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The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from

Mesenchymal-derived stromal or progenitor cells, commonly called "MSCs," have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.

Mesenchymal stem cells (MSCs) have been proven to promote tissue repair. However, concerns related to their clinical application and regulatory hurdles remain. Recent data has demonstrated the proregenerative secretome of MSCs can result in similar effects in the absence of the cells themselves. Within the secretome, exosomes have emerged as a promising regenerative component. Exosomes, which are nanosized lipid vesicles secreted by cells, encapsulate micro-RNA (miRNA), RNA, and proteins that drive MSCs regenerative potential with cell specific content. As such, there is an opportunity to optimize the regenerative potential of MSCs, and thus their secreted exosome fraction, to improve clinical efficacy. Exercise is one factor that has been shown to improve muscle progenitor cell function and regenerative potential. However, the effect of exercise on MSC exosome content and function is still unclear. To address this, we used an in vitro culture system to evaluate the effects of mechanical strain, an exercise mimetic, on C2C12 (muscle progenitor cell) exosome production and proregenerative function. Our results indicate that the total exosome production is increased by mechanical strain and can be regulated with different tensile loading regimens. Furthermore, we found that exosomes from mechanically stimulated cells increase proliferation and myogenic differentiation of naïve C2C12 cells. Lastly, we show that exosomal miRNA cargo is differentially expressed following strain. Gene ontology mapping suggests positive regulation of bone morphogenetic protein signaling, regulation of actin-filament-based processes, and muscle cell apoptosis may be at least partially responsible for the proregenerative effects of exosomes from mechanically stimulated C2C12 muscle progenitor cells.

Duchenne Muscular Dystrophy (DMD) patients often suffer from both muscle wasting and osteoporosis. Our previous studies have revealed reduced regeneration potential in skeletal muscle and bone, concomitant with ectopic calcification of soft tissues in double knockout (dKO, dystrophin-/-; utrophin-/-) mice, a severe murine model for DMD. We found significant involvement of RhoA/ROCK (Rho-Associated Protein Kinase) signaling in mediating ectopic calcification of muscles in dKO mice. However, the cellular identity of these RhoA+ cells, and the role that RhoA plays in the chronic inflammation-associated pathologies has not been elucidated. Here, we report that CD68+ macrophages are highly prevalent at the sites of ectopic calcification of dKO mice, and that these macrophages highly express RhoA. Macrophages from dKO mice feature a shift towards a more pro-inflammatory M1 polarization and an increased expression of various senescence-associated secretory phenotype (SASP) factors that was reduced with the RhoA/ROCK inhibitor Y-27632. Further, systemic inhibition of RhoA activity in dKO mice led to reduced number of RhoA+/CD68+ cells, as well as a reduction in fibrosis and ectopic calcification. Together, these data revealed that RhoA signaling may be a key regulator of imbalanced mineralization in the dystrophic musculoskeletal system and consequently a therapeutic target for the treatment of DMD or other related muscle dystrophies.

Aging-related loss of adult stem cell function contributes to impaired tissue regeneration. Mice deficient in zinc metalloproteinase STE24 (Zmpste24−/−) exhibit premature age-related musculoskeletal pathologies similar to those observed in children with Hutchinson-Gilford progeria syndrome (HGPS). We have reported that muscle-derived stem/progenitor cells (MDSPCs) isolated from Zmpste24−/− mice are defective in their proliferation and differentiation capabilities in culture and during tissue regeneration. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth, and inhibition of the mTORC1 pathway extends the lifespan of several animal species. We therefore hypothesized that inhibition of mTORC1 signaling would rescue the differentiation defects observed in progeroid MDSPCs. MDSPCs were isolated from Zmpste24−/− mice, and the effects of mTORC1 on MDSPC differentiation and function were examined. We found that mTORC1 signaling was increased in senescent Zmpste24−/− MDSPCs, along with impaired chondrogenic, osteogenic, and myogenic differentiation capacity versus wild-type MDSPCs. Interestingly, we observed that mTORC1 inhibition with rapamycin improved myogenic and chondrogenic differentiation and reduced levels of apoptosis and senescence in Zmpste24−/− MDSPCs. Our results demonstrate that age-related adult stem/progenitor cell dysfunction contributes to impaired regenerative capacities and that mTORC1 inhibition may represent a potential therapeutic strategy for improving differentiation capacities of senescent stem and muscle progenitor cells.

The primary risk factor for most musculoskeletal diseases, including osteoarthritis, osteoporosis and sarcopenia, is aging. To treat the diverse types of musculoskeletal diseases and pathologies, targeting their root cause, the aging process itself, has the potential to slow or prevent multiple age-related musculoskeletal conditions simultaneously. However, the development of approaches to delay onset of age related diseases, including musculoskeletal pathologies, has been slowed by the relatively long lifespan of rodent models of aging. Thus, to expedite the development of therapeutic approaches for age-related musculoskeletal disease, the implementation of mouse models of accelerated musculoskeletal aging are of great utility. Currently there are multiple genetically diverse mouse models that mirror certain aspects of normal human and mouse aging. Here, we provide a review of some of the most relevant murine models of accelerated aging that mimic many aspects of natural musculoskeletal aging, highlighting their relative strengths and weaknesses. Importantly, these murine models of accelerated aging recapitulate phenotypes of musculoskeletal age-related decline observed in humans.

Aging affects bones, cartilage, muscles, and other connective tissue in the musculoskeletal system, leading to numerous age-related pathologies including osteoporosis, osteoarthritis, and sarcopenia. Understanding healthy aging may therefore open new therapeutic targets, thereby leading to the development of novel approaches to prevent several age-related orthopaedic diseases. It is well recognized that aging-related stem cell depletion and dysfunction leads to reduced regenerative capacity in various musculoskeletal tissues. However, more recent evidence suggests that dysregulated autophagy and cellular senescence might be fundamental mechanisms associated with aging-related musculoskeletal decline. The mammalian/mechanical target of Rapamycin (mTOR) is known to be an essential negative regulator of autophagy, and its inhibition has been demonstrated to promote longevity in numerous species. Besides, several reports demonstrate that selective elimination of senescent cells and their cognate Senescence-Associated Secretory Phenotype (SASP) can mitigate musculoskeletal tissue decline. Therefore, senolytic drugs/agents that can specifically target senescent cells, may offer a novel therapeutic strategy to treat a litany of age-related orthopaedic conditions. This chapter focuses on osteoarthritis and osteoporosis, very common debilitating orthopaedic conditions, and reviews current concepts highlighting new therapeutic strategies, including the mTOR inhibitors, senolytic agents, and mesenchymal stem cell (MSC)-based therapies.

Hutchinson–Gilford progeria syndrome (HGPS) is caused by the accumulation of mutant prelamin A (progerin) in the nuclear lamina, resulting in increased nuclear stiffness and abnormal nuclear architecture. Nuclear mechanics are tightly coupled to cytoskeletal mechanics via lamin A/C. However, the role of cytoskeletal/nuclear mechanical properties in mediating cellular senescence and the relationship between cytoskeletal stiffness, nuclear abnormalities, and senescent phenotypes remain largely unknown. Here, using muscle‐derived mesenchymal stromal/stem cells (MSCs) from the Zmpste24 −/− (Z24 −/−) mouse (a model for HGPS) and human HGPS fibroblasts, we investigated the mechanical mechanism of progerin‐induced cellular senescence, involving the role and interaction of mechanical sensors RhoA and Sun1/2 in regulating F‐actin cytoskeleton stiffness, nuclear blebbing, micronuclei formation, and the innate immune response. We observed that increased cytoskeletal stiffness and RhoA activation in progeria cells were directly coupled with increased nuclear blebbing, Sun2 expression, and micronuclei‐induced cGAS‐Sting activation, part of the innate immune response. Expression of constitutively active RhoA promoted, while the inhibition of RhoA/ROCK reduced cytoskeletal stiffness, Sun2 expression, the innate immune response, and cellular senescence. Silencing of Sun2 expression by siRNA also repressed RhoA activation, cytoskeletal stiffness and cellular senescence. Treatment of Zmpste24− / − mice with a RhoA inhibitor repressed cellular senescence and improved muscle regeneration. These results reveal novel mechanical roles and correlation of cytoskeletal/nuclear stiffness, RhoA, Sun2, and the innate immune response in promoting aging and cellular senescence in HGPS progeria., There is increased RhoA activation and cytoskeleton stiffness in progeria cells, which mediates increased nuclear blebbing, micronuclei formation, innate immune response, and cellular senescence. RhoA activation is coupled with elevated Sun2 expression, but not Sun1, especially in nucleus with blebbing and micronuclei. Repression of RhoA and Sun2 activation was able to reduce cytoskeleton stiffness, nuclear blebbing, and micronuclei formation and delay the progression of cellular senescence.

Skeletal muscle is a highly interactive connective tissue that makes up a large portion of an adult's body mass. Muscles are integrated with tendons as well as other specialized structures to support physiologic and homeostatic functions. In the state of injury, muscles have the ability to innately repair themselves through various phases of tightly regulated healing that can result in fibrotic tissue development. However, during aging, these homeostatic processes are disrupted leading to sarcopenia and reduced muscle regeneration capacity. Several cell-based therapies and biologic therapies have been investigated to regenerate skeletal muscle tissue and reduce fibrosis following injury or during aging. These include platelet-rich plasma (PRP) and an acellular portion of blood known as platelet-poor plasma (PPP). However, current clinical practice recommendations for the utilization of different PRP preparations vs PPP are unclear. Recent efforts have strove to improve the understanding of the role of senescent cells and profiles in the presence of early to late stage skeletal muscle injury and fibrosis, yet targeted interventions to remove senescent cells and attenuate the secretory environment to improve muscle regeneration are still forthcoming. Therefore, the purpose of this article is to review the basic principles of skeletal muscle repair, the role of senescence in attenuated muscle regeneration, and discuss current standards and literature supporting PRP and PPP treatment for skeletal muscle repair. This review concludes with future directions to improve biologic therapies and ongoing initiatives to customize PRP and PPP preparations using Food and Drug Administration-approved medications.