Mesenchymal stromal/stem cells (MSCs) and their secretome are known to exert beneficial effects in many pathological states. However, MSCs therapeutic properties can be reduced due to unsuitable in vitro maintenance conditions. Standard culture protocols neglect the fact that MSCs exist in vivo in the closest connection with the extracellular matrix (ECM), the complex protein network providing an instructive microenvironment. We found recently that conditioned medium from human endometrial MSCs cultured on cell-derived decellularized extracellular matrix (CM-dECM) is dramatically enriched in a number of paracrine factors such as GM-CSF, FGF-2, HGF, MMP-1, MCP-1, IL-6, IL-8, CXCL-1, -2, -5, -6 (Ushakov et al., 2024). Given that several upregulated molecules belong to myokines that are known to participate in skeletal muscle regeneration, we hypothesized that CM-dECM may promote restoration of damaged muscle tissue. Here, we found that CM-dECM injections into barium chloride-injured murine m. tibialis anterior caused myofiber hypertrophy and promoted angiogenesis. Besides, CM-dECM significantly contributed to progression of murine C2C12 myoblasts cell cycle suggesting that muscle repair in vivo may be connected with stimulation of resident myoblasts proliferation. In this study, a role for secretome of endometrial MSCs cultured on dECM in injured murine skeletal muscle regeneration was outlined first. Our findings demonstrate that culture on dECM may be considered as a novel preconditioning approach enhancing MSCs therapeutic potential.

Muscle development is a multistep process regulated by diverse gene networks, and circRNAs are considered novel regulators mediating myogenesis. Here, we systematically analyzed the role and underlying regulatory mechanisms of circRBBP7 in myoblast proliferation and differentiation. Results showed that circRBBP7 has a typical circular structure and encodes a 13 -kDa protein. By performing circRBBP7 overexpression and RNA interference, we found that the function of circRBBP7 was positively correlated with the proliferation and differentiation of myoblasts. Using RNA sequencing, we identified 1633 and 532 differentially expressed genes (DEGs) during myoblast proliferation or differentiation, respectively. The DEGs were found mainly enriched in cell cycle- and skeletal muscle development-related pathways, such as the MDM2/p53 and PI3K-Akt signaling pathways. Further co-IP and IF co-localization analysis revealed that VEGFR-1 is a target of circRBBP7 in myoblasts. qRT-PCR and WB analysis further confirmed the positive correlation between VEGFR-1 and circRBBP7. Moreover, we found that in vivo transfection of circRBBP7 into injured muscle tissues significantly promoted the regeneration and repair of myofibers in mice. Therefore, we speculate that circRBBP7 may affect the activity of MDM2 by targeting VEGFR-1, altering the expression of muscle development-related genes by mediating p53 degradation, and ultimately promoting myoblast development and muscle regeneration. This study provides essential evidence that circRBBP7 can serve as a potential target for myogenesis regulation and a reference for the application of circRBBP7 in cattle genetic breeding and muscle injury treatment.

Efficient repair of skeletal muscle relies upon the precise coordination of cells between the satellite cell niche and innate immune cells that are recruited to the site of injury. The expression of pro-inflammatory cytokines and chemokines such as TNFα, IFNγ, CXCL1, and CCL2, by muscle and tissue resident immune cells recruits neutrophils and M1 macrophages to the injury and activates satellite cells. These signal cascades lead to highly integrated temporal and spatial control of muscle repair. Despite the therapeutic potential of these factors for improving tissue regeneration after traumatic and chronic injuries, their transcriptional regulation is not well understood. The transcription factor Mohawk (Mkx) functions as a repressor of myogenic differentiation and regulates fiber type specification. Embryonically, Mkx is expressed in all progenitor cells of the musculoskeletal system and is expressed in human and mouse myeloid lineage cells. An analysis of mice deficient for Mkx revealed a delay in postnatal muscle repair characterized by impaired clearance of necrotic fibers and smaller newly regenerated fibers. Further, there was a delay in the expression of inflammatory signals such as Ccl2, Ifnγ, and Tgfß. This was coupled with impaired recruitment of pro-inflammatory macrophages to the site of muscle damage. These studies demonstrate that Mkx plays a critical role in adult skeletal muscle repair that is mediated through the initial activation of the inflammatory response.

Photobiomodulation therapy (PBM) has shown positive effects when applied locally to modulate the inflammatory process and facilitate muscle repair. However, the available literature on the mechanisms of action of vascular photobiomodulation (VPBM), a non-invasive method of vascular irradiation, specifically in the context of local muscle repair, is limited. Thus, this study aimed to assess the impact of vascular photobiomodulation (VPBM) using a low-level laser (LLL) on the inflammatory response and the process of skeletal muscle repair whether administered prior to or following cryoinjury-induced acute muscle damage in the tibialis anterior (TA) muscles. Wistar rats (n = 85) were organized into the following experimental groups: (1) Control (n = 5); (2) Non-Injury + VPBM (n = 20); (3) Injured (n = 20); (4) Pre-VPBM + Injury (n = 20); (5) Injury + Post-VPBM (n = 20). VPBM was administered over the vein/artery at the base of the animals\' tails (wavelength: 780 nm; power: 40 mW; application area: 0.04 cm2; energy density: 80 J/cm2). Euthanasia of the animals was carried out at 1, 2, 5, and 7 days after inducing the injuries. Tibialis anterior (TA) muscles were collected for both qualitative and quantitative histological analysis using H&E staining and for assessing protein expression of TNF-α, MCP-1, IL-1β, and IL-6 via ELISA. Blood samples were collected and analyzed using an automatic hematological analyzer and a leukocyte differential counter. Data were subjected to statistical analysis (ANOVA/Tukey). The results revealed that applying VPBM prior to injury led to an increase in circulating neutrophils (granulocytes) after 1 day and a subsequent increase in monocytes after 2 and 5 days, compared to the Non-Injury + VPBM and Injured groups. Notably, an increase in erythrocytes and hemoglobin concentration was observed in the Non-Injury + VPBM group on days 1 and 2 in comparison to the Injured group. In terms of histological aspects, only the Prior VPBM + Injured group exhibited a reduction in the number of inflammatory cells after 1, 5, and 7 days, along with an increase in blood vessels at 5 days. Both the Prior VPBM + Injured and Injured + VPBM after groups displayed a decrease in myonecrosis at 1, 2, and 7 days, an increase in newly-formed and immature fibers after 5 and 7 days, and neovascularization after 1, 2, and 7 days. Regarding protein expression, there was an increase in MCP-1 after 1 and 5 days, TNF-α, IL-6, and IL-1β after 1, 2, and 5 days in the Injured + VPBM after group when compared to the other experimental groups. The Prior VPBM + Injured group exhibited increased MCP-1 production after 2 days, in comparison to the Non-Injury + VPBM and Control groups. Notably, on day 7, the Injured group continued to show elevated MCP-1 protein expression when compared to the VPBM groups. In conclusion, VPBM effectively modulated hematological parameters, circulating leukocytes, the protein expression of the chemokine MCP-1, and the proinflammatory cytokines TNF-α and IL-1β, ultimately influencing the inflammatory process. This modulation resulted in a reduction of myonecrosis, restoration of tissue architecture, increased formation of newly and immature muscle fibers, and enhanced neovascularization, with more pronounced effects when VPBM was applied prior to the muscle injury.

Our previous study indicated that whey protein hydrolysate (WPH) showed effective anti-fatigue properties, but its regulatory mechanism on recovery from exercise in mice is unclear. In the present study, we divided the mice into control, WP, and WPH groups and allowed them to rest for 1 h and 24 h after exercise, respectively. The changes in muscle metabolites of mice in the recovery period were investigated using metabolomics techniques. The results showed that the WPH group significantly up-regulated 94 muscle metabolites within 1 h of rest, which was 1.96 and 2.61 times more than the control and WP groups, respectively. In detail, significant decreases in TCA cycle intermediates, lipid metabolites, and carbohydrate metabolites were observed in the control group during exercise recovery. In contrast, administration with WP and WPH enriched more amino acid metabolites within 1 h of rest, which might provide a more comprehensive metabolic environment for muscle repair. Moreover, the WPH group remarkably stimulated the enhancement of lipid, carbohydrate, and vitamin metabolites in the recovery period which might provide raw materials and energy for anabolic reactions. The result of the western blot further demonstrated that WPH could promote muscle repair via activating the Sestrin2/Akt/mTOR/S6K signaling pathway within 1 h of rest. These findings deepen our understanding of the regulatory mechanisms by WPH to promote muscle recovery and may serve as a reference for comprehensive assessments of protein supplements on exercise.

Volumetric muscle loss (VML), a severe muscle tissue loss from trauma or surgery, results in scarring, limited regeneration, and significant fibrosis, leading to lasting reductions in muscle mass and function. A promising approach for VML recovery involves restoring vascular and neural networks at the injury site, a process not extensively studied yet. Collagen hydrogels have been investigated as scaffolds for blood vessel formation due to their biocompatibility, but reconstructing blood vessels and guiding innervation at the injury site is still difficult. In this study, collagen hydrogels with varied densities of vessel-forming cells are implanted subcutaneously in mice, generating pre-vascularized hydrogels with diverse vessel densities (0-145 numbers/mm2) within a week. These hydrogels, after being transplanted into muscle injury sites, are assessed for muscle repair capabilities. Results showed that hydrogels with high microvessel densities, filling the wound area, effectively reconnected with host vasculature and neural networks, promoting neovascularization and muscle integration, and addressing about 63% of the VML.

Developing scalable vascularized and innervated tissue is a critical challenge for the successful clinical application of tissue-engineered constructs. Collagen hydrogels are extensively utilized in cell-mediated vascular network formation because of their naturally excellent biological properties. However, the substantial increase in hydrogel contraction induced by populated cells limits their long-term use. Previous studies attempted to mitigate this issue by concentrating collagen pre-polymer solutions or synthesizing covalently crosslinked collagen hydrogels. However, these methods only partially reduce hydrogel contraction while hindering blood vessel formation within the hydrogels. To address this challenge, we introduced additional support in the form of a supportive spacer to counteract the contraction forces of populated cells and prevent hydrogel contraction. This approach was found to promote cell spreading, resist hydrogel contraction, control hydrogel/tissue geometry, and even facilitate the engineering of functional blood vessels and host nerve growth in just one week. Subsequently, implanting these engineered tissues into muscle defect sites resulted in timely anastomosis with the host vasculature, leading to enhanced myogenesis, increased muscle innervation, and the restoration of injured muscle functionality. Overall, this innovative strategy expands the applicability of collagen hydrogels in fabricating large vascularized nerve tissue constructs for repairing volumetric muscle loss (∼63 %) and restoring muscle function.

Current treatment for complex and large-scale volumetric muscle loss (VML) injuries remains a limited success and have substantial disadvantages, due to the irreversible loss of muscle mass, slow muscle regeneration, and rapid formation of non-functional fibrosis scars. These VML injuries are accompanied by denervation and the destruction of native vasculature which increases difficulties in the functional restoration of muscle. Here, reconstruction of the vascular network at the injury site was offered as a possible solution for improving the repair of muscle defects through the timely supply of nutrients and oxygen to surrounding cells. A hydrogel-based tissue construct containing various densities of the vascular network was successfully created in the subcutaneous space of mice by manipulating hydrogel properties, and then implanted into the VML injury site. One month after implantation, the mouse treated with the highly vascularized tissue had extensive muscle repair at the injury site and only spent a shorter time completing the inclined plane tests. These findings suggest that the reconstruction of the functional vascular network at the VML injury site accelerated muscle fiber repair through a timely supply of sufficient blood and avoided invasion by host fibroblasts.

Platelets have important hemostatic functions in repairing blood vessels upon tissue injury. Cytokines, growth factors, and metabolites stored in platelet α-granules and dense granules are released upon platelet activation and clotting. Emerging evidence indicates that such platelet-derived signaling factors are instrumental in guiding tissue regeneration. Here, we discuss the important roles of platelet-secreted signaling factors in skeletal muscle regeneration. Chemokines secreted by platelets in the early phase after injury are needed to recruit neutrophils to injured muscles, and impeding this early step of muscle regeneration exacerbates inflammation at later stages, compromises neo-angiogenesis and the growth of newly formed myofibers, and reduces post-injury muscle force production. Platelets also contribute to the recruitment of pro-regenerative stromal cells from the adipose tissue, and the platelet releasate may also regulate the metabolism and proliferation of muscle satellite cells, which sustain myogenesis. Therefore, harnessing the signaling functions of platelets and the platelet secretome may provide new avenues for promoting skeletal muscle regeneration in health and disease.

Injury to skeletal muscle through trauma, physical activity, or disease initiates a process called muscle regeneration. When injured myofibers undergo necrosis, muscle regeneration gives rise to myofibers that have myonuclei in a central position, which contrasts the normal, peripheral position of myonuclei. Myofibers with central myonuclei are called regenerating myofibers and are the hallmark feature of muscle regeneration. An important and underappreciated aspect of muscle regeneration is the maturation of regenerating myofibers into a normal sized myofiber with peripheral myonuclei. Strikingly, very little is known about processes that govern regenerating myofiber maturation after muscle injury. As knowledge of myofiber formation and maturation during embryonic, fetal, and postnatal development has served as a foundation for understanding muscle regeneration, this narrative review discusses similarities and differences in myofiber maturation during muscle development and regeneration. Specifically, we compare and contrast myonuclear positioning, myonuclear accretion, myofiber hypertrophy, and myofiber morphology during muscle development and regeneration. We also discuss regenerating myofibers in the context of different types of myofiber necrosis (complete and segmental) after muscle trauma and injurious contractions. The overall goal of the review is to provide a framework for identifying cellular and molecular processes of myofiber maturation that are unique to muscle regeneration.