I'd like to cover the role of biologic augmentation in rotator cuff tendon repair. These are the disclosures, support for our laboratory research. Overall message I would start with, certainly techniques and methods to positively affect the biologic events in tissue repair have tremendous potential, but I would submit to you are not yet ready for prime time. Preclinical animal studies certainly suggest promise, but have distinct limitations. Indiscriminate use of unproven therapies may in fact hinder progress in the field. So what should we use today as clinicians? I think we need to start by identifying the biologic targets that we're trying to treat. Do we want to improve cell proliferation, matrix synthesis, cell migration, angiogenesis perhaps, matrix remodeling, the accumulation of inflammatory mediators, perhaps production of nociceptive mediators. Perhaps we want to stimulate the intrinsic stem cell niche. All of these are important and biologically relevant, but they're all very different and we need to consider this in choosing the appropriate therapies for our patients. In addition to identifying the biologic target, we need to rigorously define the composition and biologic activity of these agents, and only then can we match the biologic agent with the desired biologic target. One size certainly does not fit all. The same cell-based preparation would not be expected to have a positive effect on diverse tissue types, on an acute versus chronic process. Gender may make a difference. Certainly age may play a role. All these are important factors we need to consider when we think about augmenting biology issues. What is the rationale for using cell therapy in rotator cuff tendon repair? Well, certainly numerous signaling molecules are produced by cells, various cytokines as well as anti-inflammatory and immune modulating factors. These mediators may stimulate biologic activity in these tissues, of course. The mechanism of action of cell therapy is likely a paracrine mechanism. Secreted molecules from cells we know can modulate the local environment and may affect local and distant cells. In fact, there is very little data to suggest direct engraftment of injected or implanted cells. Currently available cell therapies that we can use as clinicians, obviously we can derive cells from bone marrow. We can use peripheral blood derived cells. We can use adipose tissue. We should note that currently we can use microfragmented tissues, like the lipogenous procedure with which I have no relationship. This should be distinguished, the microfragmented fat should be distinguished from stromal vascular fraction and adipose derived cells. The minimally processed microfragmented fat is different than a stromal vascular fraction because that requires enzymatic digestion, stromal vascular fraction. That's not allowed by current FDA regulations. Moreover, the adipose derived stem cells, that requires culture expansion, which is certainly not allowed at this time. Also note that cells derived from perinatal materials, amniotic fluid, amniotic membrane, umbilical cord blood, placenta, all of these cell sources can now only be used under an IND since the end of the FDA discretionary period about a year ago, last May of 2021. So now these materials essentially can only be used in a clinical trial. So cell therapy augmentation of cough repair certainly has tremendous potential, but there are currently many unknowns in this area. I would submit to you that minimally manipulated cell preparations that are currently used in the United States should be distinguished from sorted culture expanded cells produced in the laboratory. Those are very different. In fact, the term stem cells should not even be used when discussing current techniques for cell-based therapy. Connective tissue progenitor is a more accurate term that we should use. This is a heterogeneous population of tissue resident cells that can in fact proliferate and generate progeny with the capacity to differentiate into various connective tissues, but they have a more limited differentiation repertoire than a true stem cell. Connective tissue progenitor cells can be measured by colony forming units and cultures. This is a way to quantify these cells. I submit to you that we currently have essentially zero ability to use true stem cells. The number of true stem cells by any cellular, molecular, or functional criteria in our currently used formulations is very, very small. And furthermore, there's very little evidence to suggest direct engraftment of injected cells. Again, the current paradigm is that cells work via a paracrine mechanism by production of a number of immune modulating and anti-inflammatory signals that can modify the local biologic environment and may affect both local and distant cells. This goes back to work from Arnold Kaplan, who first coined the term MSC back in 1990 and then this kind of classic paper 2017, where he suggested MSC should be standard for a medicinal signaling cell, kind of in recognition of the paracrine effect of cells. And lastly, it is not known if the multilineage differentiation that occurs in vitro even occurs in the in vivo environment. So how might cell therapy work in the rotator cuff? I'm going to summarize a lot of basic science data kind of on one slide here. Number one, stromal cells have important interactions with host immune cells. We're learning that a lot of the effects of cell therapy, stem cells if you will, acts via local immune cells in a local environment. Number two, we know that cell therapy may positively affect rotator cuff muscles. Some nice work by Christian Gerber and others have demonstrated this. Number three, cell therapy can affect the local macrophage cell populations and affect the polarization from an M1 inflammatory macrophage to an M2 more pro-reparative macrophage. And lastly, cell therapy can positively affect inflammatory mediators in the rotator cuff. This has been shown in various clinical biopsy studies. What does all this mean as far as the clinical data? Well, it's very truly not a lot of data on the use of cell therapy augmentation of rotator cuff repair. We can go back eight years to a well-done study from Philippe Pernigou in Paris where they had 90 patients undergoing rotator cuff repair. They used concentrated bone marrow derived cells, about 51,000 cells on average dosed to 45 patients versus the controls, 45 patients just undergoing routine repair. And they found that 100% of those repairs with cell augmentation had healed by six months versus just two-thirds of the repairs without cells. And in fact, at a 10-year follow-up, they found intact rotator cuffs in 87% of the cell treated group but just half that in the control group. So they found long-term cuff integrity was better in those who received a higher number of cells. More recently, a group from Seoul, South Korea, they looked at the role of adipose-derived cells in a fiber and glue carrier, 35 patients undergoing rotator cuff repair alone versus 35 with cells. And they found no difference in the patient-part outcome measures. But using MRI scan, they found that the rate of re-tear was 28% with standard repair versus just 14% in the cell therapy group. So a positive response here. This is a well-done study recently published. Patients with partial lignus rotator cuff tears treated with uncultured autologous adipose-derived cells. They were isolated from a lipo-aspirate. This is done under an IDE, an Investigational Device Exemption, so they could use enzymatic digestion to isolate the stromal vascular fraction. Patients received a single injection of cells. This is a small pilot study, 11 patients treated versus 5 controlled to get an injection of a steroid. They found good safety in the study. They found higher mean shoulder and elbow scores in both 6 and 12 months in those patients treated with cells when compared to the steroid group. This is demonstrated here, higher outcome scores. The cell group is in the green bars there. So some preliminary data here, and in fact, they now have a randomized controlled trial in 246 patients with partial lignus tears, which is currently ongoing. So we'll await there the results of this data. I think my bottom line right now is cell therapy approaches seem to be promising for improvement in structural healing, but certainly further study is required. Some cell sources we may use in the future. We can use bone marrow. It can be obtained from the proximal humerus, so it's a little easier at the time of surgery, although the cell yields are typically better from the axial skeleton, from the pelvis. We can use cells derived from cervical corneal bursa. Gus Mazzacca has popularized this. Now we certainly need to develop rapid point-of-care methods to harvest and process these cells. We have the potential in the future to use allergen egg cells, an off-the-shelf cell source. Another avenue would be stimulating the intrinsic stem cell in these, we know exists in many tissues. We can isolate cells along the walls of blood vessels, similar to parasites, that are intrinsic stem cells. Our job is to learn how to isolate and leverage these cells to induce tissue regeneration. I think most progress in this field is going to come from methods to select the desired cells and eliminate competing cells from a heterogeneous mixture of cells. Lastly, exosomes certainly have promised either extracellular vesicles that are released from cells, and these extracellular vesicles contain a number of proteins, lipids, microRNAs, and other cell contents that essentially release the cell cargo, if you will, and would allow us to achieve the benefits of cell therapy without all the issues that are related to cell culturing and use of exogenous or endogenous cells. Gus Mazzacca, University of Connecticut, now in Boston, has looked at the role of cervical cormula bursa as a source of cells in rotator cuff repair. They compared bursal-derived cells versus bone marrow aspirate, and they found that, in fact, in a higher number of colony-forming unit cells, you know, again, these connective tissue progenitor cells, higher numbers in cells derived from bursa versus bone marrow aspirate. They went ahead and used photocytometry, and here they demonstrate that bursa has a similar percentage of the cell surface markers to bone marrow aspirate, and then in a mouse study, mouse rotator cuff repair model, they found a significant increase in failure force in the tendons repaired with bursal cells, so some early positive data to support the potential use of cells in bursa. Gus has showed us how these cells can be harvested intraoperatively. They can be minced into a slurry, as you see here on the right-side picture there, and Gus reported that the size of the tear or age of the patient did not have an impact in the cellular content of the bursa, so something to look for as far as further development. Lastly, exosomes appear to be a promising strategy. These can be, again, derived from cells. This particular study from China used a rabbit model, where they did a tendon detachment and repaired the tendon. Six weeks later, these animals were treated with adipose stem cell-derived exosomes at the repair site, and they found the exosome group had lower fatty infiltration, an improved histologic score, and more newly regenerated fibrocarlids. You see the histology in the bottom left there, oral reto, staining demonstrates less fatty infiltration. On the right side, we see collagen II staining indicative of fibrocartilage, and in fact, the exosome group had a higher ultimate load to failure, so some early data, preclinical data to support the use of exosomes. There are currently no exosome products that are FDA approved for any orthopedic application at this time, however. We have two ongoing trials at HSS. One is using stromal vascular fraction cells and rotator cuff repair under an IDE, and I have another trial under an IND using, evaluating the role of gene-modified human umbilical vein endothelial cells during rotator cuff repair, phase one safety and early efficacy study. So, in summary, I think cell therapy has tremendous promise, but certainly needs further work. I think we need to identify markers of biologic activity, potency, and purity in these cell therapy formulations, ultimately changes in the regulatory environment that allows us some degree of manipulation, if you will, of these cell formulations will help us advance the field. I think the ability to use enzymes to digest adipose tissue will be helpful. I think that the ability to use cell sorting and culture expansion will help, because this will allow us to select the desired cells and, importantly, eliminate competing cells from these heterogeneous cell mixtures. I think on the horizon is use of exosomes as well as methods to stimulate the intrinsic progenitor cells that exist in many tissues. And lastly, a little bit on PRP and autologous blood products. Obviously, we can use different types of PRP. We can use plasiporoplasma, autologous conditioned serum, not currently FDA-approved, but this is a technique that allows concentration of IL-1 receptor antagonists, similarly, autologous protein solution and other formulation being developed that also concentrates IL-1 receptor antagonists. Alpha-2-macroglobulin is another molecule that can be concentrated from autologous blood, and this is a broad-spectrum MMP inhibitor. All of these have some promise but need further development. The biggest issue with all these autologous formulations is the tremendous heterogeneity. When we use a drug, it's a known drug at a known concentration. In contrast, all these blood formulations, we really need to characterize what we're actually giving to our patient. What's the data on PRP for rotator cuff tendinopathy? Kind of mixed. Andy Carr in the UK had a group of patients undergoing a chromoplasty, randomized to PRP or none, and they found no difference in their outcome scores. In fact, they did tendon biopsies in these patients at 12 weeks. They found increased apoptosis and decreased cellularity in those patients treated with PRP, so they suggested a potentially deleterious effect of PRP in tendinopathy. The study from Finland, patients received three PRP injections every two weeks versus steroid injection. Both groups improved but really no differences between groups, so again, no real support here. Group from Taiwan, patients with partial thickness cuff tears treated with a single PRP injection versus three injections of HA. They found improvements in both groups. They thought the PRP group was better as far as range of motion, so some positive data here. Another study from China here, a double-blind randomized trial of PRP versus steroid injection. Actually, this trial is from Ian Lowe in Calgary. This is with PRP versus steroid injection. They found the PRP group is superior at three months but no difference at 12 months, so sort of mixed data here. The bottom line, to my reads, the data is certainly inconclusive and I do not routinely recommend PRP at this time for rotator cuff tendinopathy. What about rotator cuff repair? Chris Joe in Seoul, Korea has a randomized controlled trial where patients received three PRP gels applied to the repair site. They found the re-tear rate in the PRP group was lower than the conventional repair, so they found some positive data here for repair. Pietro Randelli in Italy, a randomized controlled trial. They had 38 out of 53 patients they were able to follow up to 10 years and they found no difference in groups at that 10-year follow-up. And lastly, a group from China here, a randomized blinded controlled trial published just this year. 52 patients with standard repair, 52 with PRP. These patients received three injections of leukocyte-poor PRP and they found improved re-tear rates or improved healing rates in the PRP group. They found the Goutelier grade, the fat infiltration was better in the PRP group, so they report positive data here. Meta-analyses in this area of PRP do suggest improved structural healing using PRP. This particular study reported just this year, a meta-analysis looking at leukocyte-poor PRP was found to be, has significantly reduced re-tear rates in patients undergoing cuff repair and the leukocyte-poor was better than patients receiving leukocyte-rich, so positive data here. This group from the UK, another systematic review of meta-analyses, they reported PRP leads to a lower number of re-tears, improved short-term post-operative scores and functional outcomes. And they found the leukocyte content really didn't make a difference, so some positive data here. And this last group from China, systematic review, they found that PRP resulted in significantly decreased rates of re-tear, 16% versus 29%. The PRP group had improved patient-party outcome measures, and they also concluded that PRP was superior to a platelet-rich fibromatrix, so overall positive data here for the PRP, but not for platelet-rich fibromatrix. And in fact, so the bottom line here is that PRP appears beneficial for improvement in structural healing based on these recent meta-analyses. Now, if we look just at platelet-rich fibromatrix, you can see in the picture on the right there, it's taken a PRP and a second spin with calcium chloride to make this fibromatrix. We have a study we reported 10 years ago, a prospective trial, patients receiving either a platelet-rich fibromatrix or just standard repair, and we used ultrasound at 12 weeks. We found a lower healing rate in those with the platelet-rich fibromatrix, 67% versus 81% in our controls. No real differences in patient-party outcome, strength, or tendon vascularity, and using a logistic regression, we found that those patients receiving the platelet-rich fibromatrix, actually that material was a predictor for tendon defect at 12 weeks, so in fact, a negative outcome here. Perley, another meta-analysis, they compared PRP versus platelet-rich fibromatrix. PRP-treated tears had improved healing rates. In contrast, those tears treated with platelet-rich fibromatrix did not, so again, not in support of a PRFM, so the bottom line here is that platelet-rich fibromatrixes are not recommended for augmentation of rotator cuff repair at this time. My current recommendation as far as a post-injection treatment protocol if you're using PRP, note that the recommendations for rehab and activity following PRP are really largely empirical and are based on very little robust clinical or basic science data. Some have recommended avoidance of a non-steroidal medication, but there really is very little basic science or clinical data to support this recommendation of avoiding anti-platelet medications. Our current recommendation is light activity for three days, followed by a gradual progression. This just kind of makes sense kind of clinically. Eccentric exercise regimen is typically recommended for tendinopathy in other areas. We clearly need further data on the effect of mechanical load on tendon biology and the biologic response to PRP because this can help us to guide our post-injection rehabilitation protocols. This is a little review we published a year ago. The research question was, do non-steroidals and other anti-platelet therapies inhibit the therapeutic efficacy of PRP? This is a literature review. We found 12 studies. 11 of these were just in vitro analyses of growth factors and various cytokines and how those affected by non-steroidals or anti-platelet medications. One study examined outcomes in an animal model. About half of the studies showed no effect of non-steroidals or anti-platelet therapies. Half the studies showed mixed effects. No studies showed a consistent or a significant effect of non-steroidals or anti-platelet therapies on the use of PRP. So we concluded that there really is very little support at this time for the common clinical practice of withholding anti-platelet therapies in patients being treated with PRP. Lastly, we should consider some systemic factors. For example, vitamin D. We know vitamin D affects osteoblastic activity and muscle function. In fact, preclinical studies suggest vitamin D deficiency is related to impaired tendon healing. In a clinical study from Japan, they reported that lower serum levels of vitamin D was related to higher levels of fatty degeneration in rotator cuff muscles and decreased muscle strength. And some more recent large national insurance databases suggest that perioperative vitamin D levels affect the outcome of repair based on greater revision surgery or odds of failure that were higher in patients that are vitamin D deficient. And so this is a modifiable risk factor. We may consider measuring vitamin D levels and perioperative supplementation in our patients. What about PTH, parathyroid hormone? We know it affects osteoblastic activity and that's irrelevant because healing in the rotator cuff proceeds by bone ingrowth into the outer tendon. In fact, preclinical animal studies suggest that PTH can improve tendon healing. In one clinical study reported several years ago, 31 patients received daily injections of PTH for three months postoperatively. And they compared this to a propensity score matched untreated control group and they found the rate of re-tear was lower in those patients being treated with PTH. So clinically, this is certainly reasonable to consider, but practically, it's costly and usually not approved by insurance carriers. Other metabolic pathways that you consider that may be exploited for biologic augmentation in the rotator cuff, statins, there is some evidence that simvastatin reduces the risk of tendinopathy and reduces the rate of revision rotator cuff repair. I think further work in our lab and others look at the role of reactive oxygen species and oxygen-free radicals or look at mitochondrial dysfunction, which is implicated in tendinopathy. And then concentics, currently used medication to inhibit IL-17, there's some robust preclinical data from Neil Millar in Scotland and there's some clinical trials underway in this area as well. And lastly, there are some factors that have inhibitory effects on healing listed here, hypercholesterolemia, diabetes, smoking, of course. We mentioned low vitamin D and obesity. All of these should be considered. So in summary, I think we can consider PRP for rotator cuff repair. Leukocyte-reduced formulations seem to be optimal. I would not routinely use PRP for tendinopathy or subacromial impingement at this time. I think adipose-derived cells appear to be promising, but certainly needs further work. Consider vitamin D supplementation if deficient. We may consider PTH as well. I still minimize early use of non-steroidal medication, although this is controversial because we know healing proceeds by the initial inflammatory process. Consider checking serum cholesterol, again, in controlling diabetes, continuing statin therapy in those patients on statins, and of course, stopping smoking. Lastly, we know there's some data now on managing obesity that may have a negative effect on healing as well. I'll stop there. Thank you.

biologic augmentation

rotator cuff tendon repair

tissue repair

cell-based preparations

platelet-rich plasma

tissue regeneration