Objective To review the current researches of scaffold materials for skeletal muscle tissue engineering, to predict the development trend of scaffold materials in skeletal muscle tissue engineering in future. Methods The related l iterature on skeletal muscle tissue engineering, involving categories and properties of scaffold materials, preparative techniqueand biocompatibil ity, was summarized and analyzed. Results Various scaffold materials were used in skeletal muscle tissue engineering, including inorganic biomaterials, biodegradable polymers, natural biomaterial, and biomedical composites. According to different needs of the research, various scaffolds were prepared due to different biomaterials, preparative techniques, and surface modifications. Conclusion The development trend and perspective of skeletal muscle tissue engineering are the use of composite materials, and the preparation of composite scaffolds and surface modification according to the specific functions of scaffolds.
Objective To explore the effects of mechanical stimulation on the expression of autoantigens in myoblasts. Methods According to different processing methods, C2C12 cells were divided into the experimental group and control group; the experimental group was divided into 4 subgroups: 2-, 4-, and 6-day and 1-day stretch groups. In 2-, 4-, and 6-day stretch groups, mechanical loading was added on the C2C12 cells at a stretching frequency of 0.25 Hz and cellular deformation amplitude of 10%, 2 hours a day for 2, 4, and 6 days respectively by Flexercell 5000 strain unit, and at a stretching frequency of 1 Hz and cellular deformation amplitude of 15% for 1 hour in 1-day stretch group. In the control group, the cells were routinely cultured for 1, 2, 4, and 6 days (1-, 2-, 4-, and 6-day control). The cells were observed by inverted phase contrast microscope. The cell proliferation was detected by flow cytometry; the expressions of autoantigens were detected by Western blot method, including the Ku/the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), U1-70 (A part of ATP-dependent DNA helicase II), histidyl tRNA synthetase (HRS), and Mi-2 (reconfigurable components deacetylase complexes of NuRD). Results The exfoliated cells were found in 1-day stretch group, but no exfoliated cell was seen in the control group for 1-day culture. The cells proliferated more obviously in 2-day stretch group than in the control group for 2-day culture; cell differentiation was found in 4-day stretch group, and cell fusion in 6-day stretch group, which were similar to those in the control group for 4- and 6-day culture. After single stretching, cell apoptosis was found in 1-day stretch group, showing no significant difference in the relative DNA proliferation index (DPI) when compared with DPI of control group for 1-day culture (t=0.346, P=0.747). After cyclic stretching, DPIs of 2- and 4- day stretch groups were significantly increased when compared with those of the control group for 2- and 4-day culture (P lt; 0.05), but no significant difference was found between control group for 6-day culture and 6-day stretch group (t=1.191, P=0.303). Compared with the control group for 2-day culture, the relative protein expression of autoantigens (DNA-Pkcs, Mi-2, HRS, and U1-70) in 2-day stretch group decreased significantly (P lt; 0.05), but no significant difference was found between control group for 4-day culture and 4-day stretch group (P gt; 0.05). The relative protein expressions of autoantigens in 4-day stretch group significantly increased when compared with those of 2-day stretch group (P lt; 0.05), but the relative protein expressions of autoantigens in the control group for 4-day culture significantly decreased when compared with those of the control group for 2-day culture (P lt; 0.05). Conclusion Short-term mechanical stimulation can inhibit the expressions of autoantigens in myoblasts, but with the time prolonging, cell differentiation and fusion and adaptation to mechanical stimulation would result in diminished inhibitory effect.
Objective To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes. Meanwhile the myoblasts were treated with various mechanical stretch withcells tensile instrument, at the amplitude of 10% and the frequency of 0 (group A), 0.25 (group B), 0.50 (group C), and 1.00 Hz (group D) for 1 hour, 3 times a day. The myotubes morphology was observed by inverted phase contrast microscope at 5, 7, and 10 days after continuous mechanical stretch. And the expressions of mRNA for myogenic differentiation antigen (MyoD), Myogenin, Desmin, and myosin heavy chain (MyHC) were detected by RT-PCR and real-time fluorescent quantitative PCR (QRT-PCR), respectively. Results The mechanical stretch could promote the al igned fusion and increase the number of myotubes. Indeed the multilayer myotubes arranged more closely in group B at 7 days. At the same group, as the time went on, the mRNA expressions of MyoD gradually decl ined in each group. There were significant differences in mRNA expressions of MyoD between 5 days and 7, 10 days (P lt; 0.05). The mRNA expressions of Myogenin, Desmin, and MyHC were highest at 7 days. There were significant differences between different time points (P lt; 0.05), except the mRNA expression of Desmin of group B between 7 and 10 days (P gt; 0.05). At the same time, with the increase of frequency, the highest mRNA expressions of MyoD, Myogenin, Desmin, and MyHC were in group B. There were significant differences at the same time between group B and the other groups (P lt; 0.05), except mRNA expression of Desmin at 5 days between groups B and C, and mRNA expression of MyHC at 10 days between groups A and B (P gt; 0.05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.
Objective To analyze the expression changes of Pax7 and MyoD in satell ite cells of denervated skeletal muscle at the mRNA and protein level, and to explore the pathway concerning with denervation and regeneration of skeletal muscle. Methods Nine male SD rats of 2 months old, weighing 180-200 g were selected. The right gastrocnemius musclewas made denervation model as experimental sides, and the same operation was performed in the left sides as control. The immunofluorescence analysis was done for detecting the protein expressions of Pax7 and MyoD in muscles denervated 3, 7 and 21 days after operation. The RT-PCR analysis was used to explore the mRNA expression of MyoD and Pax7 in extracted tissues from denervated muscles. Results Nine rats survived after operation, with l imited motion of right hind l imbs, and normal motion of left l imbs. The result of immunofluorescence with the polyclonal antibody against MyoD and the monoclonal antibody against Pax7 showed that the number of MyoD positive cells in prolonged denervated muscles (experimental sides) increased gradually, and reached the peak 21 days after denervation, there was significant difference compared with that of control sides (P lt; 0.05). However, the total number of Pax7 positive cells decreased with the prolongation of denervation, the expression of Pax7 was b only at 3 days, then decreased gradually, and there was no positive cells in denervated muscles of 21 days. There were significant differences in Pax7 positive cells between experimental sides and control sides at 3 and 7 days (P lt; 0.05). The results of immunostaining showed that MyoD mRNA levels increased , and reached the peak at 21 days of denervation, there was significant difference compared with that of control sides (P lt; 0.05). Whereas, Pax7 mRNA expressed only in control, denervated muscles of 3 and 7 days, and its expression level decreased gradually. Conclusion One possible reason of the depletion of satell ite cell pool in prolonged denervated muscle is the down-regulation of Pax7.