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 As a bioactive material, the osteogenic activity of borate bioglass has been proved. To design a novel borate bioglass according to an improved formula and to investigate the effects of the borate bioglass on osteoblasts invitro for further research and potential cl inical appl ication. Methods The novel Na2O-K2O-MgO-CaO-P2O5-B2O3-SrO borate bioglass was prepared by melting process. The initial and secondary extracts were prepared according to ISO10993-12: 2007 respectively with different extract time of 0-24 hours and 24-48 hours. The osteoblasts (MC3T3-E1) of the 5th-15th passages from mouse were cocultured with the initial (initial extract group) and secondary (secondary extract group) extracts, respectively, to assess the effects of the borate bioglass on the cell prol iferation, protein synthesis, alkal ine phosphatase (ALP) activity, cell apoptosis, and cell migration; while α-MEM medium without addition of extract served as control group. Results The absorbance values at 450 nm were 0.356 0 ± 0.018 7, 0.331 0 ± 0.025 4, and 0.204 0 ± 0.013 8 in initial extract, secondary extract, and control groups, respectively, showing significant differences among 3 groups (P lt; 0.05). The total protein contents were (382.847 ± 9.521), (226.071 ± 5.847), and (220.248 ± 8.213) U in initial extract, secondary extract, and control groups, respectively; there were significant differences between initial extract group and control group, and between initial extract group and secondary group (P lt; 0.05), but there was no significant difference between secondary extract group and control group (P gt; 0.05). However, no significant difference was observed in the ALP activity [(0.013 01 ± 0.000 39), (0.012 93 ± 0.000 44), and (0.012 92 ± 0.000 35) U/ mg], apoptosis rate (7.03% ± 1.95%, 6.46% ± 2.88%, and 6.18% ± 2.21%), horizontal migration [(137.50 ± 11.43), (134.98 ± 10.50), (135.21 ± 8.66) μm], and transmembrane cell number [(10.92 ± 4.99), (10.07 ± 2.50), and (9.81 ± 2.64) cells/ field] among initial extract, secondary extract, and control groups (P gt; 0.05). Conclusion This novel borate bioglass has excellent cytocompatibil ity, which plays regulatory effects on the cell prol iferation, secretion, and migration.