A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation / 中国修复重建外科杂志

OBJECTIVE@#To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation.@*METHODS@#Firstly, hyaluronic acid was oxidized with NaIO 4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR).@*RESULTS@#FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05).@*CONCLUSION@#The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.

Effect of electroacupuncture on tissue elasticity and expression of muscular MyoD and myogenin in rats with acute gastrocnemius contusion / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Ashi" acupoint and "Kunlun" (BL60) on elastic modulus, histopathological changes and expression of myogenic regulatory factors in gastrocnemius(GM) contusion rats, so as to explore the therapeutic effect of local acupoint selection and acupoint selection along channel. METHODS: Male SD rats were randomly divided into blank control (n＝5), model (n＝15), Ashi-point (n＝15) and BL60 (n＝15) groups. The acute GM contusion model was established by striking (free falling) the GM with a homemade hitter. EA (0.5 to 1.0 mA, 2 Hz/10 Hz) was applied to Ashi-point (local focus) and BL60 for 30 min 24 h after muscle injury. The elasticity maximum (Emax) of gastrocnemius muscle was measured by using an ultrasonic device. Histopathological changes were observed after H．E. stain, and the number of Myogenic differentiation(MyoD)- and Myogenin (MyoG)-positive cells was detected by using immunohistochemistry. RESULTS: After mdeling, the Emax value of GM was significantly increased from the 3rd h to 7th day in comparison with pre- injury of muscle (P<0.05), and was markedly increased on the 3rd day and obviously lower on day 7 in the Ashi-point group than in the model group (P<0.05). The numbers of MyoD- and MyoG-positive cells of GM were significantly increased on day 7 in the model group than in the blank control group (P<0.05), and both further increased in Ashi-point on day 3 and 5, and MyoG-positive cells further increased in BL60 group on day 5 and in Ashi-point group on day 7 relevant to the model group(P<0.05). The therapeutic effect of EA-Ashi-point was apparently superior to that of BL60 in up-regulating Emax on day 3 and in up-regulating the number of MyoD-positive cells on day 3 and 5 (P<0.05). H．E. stain showed disordered arrangement of muscle fibers, infiltration of inflammatory cells, increase of intercellular space, and edema on day 3 after modeling (which was milder in the Ashi-point group); and gradual fusion and thickening of new born muscle fibers with obvious connective tissue hyperplasia converged to the lesioned region on day 7 in the model group (convergence of new born muscle cells to the lesion region in both EA groups, and more complete tissues in the Ashi-point group). CONCLUSION: EA of Ashi-point and BL60 can up-regulate the expression of myogenic regulatory factors MyoD and MyoG of GM tissue in GM contusion rats, which may contribute to its function in promoting recovery of muscle elasticity. The role of EA-Ashi-point is superior to that of EA-BL60.

Isolation, cultivation and identification of skeletal muscle stem cells derived from human orbicularis oculi muscle / 中华整形外科杂志

Objective@#To establish the isolation and culture methods of skeletal muscle stem cells, derived from human orbicularis oculi muscle (OOMSCs), and to identify their multi-directional differentiation potential in vitro.@*Methods@#The cellswere isolated from pretarsal orbicularis oculi muscle (OOM), obtained in routine blepharoplasty surgery.The tissue was digested using collagenase type I combined with re-plating methods. Specific cell surface antigen markers were detected using flow cytometry analysis. OOMSCs were cultured under different inductive conditions, to identify their pluripotent differentiation ability.@*Results@#OOMSCs exhibited similar fibroblast-like morphology as mesenchymal stem cells with high expression of skeletal muscle-derived stem cell surface markers. OOMSCs were able to differentiate into adipocytes, osteoblasts and chondrocytes in vitro, in the presence of lineage-specific inductive media. Moreover, after myogenic induction, the differentiated cells were fused into multinucleated myotube-like structure, and positive for myogenic-related marks, Pax3, Pax7, Myf5 and MyoD.@*Conclusions@#Muscle-derived stem cells can be isolated from human OOM with myogenic differentiation properties, showing further applications potential intissue regeneration and medical therapies of muscle diseases.

MBP-FGF2-Immobilized Matrix Maintains Self-Renewal and Myogenic Differentiation Potential of Skeletal Muscle Stem Cells

The robust capacity of skeletal muscle stem cells (SkMSCs, or satellite cells) to regenerate into new muscles in vivo has offered promising therapeutic options for the treatment of degenerative muscle diseases. However, the practical use of SkMSCs to treat muscle diseases is limited, owing to their inability to expand in vitro under defined cultivation conditions without loss of engraftment efficiency. To develop an optimal cultivation condition for SkMSCs, we investigated the behavior of SkMSCs on synthetic maltose-binding protein (MBP)-fibroblast growth factor 2 (FGF2)-immobilized matrix in vitro. We found that the chemically well-defined, xeno-free MBP-FGF2-immobilized matrix effectively supports SkMSC growth without reducing their differentiation potential in vitro. Our data highlights the possible application of the MBP-FGF2 matrix for SkMSC expansion in vitro.

Effect of electroacupuncture serum on expression of myogenic differentiation antigen and autophagy-related protein Beclin 1 in cultured muscle satellite cells under starvation / 针刺研究

OBJECTIVE: To explore the effect of electroacupuncture (EA) serum on expression of myogenic differentiation antigen (Myod) and autophagy-related protein Beclin 1 in cultured muscle satellite cells of rats under starvation conditions. METHODS: The primary multifidus muscle satellite cells of one male SD rat were isolated and cultured to obtain the 3rd generation of cells. The EA serum was got from the rat received EA stimulation of bilateral "Weizhong" (BL40, 2 Hz/10 Hz, 1 mA, duration of 20 min, once daily for 7 days). The cell suspension (2×104/well) of the 3rd generation of cultured cells was transferred to each well of a 96-well plate in medium containing 10% fetal bovine serum (FBS). Twelve duplicate wells were set up for the blank control serum (without FBS), 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups and incubated for 12 h and 24 h, respectively. Each well was supplemented with 10 µL CCK-8 reagent to be incubated for 1 h again for observing the state of cell proliferation. After culturing the primary muscle satellite cells in serum-free medium for 12 h, the cells were randomly divided into serum-free group, 10% fetal bovine serum group and optimal concentration electroacupuncture serum group, and serum of corresponding concentration was added respectively. The expression levels of Beclin 1 and cell-proliferation-related protein Myod were detected by Western blot. RESULTS: CCK-8 assay displayed that the proliferation levels were significantly higher at 12 h and 24 h after serum intervention in the 10% FBS, 10% EA serum, 20% EA serum and 30% EA serum groups than that in the blank control serum group (P0.05). As a result, 10% EA serum was selected as the optimal concentration for Western blot tests. No significant difference was found in the expression levels of Myod and Beclin 1 proteins among the serum-free, 10% FBS and 10% EA serum groups before intervention (P>0.05), and there was a marked up-regulation of Myod expression and an obvious down-regulation of Beclin 1 expression at 12 h in both the 10% EA serum and 10% FBS groups in comparison with their own pre-intervention (P0.05). CONCLUSION: EA serum can promote proliferation of cultured muscle satellite cells under starvation conditions, which is related to its functions in regulating expression of Beclin 1 and cell-proliferation-related protein Myod.

Skeletal myogenic differentiation of human periodontal ligament stromal cells isolated from orthodontically extracted premolars / 대한치과교정학회지

OBJECTIVE: To investigate the stem cell-like characteristics of human periodontal ligament (PDL) stromal cells outgrown from orthodontically extracted premolars and to evaluate the potential for myogenic differentiation. METHODS: PDL stromal cells were obtained from extracted premolars by using the outgrowth method. Cell morphological features, self-replication capability, and the presence of cell-surface markers, along with osteogenic, adipogenic, and chondrogenic differentiation, were confirmed. In addition, myogenic differentiation was induced by the use of 5-aza-2'-deoxycytidine (5-Aza) for DNA demethylation. RESULTS: PDL stromal cells showed growth patterns and morphological features similar to those of fibroblasts. In contrast, the proliferation rates of premolar PDL stromal cells were similar to those of bone marrow and adipogenic stem cells. PDL stromal cells expressed surface markers of human mesenchymal stem cells (i.e., CD90 and CD105), but not those of hematopoietic stem cells (i.e., CD31 and CD34). PDL stromal cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages. Myotube structures were induced in PDL stromal cells after 5-Aza pretreatment, but not in the absence of 5-Aza pretreatment. CONCLUSIONS: PDL stromal cells isolated from extracted premolars can potentially be a good source of postnatal stem cells for oromaxillofacial regeneration in bone and muscle.

Effects of pulsed electromagnetic fields on the expression of MyoD after acute skeletal muscle contusion / 中华物理医学与康复杂志

Objective To observe the effects of pulsed electromagnetic fields (PEMFs) on histological changes and myogenic differentiation factor D (MyoD) expression in rats with acute skeletal muscle contusion ( ASMC), and to explore the effects of PEMF therapy on rats with ASMC in its very early stages. Methods Forty-two rats were randomly divided into three groups : a treatment group, a control group and a blank control group. ASMC models were established with all the animals in the treatment and control groups. PEMF treatment was admin-istered to the treatment group immediately after the establishment of the ASMC model. Seven rats in each group were sacrificed at the 12th and 18th h after the models were set up. Their triceps surae muscles were sampled and treated with haematoxylin-eosin staining for study using immunofluorescence techniques and a fluorescence microscope. Re-suits In the control group at the 12th h and 18th h, HE staining showed pale cytoplasm and polymorphism in the cell nuclei ; in the treatment group these effects were significantly lighter, but in both groups it was more serious than in the blank control group. In the treatment and control groups, the fluorescence intensity of MyoD at the 18th h was higher than at the 12th h, and at each time point in both groups it was higher than in the blank control group. At the 18th h, fluorescence in the treatment group was stronger than in the control group. Conclusion MyoD expression in rats with ASMC is upregulated by thel8th h after early PEMF treatment. This might be one of the mechanisms ac-celerating the regeneration of skeletal muscles after trauma.