Modest progress has been made in utilizing regenerative adjuvants to improve rotator cuff healing and repair. To date, most investigative efforts have focused on improving healing at the bone-tendon interface and have yielded mixed results25-30. Less attention has been focused on the roles of muscle atrophy and fatty infiltration and their effect on the healing of rotator cuff tears. Given the efficacy of MSCs in regenerating injured tissue in other musculoskeletal applications, we hypothesized that perivascular progenitor cells could be utilized in the treatment of massive rotator cuff tears to diminish fibroadipogenic degeneration.
Utilizing a mouse model of massive rotator cuff tears, our study demonstrated that there was significantly less muscle atrophy in the groups treated with PSCs compared with the respective controls for both TT and TT + DN. These findings were supported by both wet muscle weight and muscle fiber cross-sectional area data.
The reduction in muscle atrophy observed with PSC treatment could be explained by 2 phenomena. PSCs may have a regenerative effect in which they fuse to, or differentiate into, native myofibers. This would be consistent with recent evidence suggesting that progenitor cells can contribute to myogenesis either by differentiating and fusing to growing myofibers or by entering the satellite cell pool22. At the 2-week time point in the current study, injected PSCs were found to align with native myofibers of the injured rotator cuff. Furthermore, our co-localization immunohistochemistry studies suggested that CM-Dil PSCs did not adopt a fibrotic phenotype, given the lack of expression of the myofibroblast marker α-SMA. Lastly, RNA-sequencing analysis demonstrated that pericytes and adventitial cells express myogenesis-related genes such as FGF2 (fibroblast growth factor 2), FST (follistatin), HGF (hepatocyte growth factor), IGF1 (insulin-like growth factor 1), and IL6 (interleukin 6) (see Appendix).
PSCs may also have a prolonged trophic effect on the native muscle via growth-factor expression. The ability of these cell populations to produce growth factors known to enhance tissue repair, such as heparin-binding epidermal growth factor (HB-EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, and vascular endothelial growth factor (VEGF), has been previously described23. Additionally, we found that secretion of VEGF, a growth factor known to promote angiogenesis and enhance MSC survival31,32, was high in cultured PSCs (see Appendix). Therefore, one or multiple processes may contribute to the clinical and histological differences in muscle atrophy observed in the current study.
Additionally, we found that PSC injections did not contribute to increased fibrosis in the setting of rotator cuff tears. However, the TT + DN groups that received either prophylactic or therapeutic pericyte injections or therapeutic adventitial cells had less fibrosis than TT + DN controls. The absence of increased fibrosis is promising, as a potential complication with these injections is that the cells may follow a profibrotic pathway, leading to adverse outcomes33-35. However, the decreased fibrosis seen in the TT + DN groups suggests that the cells may inhibit these profibrotic processes. This is consistent with the results reported by Chen et al., utilizing pericytes in a mouse model of myocardial infarction21.
Some of our histomorphometric results differed between the prophylactic and therapeutic treatment groups. Specifically, wet muscle weight change for the therapeutic groups following TT + DN was significantly less than for the respective controls, while the prophylactic injection groups demonstrated no such change. These findings may be attributable to an altered microenvironment and increases in myogenic transcription factor expression following rotator cuff tears36. A previous study by Frey et al. demonstrated increased expression of the myogenic transcription factor Myf-5 in the weeks following rotator cuff tenotomy in an ovine model37, which could explain the more robust clinical response observed in our therapeutic treatment groups, by which the cells were delivered into a biological milieu primed to drive myogenic differentiation.
Finally, there was a modest though significant difference in fatty infiltration in the TT groups treated with PSCs compared with controls, but no differences in fatty infiltration among the TT + DN groups. The similarities in fatty infiltration among the TT + DN groups may be secondary to denervation, which has been shown to play an important role in the development of fatty infiltration14,38,39. Furthermore, while there was significantly less fatty infiltration in the TT groups, given the small absolute difference (∼1%), it is likely not clinically relevant. Nonetheless, our results suggest that the use of PSCs may contribute to the prevention of muscle atrophy by aiding in the maintenance of muscle bulk without leading to increased fibroadipogenesis.
There were limitations to our study, many of which are inherent to using animal models to replicate human pathology. Suprascapular nerve transection was required to produce massive fat infiltrate, consistent with the original model developed by Liu et al.15. While there are data demonstrating that suprascapular nerve palsy may contribute to fatty infiltration following rotator cuff injury40,41, complete transection of the suprascapular nerve rarely occurs in human disease. Thus, while several aspects of human rotator cuff pathology are recreated in this small animal model, it may not completely mirror the human disease process. While mice of similar age, weight, and sex were used in this study, normal variation in rotator cuff muscle weight may be present in this strain. We attempted to control for this variation by comparing the treatment effect in the experimental limb with values noted for the untreated limb in the same animal.
Additionally, our study utilized a mature, but relatively young, population of mice. While rotator cuff tears are more commonly observed in an older human population, mice of this age were chosen because young mice demonstrated degenerative muscle changes similar to those seen in humans in this previously validated model15. While the number of native progenitor cells has been shown to decrease with age42,43, the myogenic potential and intrinsic functionality of satellite cells appear to be age-independent44-46. However, additional studies in aged animals are necessary to further characterize the role of PSCs in reducing muscle atrophy.
Finally, to validate the use of human PSCs preclinically, we utilized immunodeficient mice as PSC recipients. While the complex interplay of immune responses following rotator cuff injury has not been fully elucidated47-50, it is possible that this model does not mirror the muscle changes that would be seen in an immunocompetent model. However, a study by Brzóska et al. suggested that skeletal muscle regeneration in severe combined immunodeficient [SCID] mice is similar to that in immunocompetent mice with respect to muscle fiber size, fibrosis, and fatty infiltration51. Nonetheless, additional studies investigating the role of inflammatory responses following rotator cuff tears would augment our understanding of fibroadipogenesis.
In summary, we demonstrated that 2 PSC populations—adventitial cells and pericytes—exhibit regenerative potential in the setting of massive rotator cuff tears. Currently, there is great heterogeneity among methods of stem cell procurement, methods of application, and cell types utilized in rotator cuff repair studies. Ultimately, the goal is to characterize and standardize a cell population with the greatest ease and efficacy of clinical translation. PSCs provide the distinct advantages of having an abundant and readily available source in adipose tissue and of being easily isolated via cell surface markers. Further investigation is necessary to fully elucidate the regenerative capacity of these cells and to better characterize their mechanism of action. Nonetheless, these results, which demonstrate the regenerative potential of these cells in a model of massive rotator cuff tears with no significant increases in fatty infiltration or fibrosis, suggest that they may serve as a viable perioperative therapy.
Tables detailing the PSC transcript expression of growth factors related to muscle cell growth and/or differentiation and PSC secretion of VEGF as demonstrated in RNA-sequencing analysis are available with the online version of this article as a data supplement at jbjs.org (http://links.lww.com/JBJS/A10).
Investigation performed at the University of California, Los Angeles, Los Angeles, California
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