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In severe skeletal muscle damage, muscle tissue regeneration process has to face the loss of resident muscle stem cells (MuSCs) and the lack of connective tissue necessary to guide the regeneration process. Biocompatible and standardized 3D structures that can be injected to the muscle injury site, conforming to the defect shape while actively guiding the repair process, holds great promise for skeletal muscle tissue regeneration. In this study, we explore the use of an injectable and porous lysine dendrimer/polyethylene glycol (DGL/PEG) hydrogel as an acellular support for skeletal muscle regeneration. We adjusted the DGL/PEG composition to achieve a stiffness conducive to the attachment and proliferation of murine immortalized myoblasts and human primary muscle stems cells, sustaining the formation and maturation of muscle fibers in vitro . We then evaluated the potential of one selected “myogenic-porous hydrogel” as a supportive structure for muscle repair in a large tibialis anterior muscle defect in rats. This injectable and porous formulation filled the defect, promoting rapid cellularization with the presence of endothelial cells, macrophages, and myoblasts, thereby supporting neo-myogenesis more specifically at the interface between the wound edges and the hydrogel. The selected porous DGL/PEG hydrogel acted as a guiding scaffold at the periphery of the defect, facilitating the formation and anchorage of aligned muscle fibers 21 days after injury. Overall, our results indicate DGL/PEG porous injectable hydrogel potential to create a pro-regenerative environment for muscle cells after large skeletal muscle injuries, paving the way for acellular treatment in regenerative muscle medicine.

Abstract Background The capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies. Methods In the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile. Results We observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake. Conclusions Overall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

Abstract Satellite cells are tissue resident muscle stem cells required for postnatal skeletal muscle growth and repair through replacement of damaged myofibers. Muscle regeneration is coordinated through different mechanisms, which imply cell‐cell and cell‐matrix interactions as well as extracellular secreted factors. Cellular dynamics during muscle regeneration are highly complex. Immune, fibrotic, vascular and myogenic cells appear with distinct temporal and spatial kinetics after muscle injury. Three main phases have been identified in the process of muscle regeneration; a destruction phase with the initial inflammatory response, a regeneration phase with activation and proliferation of satellite cells and a remodeling phase with maturation of the regenerated myofibers. Whereas relatively minor muscle injuries, such as strains, heal spontaneously, severe muscle injuries form fibrotic tissue that impairs muscle function and lead to muscle contracture and chronic pain. Current therapeutic approaches have limited effectiveness and optimal strategies for such lesions are not known yet. Various strategies, including growth factors injections, transplantation of muscle stem cells in combination or not with biological scaffolds, anti‐fibrotic therapies and mechanical stimulation, may become therapeutic alternatives to improve functional muscle recovery.

Abstract Background The bicaval drainage under veno-venous extracorporeal membrane oxygenation (VV ECMO) was compared in present experimental study to the inferior caval drainage in terms of systemic oxygenation. Method Two mathematical models were built to simulate the inferior vena cava-to-right atrium (IVC → RA) route and the bicaval drainage-to-right atrium return (IVC + SVC → RA) route using the following parameters: cardiac output (Q C), IVC flow/Q C ratio, venous oxygen saturation, extracorporeal pump flow (Q EC), and pulmonary shunt (PULM-Shunt) to obtain pulmonary artery oxygen saturation (SPAO2) and systemic blood oxygen saturation (SaO2). Results With the IVC → RA route, SPAO2 and SaO2 increased linearly with Q EC/Q C until the threshold of the IVC flow/Q C ratio, beyond which the increase in SPAO2 reached a plateau. With the IVC + SVC → RA route, SPAO2 and SaO2 increased linearly with Q EC/Q C until 100% with Q EC/Q C = 1. The difference in required Q EC/Q C between the two routes was all the higher as SaO2 target or PULM-Shunt were high, and occurred all the earlier as PULM-Shunt were high. The required Q EC between the two routes could differ from 1.0 L/min (Q C = 5 L/min) to 1.5 L/min (Q C = 8 L/min) for SaO2 target = 90%. Corresponding differences of Q EC for SaO2 target = 94% were 4.7 L/min and 7.9 L/min, respectively. Conclusion Bicaval drainage under ECMO via the IVC + SVC → RA route gave a superior systemic oxygenation performance when both Q EC/Q C and pulmonary shunt were high. The VV-V ECMO configuration (IVC + SVC → RA route) might be an attractive rescue strategy in case of refractory hypoxaemia under VV ECMO.

Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.

Objective Low-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice.Methods Experimental OA was simulated on cartilage explants using interleukin-1β (IL-1β) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry.Results Whole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/- and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1β increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM.Conclusion NOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.

Abstract Satellite cells, localized within muscles in vivo, are Pax7+ muscle stem cells supporting skeletal muscle growth and regeneration. Unfortunately, their amplification in vitro, required for their therapeutic use, is associated with reduced regenerative potential. In the present study, we investigated if human myogenic reserve cells (MRC) obtained in vitro, represented a reliable cell source for muscle repair. For this purpose, primary human myoblasts were freshly isolated and expanded. After 2 days of differentiation, 62 ± 2.9% of the nuclei were localized in myotubes and 38 ± 2.9% in the mononucleated non-fusing MRC. Eighty percent of freshly isolated human MRC expressed a phenotype similar to human quiescent satellite cells (CD56+/Pax7+/MyoD−/Ki67− cells). Fourteen days and 21 days after cell transplantation in immunodeficient mice, live human cells were significantly more numerous and the percentage of Pax7+/human lamin A/C+ cells was 2 fold higher in muscles of animals injected with MRC compared to those injected with human myoblasts, despite that percentage of spectrin+ and lamin A/C+ human fibers in both groups MRC were similar. Taken together, these data provide evidence that MRC generated in vitro represent a promising source of cells for improving regeneration of injured skeletal muscles.

Myoblast transplantation represents a promising therapeutic strategy in the treatment of several genetic muscular disorders including Duchenne muscular dystrophy. Nevertheless, such an approach is impaired by the rapid death, limited migration, and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant for various cell types. Therefore, we investigated whether DXS could act as a “myoblast protectant” either in vitro or in vivo after transplantation in immunodeficient mice. In vitro, DXS bound human myoblasts in a dose dependent manner and significantly inhibited staurosporine-mediated apoptosis and necrosis. DXS pretreatment also protected human myoblasts from natural killer cell-mediated cytotoxicity. When human myoblasts engineered to express the renilla luciferase transgene were transplanted in immunodeficient mice, bioluminescence imaging analysis revealed that the proportion of surviving myoblasts 1 and 3 days after transplantation was two times higher when cells were preincubated with DXS compared to control (77.9 ± 10.1% vs. 39.4 ± 4.9%, p = 0.0009 and 38.1 ± 8.5% vs. 15.1 ± 3.4%, p = 0.01, respectively). Taken together, we provide evidence that DXS acts as a myoblast protectant in vitro and is able in vivo to prevent the early death of transplanted myoblasts.