Ethical statement
We get the ethical review permission from Capital Medical University, Beijing, China and obey the guidelines for the care and use of animals. There is a statement of the ethics committee indicating approval of the research (2014SB-011-01).
Design of the hydrostatic pressurization chamber
The hydrostatic pressurization chamber was manufactured by the company (Beijing Century Senlong Experimental Apparatus Co., Ltd) was used in this study. The machine is a convenient and completely used in in vitro-operated hydrostatic pressure system (Fig. 1) and the same system was used in previous studies [10,11]. The major components include the pressure container, smart temperature control device, pneumatic supply and pressure indicator, as well as a piston device; distilled water is also used. The experimental system has a stable pneumatic supply, and it can monitor and regulate pressure in real time through a pressure gauge. The designed pressure range is 0-5 MPa. This system not only has the advantage of a general liquid chamber, but also the hydrostatic pressure can be altered as needed; therefore it can provide conditions of hypoxia and constant temperature which are similar to that in the human IVD, without changing ion composition and osmotic pressure in response to pressure.
Isolation and culture of rabbit intervertebral cartilage endplate cells
Intervertebral cartilage endplate cells were isolated from the adult Japanese white rabbit, Jw-Nibs strain, with an average weight of 2.5 kg and 50% male and 50% female (Beijing Vital River Lab Animal Technology Co., Ltd.). We can get sample from 4 discs in every rabbit. The number of animals in each experimental group was 2. The experiment is divided into 4 groups, each group has two times, so a total of 16 rabbits was required. The housing of animals is cages with wood floor and hay. The number of cage companions is 4. The light/dark cycle is 12 with 22℃. They were feed with fresh water and forage in our animal center building. The animals are uthanized during the experiment by air embolization after injecting the phenobarbital sodium in rabbit ear veins. The second generation of cartilage cells cultured in vitro and the culture medium were randomly divided with random number table into 4 groups: the control group (0.1 MPa), the low pressure group (0.7 MPa), the medium pressure group (2 MPa) and the high pressure group (4 MPa). Culture medium was added to each group and then the cells were placed in the pressure vessel (loaded with 37°C distilled water) using a 20ml-plastic injector. Hydrostatic pressure was applied at 0.7 MPa, 2 MPa, and 4 MPa for 4 or 24 hours. The pressure vessel temperature was maintained at 37°C during loading by using the smart temperature control device. After the experiment, the animals are put into the special plastic bags and handed over to the animal center. Another researchers of our team assess results of the experiment independently.
Gene expression
Immediately after pressure application, the cartilage cells were removed from the sodium citrate dissolution. mRNA was isolated using an mRNA extraction kit (R2050; Zymo Research Corp., Irvine, CA). According to the kit instructions, OD260/OD280 was measured and RNA was detected by 1% agarose gel electrophoresis. For cDNA synthesis, 1 μg total RNA in each sample was used according to the instructions of Bios Script First Strand cDNA Synthesis Master Mix (Thermo Fisher Scientific KK, Tokyo, Japan). The designed primers are shown in Table 1. Real-time polymerase chain reaction (RT-PCR) was used for the analysis of gene expression. The tested genes included markers of inflammation (iNOS, COX-2), matrix metabolism (MMP-3), anticatabolic metabolism (TIMP-1), structural component (aggrecan) and reference gene (GAPDH). The ΔΔCt method was used to calculate relative gene expression. The primary experimental outcomes are the gene expression of aggrecan and Cox-2, and the secondary experimental outcomes are the expression of iNOS, MMP3 and TIMP.
Statistical analysis
The relative gene expression was calculated as a percentage in order to compare the effect of different intervention factors on the same gene. In the figures, the positive and negative values respectively represent augmentation and inhibition of gene expression compared to that in the control group. Values represent the average of three trials ± standard error, with 95% confidence intervals calculated to determine the statistical significance. The confidence interval was calculated based on the t distribution due to the small sample size. One-way ANOVA was performed to compare groups. All data were analyzed with SPSS18.0 and p<0.05 was considered as statistically significant.