This is a study with cross-sectional design.
Development of a novel cell mechanical loading system
A newly developed mechanical pressure loading system for living muscle cells is shown in Fig. 2. The system consisted of a biological material test system (MTS, type 858; MTS Company, Eden Prairie, USA), a jig, a connecting rod shaft, a piston with two silicone compressing rings for sealing, a stainless steel cylinder as pressure vessel, and a pressure sensor (EVT100A; Yuran Sensor Technology Company, Shanghai, China). The pressure was generated within a stainless steel cylinder interfaced to MTS, a servo-hydraulic loading frame. The cell-culture dish was put on the bottom of the chamber. The chamber was completely closed to create a pressure chamber.
Curve fitting of rolling manipulation
The pressure-time curve of rolling manipulation was recorded in the Manipulation Technique Parameter Analyzer (TypeII, Shanghai Research Institute of Traditional Chinese Medicine, China) when the operator was performing rolling manipulation. The data of the pressure-time curve were imported into the Waveform Editor in MTS, and MTS output the load to the Cell Mechanical Loading System imitating rolling manipulation.
The full curve in Fig. 3 was monitored by the pressure sensor in the Cell Mechanical Loading System, which showed that the cells were cyclically exposed to the 9.5-12.5 N/cm2 of intermittent pressure imitating rolling manipulation (IPIRM) at a frequency of 1.0 Hz. The dotted curve in Fig. 3 showed that the cells were loaded at a continuous pressure of 12.5 N/cm2.
Cell culture and establishment of injured cell model
The human skeletal muscle cells (HSKMCs; US Type Culture collection warehousing, San Diego, USA) from the 4 – 8th generation of skeletal muscle cell strain were used for this study. All cells were kept in CO2 cell culture box (Biorad Company, Hercules, USA) at 37 °C in a humidified atmosphere containing 5 % CO2. HSKMCs were cultured in DMEM high-glucose medium (HyClone Company, Logan, USA) containing 4.5 g glucose, 100,000U penicillin, 100 mg streptomycin, and 3 % fetal calf serum (FCS; HyClone Company, Logan, USA) per liter. They were considered to be cultured successfully when the following four criteria were identified under an inverted microscope: 1. The shape of human skeletal muscle cells was spindle-shaped. 2. No floating cells were found which indicated that the cultured cells had a good capacity of cellular adherence to wall of the culture bottle. 3. The cell nuclei were oval-shaped without any sign of breaking out, dissolving, or pyknosis. 4. The culture bottles were clear without pollution. Once the HSKMCs grew up to the whole bottom of each culture bottle, they were harvested and divided into two portions in order to re-proliferate further down.
The human skeletal muscle injury modeling cells were induced by dexamethasone according to the reference literature [26]. Some researches conformed that excessive dosage of dexamethasone could produce injury to muscle cells by suppressing cells proliferation, reducing SOD level, increasing MDA, and causing intracellular Ca 2+ overloading [27, 28].
When HSKMCs in the culture bottle showed good adherence, i.e., covering 80-90 % on the bottom of the bottle observed under microscope, the cultured cells were harvested and then the medium in the bottle was abandoned. The mixture of the fresh DMEM high-glucose medium and dexamethasone sodium phosphate injection (The 3rd Pharmaceutical Factory, Jiangshu, China) was added into the culture bottle, and the final concentration of Dexamethasone sodium phosphate injection was 2.5 g/L. The cells were then cultured in the incubator containing 5 % CO2 for 24 h at 37 °C.
Groups and treatment of HSKMCs
The normal HSKMCs were used as control normal group (CNG), and they were cultured in 12 dishes. The injured HSKMCs were future divided respectively into 5 following different groups with 12 dishes per group: control injured group (CIG), rolling manipulation group (RMG), rolling manipulation-verapamil group (RMVG), Static pressure group (SPG) and Static pressure-verapamil group (SPVG).
CNG (control normal group) and CIG (control injury group) cells were cultured in the same conditions as RMG, RMVG, SPG and SPVG cells except being loaded pressure. RMG and RMVG cells were cyclically exposed to 9.5-12.5 N/cm2 of IPIRM at a frequency of 1.0 Hz for 10 min. SPG and SPVG cells were loaded to a continuous pressure of 12.5 N/cm2 for 10 min. In both RMVG and SPVG, verapamil hydrochloride injection (Ver; Wellhope Pharmaceutical Company, Shanghai, China), a calcium ion influx inhibitor, was added into the culture medium with a concentration of 10-5 mol/L.
Procedure of measuring SOD activity, MDA content, and CK activity
The medium were removed by aspiration from culture vessels of each group cells described above. 1 ml of 0.25 % trypsin (Jibco Company, Grand Island, USA) was added into culture vessels, which were placed in 37 °C incubator for approximately 1.5 min. The trypsin was removed by aspiration until HSKMCs appeared rounded when they were observed using an inverted microscope. 6.0 ml of DMEM high-glucose medium were added into culture vessels to terminate trypsinization process. HSKMCs were collected at 1000 r/min for 10 min, and preserved in the refrigerator at -20 °C.
SOD activity, CK activity, and MDA content were quantified in the same experiment and in duplicates with the use of commercially available SOD, CK and MDA kits (Nanjing Jiancheng Bioengineering institute, Jiangsu, China).
Statistical analysis
All data were continuous data that were expressed as mean ± standard deviation \( \left(\overline{x}\pm \mathrm{s}\right) \). One-way Analysis of Variance (ANOVA) with post-hoc multiple comparisons was conducted to analyze the differences between different groups. All the data were analyzed with software (Statistical Package for the Social Sciences, version15.0). A p-value of less than 0.05 was considered to be statistically significant.
