One of the nice sets of stage here for some of the basic concepts we'll go over, kind of focus more on cells here in rotator cuff. This is our support for our laboratory research program. So I think as you've seen here, it's clear the techniques and methods to affect biologic events in tissue repair, certainly great potential, but a lot of these frankly are maybe not yet ready for prime time. No doubt that a lot of preclinical studies support the potential here, but we do see a lot of indiscriminate use and indiscriminate use of unproven therapies may actually hinder progress in this field. So kind of how do we move forward here? Think about tendon repair in particular on this particular talk. Certainly cells produce a number of signaling molecules that can have an important role in biology. And you're kind of seeing all these listed here and we know that mediators produced by cells. When we talk about cells, we're going to keep this generic right now. That could be cells derived from adipose, from bone marrow, or other sources. Certainly the signaling molecules produced by cells stimulate relevant biologic activities, cell proliferation, matrix synthesis, chemotaxis, angiogenesis, I mean a number of factors that the cells will impact. The important point is that cells, exogenous cells that are implanted work via a paracrine mechanism by producing these various signaling molecules. It's these secretive molecules that affect the local environment and maybe affect local cells and perhaps even distant cells. Understand there is very little data to suggest that the cells we inject actually engraft at all. These cells do not reliably intercalate into the healing tissue or into the joint where we inject these. What do we have available? You've kind of heard all this. We can move right through this. Obviously we can use cells derived from bone marrow. There are peripheral blood-derived cells we could talk a little bit about that has a potential. Obviously you've heard a lot about adipose tissue here. We can use currently the microfragmented fat, the minimally manipulated fat. Distinguish that from stromal vascular fraction and certainly adipose-derived cells. The stromal vascular fraction requires enzymatic digestion. That's not currently allowed by the FDA. That needs to be done under an IND, Investigation of New Drug, or IDE, device exemption. Certainly adipose-derived stem cells, that implies cultured cells. Again, those of course are not something we can do currently outside of a clinical trial. Understand now that any of these perinatal tissues, cells from amniotic membrane, amniotic fluids, umbilical cord bloods, those can only be used now under an FDA-approved trial since May 31st, 2021. Those perinatal products now are essentially for you and I, they're kind of off the market unless you're in a trial here. Let's talk a little bit about tendon repair here. Certainly again, great potential. I would submit to you that the minimally manipulated cell preparations that are used by us, bone marrow, adipose tissue, that needs to be distinguished from sorted culture-expanded cells produced in the laboratory. Look at all the basic laboratory science data on MSCs and cell therapy. It's all based on, much of it based on in vitro cultured cells. That's very, very different. In fact, the term stem cells should not even be used when we're talking about cell therapy. Most agree that the term connective tissue progenitor is probably a better term to use. I would just emphasize here, there are no stem cells. There are zero stem cells that we can use, none. Again, terminology gets complex, but be careful using that term. So connective tissue progenitor, that refers to cells that are heterogeneous population of cells contained in many tissues. We can, in fact, proliferate and generate progeny to differentiate various tissues, but it's a more limited sort of repertoire certainly than a true stem cell by any formal cellular or molecular criteria. You can measure connective tissue progenitor cells by measuring colony forming units in cultures. You can culture a population of cells and each founding cell for a colony is a connective tissue progenitor cell. This is probably a better terminology for us to use. Again, we have essentially no ability to use stem cells at this time and the number of true stem cells by any formal cellular or molecular or functional criteria in any of these formulations is very, very, very small. The classic definition of an MSC, an MSC is a mesenchymal stromal cell. Again, it's based on culture-expanded cells. That is very different than the cell populations that are contained in freshly harvested bone marrow or adipose that we can use today. Again, the term MSC should only be used to refer to culture-expanded populations of cells that meet these specific criteria that are defined. It certainly should not be used to describe these heterogeneous populations of cells that we're all using. Again, as I mentioned, very little evidence to suggest that the cells we inject even in graft rather they act via parachromagnesin. These cells produce a number of immune-modulating and anti-inflammatory signaling molecules that certainly have an effect. That's certainly how they can be symptom-modifying, improving symptoms. Next question is, can they really be structure-modifying? Can we truly regenerate tissue? Again, we don't even know if this multilineage differentiation that we know happens in culture. We don't even know if that happens in vivo. We have no data to suggest the cells we inject go through this differentiation phase. Again, rather it's this paracrine mechanism here. And in our current regulatory environment, we can't take the cells out of the operating room and do any kind of cell sorting or culture expansion with these products here. So we can't. You've heard a little bit about this already here. We can't perform ex vivo manipulation. Culture-expanded populations, that's very different. They're highly selected cells, and they're fundamentally different from these mixed starting populations that we all use. And that, I think, is the biggest or most critical issue right now in cell therapy is heterogeneity. These cell populations we use are poorly characterized. The identity and the biologic activity of these cells is really unknown. It's a mixture of cells. It's a mix. It's very heterogeneous. We clearly need markers of cell purity, potency, quality, biologic activity. We need to characterize what it is we're putting in patients. Right now, that's not being done. We don't have a good way to do that on any regular basis. Taking all that, now we talk about tendon and kind of focus on one of the clinical aspects, suggests to me that use of cells to improve tendon healing, a little unpredictable. And there's a lot we need to learn still. So how might it work? If we kind of focus on rotator cuff, I thought I'd go through some basic mechanism for how cells might work in tendon. I'm going to focus on the rotator cuff and what we know there, but a lot of this will apply to other types of tendons. Number one, implanted cells have important interaction with host immune cells. This is kind of an important paper, I think, from a couple of years ago from the Netherlands, where they just injected human MSCs, and then they tracked these in mice. And what they see, basically, the punchline here is cells that we inject are phagocytosed by monocytes. So the MSC is red, it's phagocytosed by green, the monocyte there. That induces a change in the monocyte. That monocyte undergoes a phenotypic change, becomes more of kind of a pro-regenitive or anti-inflammatory cell, if you will. The CD206 is just a marker for an M2 type of phenotype. So the point is it modulates the local immune cells. These monocytes then express more kind of an anti-inflammatory phenotype. So this suggests that monocytes play a crucial role in mediating the whole effect of cells we inject. So the point here is very important interaction between cells and the local immune cell sub-populations. Cell therapy can certainly affect muscle. In addition to the tendon, this is some nice work from Christian Gerber's group. They have a sheet model. They release the infraspinatus tendon, and they come back four months later. And they compared just injecting it with, or after the repair, putting bone marrow-derived cells versus just doing a standard repair. As you expect, after 16 weeks of release of the tendon, the infraspinatus muscle volume is decreased. The fat fraction is increased. There's some muscle atrophy there. The fat-free mass and the fat fraction was improved in the cell-free group. In fact, looking at gene expression, those animals that received cell injection had increased levels of an important proteinogenic marker, tenacin C, reduced levels of this PPAR gamma, which is more of a dipogenic gene. So just demonstrating here that the implanted cells had a positive effect on muscle. So we all talk about tendon here. Clearly, tendon healing is a challenge for us, but in the rotator cuff, it's made to the muscle needs to be considered as well. That's another unsolved frontier as far as the atrophy and fatty infiltration in muscles. So they concluded that injection of cells in the degenerative rotator cuff here during repair may affect this muscle-to-fat conversion, may have a positive effect on the fatty infiltration. That's important because, clearly, we need clinical translation, but none of our therapies really can reverse the fatty changes in muscle. Number two, cell therapy, or three, cells probably interact with local macrophage cell population. I mentioned monocytes a moment ago. Macrophages play a critical role in the initiation and regulation of healing. This particular study, they looked at adipose stem cell-derived exosomes. So this is exosomes derived from adipose cells. This is a tendinopathy model. So mice assigned to treadmill overuse running. So it induces a tendinopathy. Essentially, animals treated with these exosomes derived from adipose stem cells inhibited the M1 macrophage, which is more of your pro-inflammatory macrophage, and augmented polarization to an M2 phenotype. The M2 macrophage phenotype is more of a pro-regenerative, anti-inflammatory macrophage. So there's this phenotypic switch in animals that are treated with these exosomes here. Those animals also show less tendinopathy on histology, improvements in MMP levels, changes in biomechanical testing. So really a positive effect in these animals. And then they conclude here that it may be that the regulation of that, the macrophage polarization, the balance between M1 and M2 macrophages, may be how cells work as well. So again, basic principle or point here being, again, how cells interact, cells that we inject, or exosomes in this case, how that interacts with local immune cell populations. A lot of the effects of cells that we inject, a lot of these biologics may act through local immune cells. We know that cell therapy may affect inflammatory meteors in the rotator cuff. This is some work from Pietro Rondelli in Italy that took tendon cells from injured supraspinatus tendon. They co-culture these with the autologous microfat that Jason talked about. This is from the use of lipogenous system here. And essentially, cells cultured with this microfat showed reduction of catabolic and inflammatory marker expression. So MMP3, COX-2, an important inflammatory meteor, IL-1 receptor antagonist, there was a reduction in all of these inflammatory, catabolic, and fibrotic marker genes in those cells that are exposed to the microfat. So they concluded that microfat may have an anti-inflammatory action on tendon cells. So again, supporting the positive effect of adipose-derived cells on tendon. And lastly, cell transplantation. This allows us eventually to use gene therapy approach. Gene therapy is sort of making a comeback. We can insert certain genes in our exogenous cells, some work we've done in our laboratory some years ago, just kind of proof of principle. We used scleraxis, an important kind of master gene for tendon formation, involved in tendon formation in embryologic development. So we transfected bone marrow-derived cells with the gene for scleraxis. We use a mouse rotator cuff repair model and inject these cells that are these transfected cells into our model and look at these animals at various time points. Essentially, what we see is it really has an effect on healing. We see improvements in fibrocollagen formation, better load to failure, better stiffness. So these gene-transfected cells did have a positive effect compared to just untransduced cells. Again, potential there for gene therapy approaches when we're using cells. But that is a backdrop. Where are we clinically? So what is the clinical data? Well, there's a little bit of data out there on cell therapy augmentation for a cuff repair. We go back to E. prunergu nine years ago, a study of patients. You had 90 patients undergoing rotator cuff repair. Half of them received bone marrow-derived cells. Put the number up here, number of MSCs, 51,000. Kind of keep that in mind. That's the number of MSCs. They did that based on colony forming units in culture. Basically, that's a low number, but that's the number of cells here. But all the repairs with MSC augmentation did heal by six months versus about two-thirds of those without cells. And in fact, a 10-year follow-up, they found intact repairs in 87% of their patients versus about half that in the control group. In fact, long-term cuff integrity is better in those patients who receive more cells. So here's some early data supporting the use of bone marrow-derived cells. A group from Seoul, South Korea used adipose-derived cells. Here's 70 patients undergoing cuff repair. Again, half of them received cells. And again, no real differences in patient-reported outcomes, your subjective measures, but the failure rate was about cut in half, again, 28% without cells, 14% with cells, so demonstrating the potential for cells to have a structural effect. And then the Rush Group recently published this study just in our journal in recent months here. Prospective study, 91 patients, half of them received concentrated bone marrow aspirate, kind of standard cuff repair. I want to show you here. So the average will be 44,000 cells. Again, that's kind of the number of cells we can put in. They did this based on flow cytometry. So these are the number of cells we try to define what we're actually putting into our patients. And they found an improvement in structural healing. There was a higher re-tear rate in those patients that did not receive cells, or conversely, a lower re-tear rate or lower defect rate in those patient-treated cells, but no real differences in the patient-reported outcomes, no difference in overall failure rate. So they concluded that concentrated bone marrow aspirated bone marrow augmented repairs may result in a structurally superior repair, but did not really improve treatment failure rates or the patient-reported outcome measures. But I submit to you over a long-term follow-up, those patients with a structurally intact repair, I think we would all agree that would be favorable and may have much better durability over that longer-term follow-up. Pietro Randelli in Italy, a randomized trial here using autologous microfats or the LipoGEM system. A small group of patients, 22 patients in each group, simple, again, cuff repair, used a microfat. The LipoGEM system, as Jason showed you earlier, they found an improvement at the six-month time point. Essentially, no real differences in outcome measured at later time points, but at six months, they found that there was an improvement. You see this graph at the bottom. Only separation is that six-month time point. So a little data here to support positive effect, but just at that one early time point. Lastly, there's some work being done using adipose-derived cells nonoperatively. This is a group of patients undergoing, being treated for partial ligninus cuff tears. No surgery. This is just an injection. So these are partial ligninus cuff tears. These are uncultured cells, so adipose-derived cells, but they use enzymatic digestion to isolate the stromal vascular fractures. This is done under investigational device exemption. They can use enzymes to isolate the stromal vascular fraction. Small sort of proof of principle study here, but I want to point out, so 11 million cells. So there, remember, you have the studies I showed you, 51,000, 44,000, and with stromal vascular fraction, we can use enzymatic digestion to isolate cells. Much logarithmically different numbers of cells here. Small groups, 11 patients treated with cells versus five controls. This is just an early phase one safety study, but they did find higher ASCS scores, American Schrodinger Society scores, in those patients treated with cells. So very preliminary data, and they now have an ongoing clinical trial, randomized trial, which is actually like 180 patients, but they've completed the trial, I hear. Company's called Ingeneron. I have no relationship with them, but they've basically completed this trial. This is again for partial ligninus cuff tears. So bottom line, I think cell therapy approaches are promising for improvement in structural healing. Certainly, we need further study in this area. What can you and I use today, and where are we in this whole area? I think if you're using any biologics, this will probably hold for cartilage, for muscle, for tendon, anything. Start by identifying the biologic targets, and what are we trying to treat, and I kind of list just briefly here all the different things that cells might treat. These are all important biologic targets, but they're all different, you know, and so this may ultimately identify what we're trying to accomplish, may help us choose what are we going to use. Do we want to use bone marrow, PRP, microfat? We need to really characterize these things. In addition to identifying the biologic target, we need to define, again, the composition of the biologic activity of these various agents, and only then can we match our treatment with the desired biologic target. Certainly, one size does not fit all. It doesn't make sense that the same microfat or bone marrow would have the same positive effect on different tissue types, acute versus chronic process, gender, age, all those things obviously make a difference. We need much better data in this area to be able to match our treatment to the pathology. If you are using biologics, ideally we take a, you put the patient in registry, you collect outcomes data. We need to identify biomarkers to characterize, you know, what we're putting in the patients. I mentioned that at the beginning. I think the single most important issue is the heterogeneity of what we're putting in our patients and the undefined nature of what we're putting in the patient. Ideally we take a small aliquot of what you're putting in the patient, take it to the laboratory, store it for later analysis once we know what, you know, sensible markers we might want to measure. Ultimately, the goal would be to correlate the patient's outcome with the biologic activity or composition of whatever the heck I put in my patient. And only until we do that can we really understand what is the ideal formation for different patients. So we need to identify some type of markers to characterize whether it's PRP or cell therapy formulation. In the future, just going to finish up here, we may be able to use bone marrow from the proximal humerus. If we're doing rotator cuff repair, Gus Buzak has done a lot of work looking at cells derived from subacromial bursa. Ideally we can have some rapid point-of-care methods. You could harvest cells from bursa. The potential for allogeneic cells, you know, cell banks has potential. I think a whole other area will be stimulation of the intrinsic stem cell niche that exists in many tissues. Many tissues have a niche of progenitor cells, polypericytes, perivascular cells in the walls of blood vessels. We need to learn how to mobilize and leverage those cells for tissue regeneration. I think progress will come from methods to select the desired cells and importantly eliminate competing cells from these very heterogeneous mixtures of cells. And then, of course, exosomes have great potential. These are extracellular vessels that are released from cells. The exosomes essentially have all the goodies of the cell, the cell cargo, microRNAs and cytokines. By now there are no exosome products available in orthopedics. To produce these at scale is a huge challenge right now, but there's preclinical data in animal models to suggest the potentials here. Essentially, use of exosomes allow us the benefits, if you will, of cell therapy without all the hassles that go with processing and manufacturing cells. Just bursa, I mentioned briefly. Gus Mazzocchi did a lot of work in this area. He's here comparing bursa to bone marrow aspirate concentrate. So measuring colony forming units, higher numbers from bursal-derived cells. They went ahead and, you know, this is their culture material to look at colony forming units. If you look at flow cytometry, they find that bursal cells have similar cell surface markers to cells derived from bone marrow. In fact, in a small animal model, a mouse model of carfree pair, just demonstrating that bursal cells had a positive effect. So again, that's another potential source of cells for us. Again, needs to be further characterized. Exosomes I mentioned, to kind of go through this really quickly, this is just an animal study. Again, I mentioned they're animal studies. They've done a ton of work demonstrating the efficacy of exosomes, and in this particular animal model, rotator cuff repair model, they improved fatty infiltration, which again, as I mentioned, that's one of our unsolved challenges. Improvements in tissue formation at the healing site. So again, the potential here for exosomes to improve healing. We have two ongoing trials at HSS. One is just completed. One, we're using stromal vascular fraction cells under an investigational device exemption. So we're using enzymes to digest fat. So that's ongoing. We've completed a phase one trial using gene-modified cells derived from human umbilical vein. Phase one safety study. So we'll be reporting that, or that's been reported, but trying to finish the manuscript now. So this is some ongoing work. And lastly, where is this field headed? I think we're going to learn more about the role of immune cell subtypes in tissue repair. I mentioned briefly the role of monocytes and macrophages. I think that's where we're going to learn a lot. Techniques such as single cell analysis, gene editing. We learn more about epigenetics. This is kind of the future to really understand much more about the biology of cells. Induced pluripotent cells. Another area we haven't talked about here. That's taking mature cells and using four specific genes, these Yamanaka genes, to essentially make that cell tantamount to an embryonic stem cell. It's a Nobel Prize work from years ago. Again, not clinically available at this time. So in summary, cell therapy, great potential. We need markers of biologic activity and potency. Hopefully changes in the regulatory environment eventually will allow us manipulation of these products. I think advance in the field will come from enzymatic digestion of adipose from cell sorting and culture expansion once the FDA allows us to do that. I think importantly methods to select the desired cells and importantly eliminate competing cells from these very heterogeneous mixtures. On the horizon I think the role of exosomes. I mentioned the potential to stimulate the intrinsic cells in tissue has tremendous potential for us. And ultimately manipulating immune cells and understand the interaction between cells we transplant and the local immune cell populations. So I'll stop there. And we can take a question or two and see where we are on time. We have 15 minutes and yes. Can I ask as the private practice, I have two questions. One is how to deal in the community with, I do a reasonable amount of adipose and having Regenexx on one side of me and US stem cell on the other side of me, which means the patients have already been there and they've come in with their $10,000 charge for cell regeneration. That's the how to deal with society. And then the other is a question about adipose. Most of these adipose have like a bible pain relief for the first two weeks. They actually feel pretty good. That tapers away and then around six weeks they start improving. How does that happen? I don't know. I'm not, I'm not, that's interesting. I've not heard or seen that. Have you seen that? That's interesting. It's a lot we don't know about the biology, you know, and it just shows how much we don't know. Kind of my answer quite honestly. Especially in the biology environment evolves. So injecting cells at surgery here versus, may I actually inject my cells a week later or four weeks later or eight weeks later. Who knows? I mean, and it's biological. I mean, we was evolving. So you kind of implied that something had something different in that joint at two weeks versus six weeks. Cool. I don't know. We face the issue of the scopes on the same day, on the same day injection versus two weeks later. You're not paying for the room and you're not paying for anything else. You can decrease the cost of the patient on injection, but you worry, you know, is this all being washed away with my saline at the very end. So, but most of the patients have gone back to them. We'll take the 2,000 off and take it at that time. Exactly. And the adipose, they've been happy. But it's hard trying to clarify to them that this isn't a stem cell. This isn't, you know. And I think most of them walk away. Paul, thanks for listening to the FDA view of musculoskeletal. No, that's a stem cell, but I just can't say it. And no matter how much you tell them that, they've just been so sold on it by eugenics and these other things. Like U.S. stem cell? Yeah. I mean, they should take that out of their right now. The FDA, that's, they're pulling the, tugging the tiger's tail doing that at the FDA. One more question, then maybe we'll go ahead and Dr. Webber's talk. It's about a 10-minute talk, so. Yes. I really appreciate the very interesting data with the monocyte stuff. So if we know that these cellular therapies are working via the inflammatory pathways, would it not make more sense to do like a monocyte-rich therapy? I'm interested to hear your opinion. Yeah. Good question. Yes. I think we need a lot. You know, we talk about leukocyte-rich versus leukocyte-poor. I think we need a much more refined analysis. I mean, what type of leukocyte? So we should consider the leukocyte differential. Probably more important may be the ratio of platelets to leukocytes. I mean, not just, you know, leukocyte, yes, no. So maybe so. Maybe techniques to enrich monocytes and, frankly, lymphocytes, but deplete neutrophils. Yeah, that's what the data would suggest, so. Why don't we go ahead, then? We've got 10, 15 minutes here. Steve Webber.

cell therapy

tissue repair

tendon repair

adipose cells

bone marrow cells

perinatal tissues