All right, thank you. This is a bit of a whirlwind, a hodgepodge of kind of some thoughts on, you know, one man's thoughts on where this field's going. He could talk for an afternoon on this topic, so we'll kind of go through just a little bit here. This is our support for our laboratory work, and I will mention some different products here. I have no relationship with any of these companies that'll be mentioned throughout this talk here. So my goal is to just brief overview, a 30,000th of a view, of some evolving concepts and approaches both the near and longer-term future. And we'll talk brief about various scaffold materials, cell therapy approaches, and then some emerging or novel cytokines, small peptides that are being developed, starting with materials. You know, materials are a critically important part here. It's where we learn a lot from our colleagues in the biomaterials space. Important factors that we need to think about in designing new materials in this whole area. Surface area, surface chemistry, and surface texture are important. Obviously, poor size and poor geometry. The overall architecture of how this is all put together. And then, of course, the degradation properties. All those are important and critically important factors that need to be considered. Various scaffolds have been combined with microfracture, the so-called AMIC technique. I'll list a couple here. Again, no relationship with these companies. These are essentially used in a scaffold on top of a microfractured bed. You see different materials here. Collagens and hyaluronans have been used. Hydrogel materials are being developed. I think you may see, as new materials and scaffolds are developed, they may be combined with these microfracture approaches. If you're using a cell matrix composite, the environment created by the matrix needs to be compatible with survival of cells. So important factors are listed here. The pH needs to be an isoosmotic environment. Importantly, the balance between the local metabolic demand of the cells and the ability for oxygen and nutrients to get there. And that kind of relates to vascularity and revascularization of these tissues. So these are all important factors that need to be considered. Biphasic scaffolds are important. That's kind of where this field is going. Articular cartilage, of course, has a zonal architecture and this biphasic mechanical properties. It's a very complex tissue. And our challenge is recapitulating surface lubrication properties while still maintaining the high compressibility of the deeper layers. So the goal should be to store this hierarchically organized architecture, which has been very, very difficult to do with most existing techniques to date. Some examples here, the JILI-C as a recently approved off-the-shelf implant. This is basically a porous material. This is a resorbable. It's biphasic. It's a calcium carbonate material, aragonite is this particular material. FDA approved last year. And this is based on some well-done data. A multi-center study here, Elisa V. Tacone is the senior author. 250 patients up to three defects treated. These are really early arthritic knees. Patients randomized to this implant versus a microfracture, two to one. And essentially, they found that the implant group had better outcomes based on their patient-reported outcome measures. And if you look at MR at two years, the fill was better in this group. So this is one example of a biphasic implant now available for us. I guess we're using one screen here, right? The next generation, kind of a cool paper here. This is the concept here is creating zone-specific chondrocytes. Again, back to that picture of the histology of cartilage. Very complex tissue. So can we create zone-specific chondrocytes by different culture protocols? In this study, they took cells, and they differentiated them by culturing on surfaces with different topographic patterns. You see this grill and this other pattern here on the right side here. And essentially, they create a bilayered, stratified construct that would hopefully recapitulate the zonal architecture of hyaline cartilage. And then they went ahead and used this in a smaller ABBA model. They compared this to implantation with just a cell-free hydrogel. And essentially, this stratified bilayered hydrogel construct did in fact improve repair. So this is kind of a neat area that may move toward clinical development. Cells. This is my own passion. What about cell therapy? Where are we in this whole field? Here's some thoughts on improved ways to use bone marrow or adipose cells as we move forward in the future here. Number one, we need to characterize these very heterogeneous cell populations. We need to measure cell composition and the biologic activity. We certainly need markers of cell identity, purity, potency, biologic activity. All these things are so poorly characterized. Importantly, I think we need to identify the computing or inhibiting cells in these heterogeneous mixtures of cells. And ultimately, think about identifying the biologic targets so you can match the therapy to what we're trying to achieve here. Second area, I think changes in the regulatory environment will allow us potentially to eventually do cell sorting and culture expansion. Current regulations, of course, do not allow us to do the so-called ex vivo manipulation. We can't take these to the laboratory and isolate a desired cell type, but maybe in the future. Lastly, the ability to mobilize cells prior to harvest. Anna Manz has done some nice work in this area, showing, for example, exercise. Blood flow restriction training increases the circulating cells, the CD34 cells, so something you might do before you do a harvest. Similarly, granulocyte colony stimulating factor, filgarastin or neupogen, this has been used to mobilize cells prior to bone marrow transplants. We might think about using something like this. It definitely increases the circulating cells. I think we'll make progress in the area of stimulating the intrinsic stem cell niche. We know that many tissues, probably most of the body, harbor this population of intrinsic progenitor cells. These are the cells probably on the walls of blood vessels, pericytes or perivascular cells. And pericytes can be induced to differentiate into various connective tissue phenotypes. And it's these quiescent cells that are probably stimulated during tissue injury and repair. We know there are tissue-specific factors, these so-called angiokine factors produced by endothelial cells, that stimulate and control these intrinsic progenitor cells. I think a lot of progress will be made in years to come on our ability to stimulate and leverage these intrinsic cells already present in tissues. Concept here, human skeletal stem cells. This is kind of seminal work from Charles Chan and the group at Stanford, Michael Longacre. The whole concept of MSCs, frankly, is being replaced by this is a more contemporary concept, human skeletal stem cells. These are a population of self-renewing and multipotent human-derived skeletal stem cells that generate progenitors of bone and cartilage. These can be cells identified by a specific cell surface marker pattern, a very specific cell type they've isolated. These can be found in both fetal and, importantly, adult bones. And these human stem cells undergo local expansion in response to skeletal injury. So a lot more to come in this area, but this is all basic laboratory work that has tremendous potential in moving this whole field of cell therapy forward. Other emerging options, briefly, exosomes, of course, these are vesicles that are released from cells upon fusion of a, you know, a multivesicular body with the membrane that releases the goodies of the cell, the cell cargo, microRNAs and DNAs and proteins and lipids, which in exosomes may allow us to have the benefits of cell therapy without all the logistic hassles that go with cell manufacturing and processing everything. And then iPSCs, induced pluripotent cells, this is essentially reprogramming a mature, differentiated cell with four different genes to make that mature cell go backwards toward essentially tantamount to an embryonic stem cell. This is Yamanaka's work from 2006. That's a Nobel Prize work in 2012. There are currently no induced pluripotent stem cell products in orthopedics, but an area that has tremendous potential. Gene therapy, making a bit of a comeback here. The gene therapy techniques are out there. Chris Evans up at Mayo Clinic has worked probably for 25 years on IL-1 receptor antagonists, a gene therapy approach, and now they have a clinical trial going using a recombinant adeno-associated virus, essentially treating patients with moderate knee OA with this IL-1 receptor antagonist. So this is ongoing work. Another example of a gene therapy product that's being developed from a company in Korea. Chondrocytes here, they're retrovirally transduced with TGF-beta, an important cytokine that can have a positive effect on chondrocytes, and their preliminary data is favorable based on imaging and some patient-reported outcome measures. They've published this data. This is the company that's in South Korea. Again, no relationship with these, but these are all potential avenues for the future here. Beyond gene therapy, the really cool area is gene editing. Farsh Gilak now at Wash U in St. Louis has done some very neat work in this area, essentially using gene editing techniques now, so CRISPR-Cas9, to produce what you call smart cells. Essentially, they can turn on or off gene expression of a particular transgene in response to changing levels of endogenous inflammatory mediators. So essentially, it's like a gene switch. If there's inflammation, it turns on the gene. If the inflammation goes down, it turns off the gene. And the strategy they've used is two different antagonists, IL-1 and TNF inhibition. Essentially, what they do, just very briefly, use CRISPR-Cas9 to insert the gene for a TNF antagonist at this CCL2 locus. That's just a certain locus that's turned on instead of inflammation. So put this in an inflammatory environment, cell sees TNF, it turns on this gene, and produces this soluble TNF antagonist, essentially. And these cells, they'll undergo rapid activation in an inflammatory environment, and then they turn it off when inflammation goes down. In fact, small animal work demonstrates that these have a positive effect on mitigating disease severity. So neat, novel concept. Certainly, you know, this is for the future. We're learning that the activity of MSCs really is mediated by cells of the innate immune system. I think as we gain further understanding of the interaction between stem cells and immune cells, we're going to be able to move this field forward. This is an important paper from the Netherlands, where they administered MSCs, and they found that these are rapidly phagocytosed by monocytes. But that leads to change in the phenotype of the monocyte to more of an M2 sort of pro-regenitive-like cell. And these prime monocytes then go on and upregulate T-regulatory cells. Essentially, what that does, it induces long-term adaptive immunity in that individual. So you start with an innate immune response, and you turn on the adaptive immune response, this could lead to a durable approach, a much more durable long-term symptom relief. And this data importantly shows the biologic activity of a few cells may be, in fact, independent of their cellular activity, and challenges the hypothesis that effective MSCs are just mediated by the secretome. And in fact, rather, the mechanism of action may be mediated by local or systemic immune cells. So we're learning that this is critically important interaction between the cells we put in the body, whether that's marrow, adipose, whatever, and immune cell populations. And lastly, a little bit on cytokines and small peptides, just to give you a brief overview here. FGF-18, fibroblast growth factor 18, has been evaluated as a drug called sprefermin. FGF-18 does certainly stimulate chondocyte proliferation, matrix synthesis, and does have some inhibitor effects on MMPs and various proteolytic enzymes. A particular trial, Mark Hochberg, nearby here in the University of Maryland, led this trial, a five-year multicenter trial, a patient with grade 2 to 3 in EOA. They received one injection of this material every six or 12 months. And essentially, they found improvement in total joint cartilage thickness. So they do see some structural improvements. Now, it was statistically significant. Whether that's clinically important, we'll see over the long term, but this is one of the few studies to demonstrate regeneration of tissue. So this is undergoing further FDA trials. Similarly, loracivavent is another potentially disease-modifying drug, and not just symptom-modifying, but can you actually be structure-modifying? This is a — this particular molecule modulates the Wnt signaling pathway. It's just a company in San Diego producing this. And here's another study, Mark Hochberg, again, his group, a six-month multicenter randomized trial demonstrated efficacy in this early phase, phase two trial. So this is an ongoing evaluation as well. And lastly, can't talk about arthritis without mentioning subchondral bone. There's been increasing recognition of the role of subchondral bone abnormalities, of course, and marioedema and the reaction that we see in the subchondral bone, and we're recognizing critically important interaction between subchondral bone and cartilage, of course. And there's a number of these subchondroplasty techniques that are being used. There's some early data, which seems promising. Poly works via decompression of the intraosteous hypertension that occurs in the sediment of these bone marioedema lesions, and a number of different scaffold materials are being used. Calcium phosphate, ceramics, and other things, just goes back to the beginning there. Scaffolds and materials will have an interaction in this area as well, but certainly we need further data. So to summarize here some of my own conclusions, I think advances in treatment options for cartilage is going to come from improved understanding of the cellular and molecular mechanisms of cartilage injury and repair. Cells, nothing heals without cells. We need to learn more about the basic cell physiology and the cell biology and the interaction of the cells that we put in with other local cells. I think we're going to learn that cell therapy approaches largely work via stimulation of the intrinsic stem cell niche, these intrinsic progenitor cells that reside in many tissues. We're learning there are important interactions between exogenous cells and both innate immune cells, as well as these intrinsic tissue resident progenitor cells. And lastly, and I haven't even touched on this, but we need to broaden our thinking beyond just the cartilage. We need to consider the entire inter-articular milieu, the synovium, the subchondral bone, the whole joint, and that's a whole other topic for another day. I'll stop there. Thank you.

cartilage repair

scaffold materials

cell therapy approaches

biphasic scaffolds

cell therapy advances