify conditions that have been difficult to identify in the general literature. So if you look here on the upper left, what you're seeing are conditions where the DTT represents a degradation, so we include -- these gels are capable of degrading over time. It's not important basically what the DTT specifically means, but essentially it means that the gels are degrading over time. So on the upper left what you're seeing is low concentrations of two different cell adhesion ligends. RGDSP is a peptide sequence derived from fibernectin which is an ECM protein. An IKVAV is a peptide sequence derived from laminin, another ECM protein. So if we expose the cells to those factors, we actually see very good viability. 

The cells survive quite well. But they don't spread out. The cells are rounded up. They don't interact with the material very strongly. They're not -- they don't look like they do in two-dimensional culture, they essentially look like they're confined. If we then add higher concentrations of both of these ligends on the bottom here, you can see that the cells are starting to spread out, starting to interact with their local environment and then if we add a soluble factor, fibroblast growth factor-2 on the upper right here you can see that the cells are now starting to spread out more significantly. You can see them sending out extensions. And this is something in synthetic gels that you just don't see very often. So identifying these particular concentrations of multiple signals and combination as you can imagine would be very difficult to guess. It would be very difficult for us to guess which concentrations were going to work, but because we have this high throughput screening format we can identify which conditions are appropriate. 

So this general concept extends to other potential applications. A second challenge that I want to describe is one that really a lot of investigators in tissue engineering are starting to work on and that is the idea that there is very poorly controlled signaling in standard tissue engineering constructs. So what you're seeing here is a mammalian embryo at day seven and you can see that this is a tremendously complex organism even at day seven during early development. Spatially in different parts of the embryo you have different cell and tissue types forming, organizing, interacting with one another. You have heterotypic cell-cell interactions taking place and this is a snapshot at day seven. If you were to look at the same embryo at day nine you would see an entirely different sort of organization. So time and space are very important during development. Very similarly, time and space are likely to be important during regeneration of organs. So in contrast, typical tissue engineering constructs don't localize signals in a way that mimics natural signaling. It's just very difficult to do. 

It's also difficult to turn signaling on and off in a predictable manner in time. You're working typically against natural regulation of signaling. So even if you're upregulating signaling, it could be that the cells are becoming desensitized to that signal, so it's a difficult dynamic process to deal with and the developing tissue is a moving target. So what you don't want likely is one factor being upregulated in one spot in a developing tissue for the entirety of tissue development. You'd like to be able to really highly control things. So this is really a tremendous challenge and one that has no clear solution at this stage. What engineers have typically done is use engineering technologies to build up materials layer by layer that have different properties at each layer, so this is a mechanism for trying to pattern tissues during early development and trying to pattern stem cells into materials so that over time the concept is that each layer contains a different set of signals that cells can be exposed to. I'm not going to go through each of these processes but some of them are really very familiar processes, so heat molding, the idea of essentially spot welding portions of a material together. Inkjet printing is becoming a very common mechanism for creating materials. Photo encapsulation of cells, so the idea that you can shine light on cells in a solution and it solidifies around the cells in response to light. 

And then pattern encapsulation again the idea that you've surrounded cells with a polymer environment and it either self assembles around the cells or somehow the cells assemble the material. So there is a variety of engineering technologies that are being used. But part of the issue here is that you're really only patterning things at day zero. As you go down the line, there's not yet a mechanism for controlling timing as well. So a significant challenge. So what I'd like to describe a bit about is some things we've been doing to try and address this issue that might be generalizable to other material types, but we've started with hyroxyappetite substrates because these are very important in bone tissue engineering. So the general concept is shown here. If you can imagine that this entity here is a stem cell and these receptors on the surface are binding to external ligands. So this blue receptor would represent a cell adhesion receptor like and integrin receptor. This purple molecule would represent a growth factor receptor, in this case a receptor for bone morphogenetic protein-2 or BMP2. 

So the idea here is that we engineer a growth factor that has two functional components. One portion of the growth factor binds to the material and the other portion of the growth factor binds to the cell surface receptor. So that then if this molecule is coded onto the surface or painted onto the surface or spotted onto the surface, that portion of the surface retains the activity of that growth factor. So it's a way of spatially patterning growth factor activity on materials. So how do we do this? This is a bio-inspired mechanism. There is a protein that's present in the body called osteocalcin that binds very strongly to hyroxyappetite mineral and there is in particular on the N-terminus of the protein a nine amino acid sequence that contains three carboxy glutamic acid residues. And those are the residues that primarily promote binding to hyroxyappetite. So our idea was to essentially pull that sequence out, link it to a growth factor and see whether we could get a growth factor that does two things, has biological activity and binds to cell surface receptors and also binds to a material surface. So the protein we were interested in is bone morphogenetic protein-2. 

The reason we're interested in this is really twofold. The first is that it induces osteogenic differentiation of mesenchymal stem cells, these are stem cells that are isolated from bone marrow. Osteogenic differentiation means that these cells are differentiating into osteoblasts that form bone, so there is evidence for this in the literature, some of which is shown here. This factor also promotes bone formation at both ectopic and orthotopic sites meaning both sites in a bone defect and sites away from bone. So you end up getting bone formation in vivo in response to this signal. And this is important because in some cases in tissue engineering we can think about trying to incorporate both cells and molecules into a material construct to promote regeneration but we'd like to also be able to promote regeneration in vivo in the absence of stem cells so trying to promote essentially regeneration by native stem cells in the body is an important mechanism as well. Okay, so we have these growth factors that we synthesize and this isn't really an important slide, just indicates that we've generated a library of engineered growth factors that contain this active sequence and mineral binding sequences and two of these mineral binding sequences we've named E3-glue and E3-gla, so E3-glue essentially is -- contains the growth factor component but contains a mineral binding component that doesn't bind very strongly at all. E3-gla contains both functional components and so if we spot these onto a hydroxyapatite surface, a mineral surface that's shown in the upper left here, you can see that the E3-glue doesn't bind in any appreciable way to this surface. We don't see very much of it on the surface whereas the E3-gla does bind and if we spot it out and then wash it copiously we actually see that the molecule doesn't come off the surface so this is a nice mechanism for dip coating or spot coating materials with a growth factor. So who cares right? 

Is the molecule actually active, does it matter? So our next step was to try to figure out is it active on these surfaces and so we did some experiments looking at osteogenic differentiation of human mesenchymal stem cells so these are stem cells that are isolated again from bone marrow and if we look at -- if we compare control surfaces that contain no peptide at all, surfaces that contain the E3-glue peptide that doesn't bind very strongly and surfaces that contain the E3-gla peptide that does bind strongly, you can see that there is an upregulation in alkaline phosphatase activity in alizarin red S staining which stains for mineralized tissue. So these are two hallmarks of bone differentiation. There is also upregulation in secretion of native bone morphogenetic protein-2 by the cells. There is an up regulation in osteocalcin secretion by the cells which is again a bone marker. And if we look at the expression of osteocalcin as well we see a significant increase in osteocalcin expression in response to that molecule when it's immobilized. So the short take home message from these data are that the growth factors remain active when they're immobilized on the surface, so it creates a really nice mechanism to control local growth factor activity and potentially to spatially control differentiation of stem cells. And so these are just a few examples of how one might consider spatially controlling the stem cell, or the peptides on the surface. So I already showed the spotting on the lower left. On the lower right, this is literally taking a paintbrush, dipping it in the peptide and writing a "W" on the surface. 

And you can see that it sort of looks like a "W" at the end of the process. So this is a rather crude way of showing that an orthopedic surgeon could literally dipcoat an implant, load it with stem cells and get bone differentiation potentially in vivo. So another sort of bio-inspired mechanism for controlling spatial -- spatially controlling new tissue formation involves growth factor gradients, so any of you who are developmental biologists are certainly familiar with the concept of spatial gradients and growth factor activity. They're really ubiquitous during early development and effect development of just about every organ type in the body in a very significant way. Here what you're seeing is data from John Fallon's lab actually here at the University of Wisconsin, and what they've done is they've resected the apical epidermal ridge from the limb bud during early development in the chick model. And so what you can see is in the upper left, in the case where they resect the ridge, they get poor development of the limbs, so the limb becomes truncated, it doesn't fully develop. If they then replace the ridge with a bead that releases fibroblast growth factor-2 or FGF2, you can see on the upper right that they get better development of the limb if they replace the ridge with two beads that are each releasing fibroblast growth factor-2 then they get near fully development of the limb, a much better development of the limb. So what's clear here is two things. One is that on the upper left it's clear that the ridge that releases fibroblast growth factor-2 in typical scenarios during development is necessary to promote full limb formation during budding. So you don't get full regeneration of the limb in the absence of the AER. 

What's also obvious is that if you replace just that function of the ridge -- not all of the functions of the ridge but just the ability to secrete fibroblast growth factor-2, then you essentially replace, you can replace the full function of the ridge. So this kind of gradient is vital during early development and also can potentially be replaced. So this is a motivation for developing materials that can essentially do the same thing. And that's one of the things that we've been working on in the lab. So here what we've done is we've created materials that contain specific regions that release growth factors, so these particular regions that are shown in gray here are releasing growth factors again into a hydrogel, so these hydrogels are similar to the ones that I described earlier. And this is a top view of what these look like so on the left here is where we're releasing the protein and on the right is the area where cells would be. And if we look at sort of a heat map of protein concentration going away from the source, you can see that there is a spatial gradient, so there is a very high concentration close to the source and as you go away, because you're diffusing away from a source, you're getting decreases in concentration with distance. So this is a mechanism for sort of mimicking what happens during early development. And by varying the properties of the material that's releasing the protein as well as the hydrogel construct, you can control then the shape of the gradient. So these are examples on the right where the slope of the gradient and sort of in general the persistence length of the gradient ultimately also the persistence time of the gradient can be very highly controlled by changing the material properties. 

So this is something that engineers can do, sort of controlling specific parameters that are associated with in vivo gradients. Now one of the issues here comes back to what we described earlier. If we get to the end of this process and we can control the parameters associated with the growth factor gradient, the next question is what do we use it for? So what type of gradient would we want to expose stem cells to? And this is a complex question. It's not an easy question to guess the answer to so if we basically say well why don't we use a gradient that is about ten nanograms per mil to one nanogram per mil and it's over about 500 microns. Now that's a guess, so we don't know that that's going to have an impact. And typically the in vivo gradients that have been characterized haven't been characterized in a way that's useful to engineers. So there is sort of a qualitative representation of how the gradient dissipates over distance but it's not quantified in a way that would be helpful in designing these kinds of materials. So we're starting to apply this concept to the high throughput cultures described earlier to try to ask a lot of questions at once related to the effects of growth factor gradients. 

And this is just one example with fibroblasts that respond to fibroblast growth factor-2 if we put them in a spatial gradient of fibroblast growth factor-2 they can respond to it and you can see that on the left here where they're at higher concentrations of the growth factor there are more cells alive, so the green represents a lot cells that are alive, and as you move to the right you have less cells that are alive, so it's preliminary evidence that these kinds of gradients can be active and effect cell behavior, so a lot of potential directions to head in from here. Okay. The final challenge that I'll go through quickly because we're running out of time involves transport, so mass transport in developing tissues is a huge challenge. What's very common is that you get substantial cell death if you're more than about two hundred microns from the boundaries of a scaffold. So what you're seeing here is a modeling representation of oxygen concentrations going away from the surface of a scaffold into the center of a scaffold and you can see that oxygen drops off really quickly, so cells, this is the major reason why cells don't survive as you get deep within a construct. So there's a need for a surrogate vasculature if the idea is to try and form the tissues ex vivo. So there has to be something that acts like a vascular supply outside the body to feed stem cells as they develop a new tissue. If the idea is to try to form the tissue in the body then there is a requirement that you get vascular tissue to grow in very quickly. It has got to happen over days not weeks if the idea is to try to develop the tissue in vivo. So vascular engrowth is a substantial issue. 

And there are a variety of methods that are potentially available for vascular tissue engineering. Many of them really rely on natural development of vascular structures and sort of mimicking natural development of vascular structures. And I don't want to go into too much detail on vascular development, but I'll just describe a couple of different processes here. The first is called vasculogenesis, so this is formation of a primitive endothelial network and ultimately formation of a vasculature from endothelial cell precursor cells. So this would be sort of an example of stem cell differentiation into an endothelial network. And the second is angiogenesis which is the formation of a vasculature from a preexisting vascular supply, so this would be mature endothelial cells sprouting off from an existing vasculature. So each of these processes can potentially be mimicked in engineered systems to try to promote tissue regeneration. One growth factor that's of particular interest in both of these processes is called vascular endothelial growth factor or VEGF, so VEGF induces differentiation of multiple stem cell types down the endothelial lineage. So this is a potential mechanism for promoting vasculogenesis. It also is an endothelial cell mitogen and angiogenesis promoter. 

So it can promote angiogenesis or development of blood vessels from a preexisting supply, and this has been shown in a variety of studies as well. So this is a factor that's of substantial importance. And so one mechanism that we've been trying to generate for regulating cell behavior, particularly regulating growth factor activity and more specifically regulating VEGF activity involves colocalization of cells and molecules on a material surface, so I'll page through this a little quickly. If you look at the lower left here, what you're seeing is the concept that I'm talking about, so the idea here is you have a material that without the presence of some ligand, doesn't interact very strongly with a growth factor, so the growth factor is just floating in solution, it isn't interacting with the material. If we then immobilize a ligand on the material, it binds to that growth factor with a controlled affinity so the material is now part of the signaling system, it's not a passive observer. It's actually actively regulating signaling. And on the lower right is the concept that we're thinking of so the idea here is that this is -- let's see if I can get this pointer -- so at any rate the blue signals that are shown there are cell adhesion peptides, so signals that promote cells to attach to a surface and then the green signals are sequestering growth factors. So the idea here is that growth factors and stem cells are placed in the same place at the same time and therefore we're affecting the activity of the growth factor. In some cases what we want to do is down regulate the growth factor and therefore we can use ligands that interact with the active site of the growth factor and pull it out. In other cases we want to upregulate growth factor activity so there we would want to either sequester the growth factor via a site off of the active site or we would allow it to pop on and off the surface at low affinity so the cells can interact with it. 

So we're creating a material that essentially acts like a signaling system rather than a passive observer. So this is the concept. And so if we use a ligand that has a high affinity, a relatively high affinity for a growth factor and we place it on a surface at relatively high density then this is what the situation looks like. So we have an integrin receptor binding to RGDSP which is a fibronectin derived cell adhesion ligand. And then we have growth factors that are binding to the surface. And then on the right we have a scrambled VEGF ligand, so a ligand that doesn't interact with VEGF on the surface and if we compare quantitatively how endothelial cells act in response to this -- as I mentioned before, endothelial cells proliferate in response to VEGF so that's why we're interested in using these. And you can see that in each case where we have the ligand immobilized on the surface that is the ligand that binds to the growth factor, we get downregulated activity of the growth factor. So the cells are actually decreasing in number. They're not proliferating as they normally would in response to VEGF. And if we look at controls on the right hand side where there is no ligand on the surface at all then in that case we do get proliferation in response to the growth factor. 

So effectively this is a concept where the material is knocking out growth factor activity. The material is actively regulating cell behavior. So you can imagine where that might become useful in tissue engineering strategies if the idea is to control in space and time what cells are being exposed to. So as I mentioned earlier, this is sort of the general goal of the field, how can we use materials to try and promote stem cell based tissue regeneration. How do we first identify which signals promote particular cell functions and then how do we use the material to actively deliver those signals to cells. And hopefully I've described a little bit about how we've done that in our lab and how the field has begun to start to consider these questions and more importantly what are the major challenges that need to be addressed by the current field as well as the next generation of researchers in the field and hopefully several of you in the audience are among those that will solve some of these problems. So in the interest of time I'll skip through the very end here and just indicate that these sorts of challenges that I mentioned earlier are the ones that the field is going to be addressing, so it's a nice short list and hopefully the list is going to get shorter in the coming years. I just want to acknowledge members of my group that actually did all of the work that I described today, and I'm happy to discuss anything that's of interest to the people in the audience. Thanks very much.