The experimental protocol was performed in accordance with the Declaration of Helsinki and approved by the human subjects Institutional Review Board of the Chang Gung Memorial Hospital. Written informed consent was obtained from all patients. Demographic and clinical data such as age, gender and surgery reason were collected.
Patients and surgical procedures
MSCs were harvested from 12 patients (5 females and 7 males) who underwent iliac bone grafting for spine fusion. The mean age was 58.3 years-old, where the age range from 39 to 77 years old. The cells from each patient were separately evaluated. The cells from 3 or 4 patient were subjected to each treatment. During bone graft harvesting, 10 mL of bone marrow was aspirated and collected in a sterile heparin-rinsed syringe.
Isolation and cultivation of MSCs
Each marrow sample was washed with PBS. Up to 2 × 108 nucleated cells in 5 mL of PBS were loaded onto 25 mL of Percoll cushion (Pharmacia Biotech). A density gradient was used as the isolation procedure to eliminate unwanted cell types. A small percentage of cells were isolated from the density interface at 1.073 g/mL. The cells were re-suspended and plated at 2 × 105 cells per T-75 flasks. The cells were maintained in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG; Gibco, Grand Island, NY) that containing 20% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 7 d of primary culturing, the non-adherent cells were removed by changing the medium. The MSCs grew as symmetric colonies and were subcultured at 10 to 14 d by treatment with 0.05% trypsin (Gibco) and seeded into fresh flasks.
Flow cytometric analysis of surface antigen expression
When confluent, the MSCs were passaged 1 in 3, and a sample was analyzed for MSCs marker expression by flow cytometry. The cells were washed in phosphatebuffered saline (PBS), and then removed from the flask by 0.05% trypsin (Gibco). 1 × 105 cells were incubated with each mouse monoclonal primary antibody at 4°C for 30 minutes. Mouse FITC–conjugated anti-CD105 antibody (1:100 dilution), mouse PE–conjugated anti-CD146 antibody (1:100 dilution), and mouse FITC–conjugated anti-CD34 antibody (1:100 dilution) were purchased from Beckton Dickinson (Oxford, UK). Mouse PE–conjugated anti-STRO-1 antibody (1:50 dilution) was purchased from Santa Cruz (CA, USA). After wash, the cells were resuspended in 500 μl wash buffer and analyzed on a BD flow cytometer (Oxford, UK).
Cell exposure to intermittent HBO
The cells were cultured in 100 mm culture dishes (2 × 105 per dish) in complete medium (DMEM-LG containing 20% FBS and antibiotics) or in osteogenic induction medium (DMEM-LG containing 20% FBS, antibiotics, 100 μM ascorbate-2 phosphate, 100 nM dexamethasone, and 10 mM β-glycerophosphate). The cells were either maintained in 5% CO2/95% air throughout the experiment or were HBO treated by exposure to 100% O2 for 25 min and then to 5% CO2/95% air for 5 min at 2.5 ATA (atmospheres absolute) in a hyperbaric chamber (Huxley Corporation, Taipei, Taiwan) for 90 min every 36 h.
RNA preparation and real-time quantitative polymerase chain reaction analysis
After culturing for 1, 4, and 7 d with or without HBO treatment, total RNA was extracted using a Qiagen RT kit (Qiagen, USA) according to the manufacturer’s instructions. The RNA concentration was evaluated by A260/A280 measurement. To detect Wnt3a, GSK-3β, β-catenin, Runx2, and GAPDH RNA transcripts, cDNA was analyzed on an ABI PRISM 7900 sequence detection system using TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA). The cycle threshold (Ct) values were obtained, and the data were normalized to GAPDH expression by using the ΔΔCt method to calculate the relative mRNA level of each target gene.
Small interfering RNA transfection
On day 1, 2 × 105 MSCs were plated onto a 6-well tissue culture plate in 2.5 mL of OPTI-MEM (Invitrogen, Carlsbad, CA) medium without antibiotics and serum. The cells were then transfected with human β-catenin small interfering (si)RNA or scrambled siRNA (Stealth RNAi, Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. After 8 h of transfection, the culture medium was changed to osteogenic medium with 10% FBS and the cells were exposed to HBO treatment. On days 4 and 7, the cells were re-transfected once and exposed to HBO. After an additional 24 h of culturing, the cells were harvested for analysis. The silencing effect on β-catenin and downregulation of Runx 2 was detected by real-time PCR after the treatments.
Western blot analysis
After culturing for 7 d with or without HBO treatment, the cells were washed with PBS and extracted using M-PER mammalian protein extraction reagent (Thermo, USA). The protein content was quantitated using a protein assay kit (Pierce Biotechnology, IL), separated by 7.5% SDS-PAGE for Wnt3a, GSK-3β, β-catenin, Runx2, β-actin, and α-tubulin, and transferred onto membranes using a transfer unit (Bio-Rad, USA). After blocking with 10% non-fat milk, the membranes were incubated overnight at 4°C with 1000-fold diluted rabbit antibodies against Wnt3a, GSK-3β (Cell Signaling, MA, USA) or mouse antibodies against β-catenin (Millipore), β-actin (Millipore), and Runx2 (Millipore). After washing, the membranes were further incubated for 2 h with 10000-fold goat anti-mouse IgG (Calbiochem, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Amersham Pharmacia Biotech, UK). The band images were photographed using ECL Hyperfilm (Amersham). The intensity of each stained was quantified using an image-analysis system (Image-Pro plus 5.0, Media Cybernetics, USA).
Preparation of cytosolic and nuclear fractions for β-catenin detection
After culturing for 7 d with or without HBO treatment, the cells were rinsed with ice cold PBS, treated with 0.05% trypsin, and then collected by centrifugation at 800 g. NE-PER nuclear and cytoplasmic extraction reagents (Thermo science, USA) were used to isolate cytoplasmic and nuclear extracts from the cells. The protein content was quantitated using a protein assay kit (Pierce), and separated by 7.5% SDS-PAGE to detect β-catenin (Millipore) and TATA binding protein (TBP; Abcam, Cambridge, UK). The silencing effect on β-catenin and downregulation of Runx 2 was detected by western blotting after the treatments.
Quantitative measurement of alkaline phosphatase activity
After culturing for 7, 14, and 21 d with or without HBO treatment, the cultured cells were washed with ice cold PBS. A 5-mL of alkaline phosphatase (ALP) substrate buffer (50 mM glycine, 1 mM MgCl2, pH 10.5), containing soluble ALP substrate (2.5 mM p-nitrophenyl phosphate), was added at room temperature. Twenty minutes after adding the substrate, 1 mL of the buffer was removed from the culture and mixed with 1 mL of 1 N NaOH to halt each reaction. The absorbance of each mixture was determined on an ELISA plate-reader (MRX; Dynatech Labs) at 405 nm. Enzyme activity was expressed as n mole p-nitrophenol/min.
Calcium level quantification
After culturing for 7, 14, and 21 d with or without HBO treatment, the cultured cells were rinsed with ice cold PBS and placed into 5 mL of 0.5 N HCl. Calcium was extracted from the cells by gently shaking the cultures for 24 h. Cellular debris was centrifuged and the calcium in the supernatant was measured using a Quantichrom calcium assay kit (DICA-500, Bioassay systems, USA).
von Kossa staining
After culturing for 21 d with or without HBO treatment, culture dishes were rinsed twice with 5 mL of Tyrode’s balanced salt solution, and fixed in 10% buffered formalin for 1 h. A 10-mL aliquot of freshly prepared 2% (w/v) silver nitrate in water was added, and the dishes were kept in dark for 30 min. The plates were then washed thoroughly with distilled water and exposed to bright light for 30 min. The presence of mineral deposits was indicated by the development of a black precipitate on the mineralized matrix. The matrix intensity was quantified by image-analysis system (Image-Pro plus 5.0).
Dedicated Wnt secretion factors assay
After culturing for 1, 4, and 7 d with or without HBO treatment, the culture medium was collected and the cells were washed with ice cold PBS and cellular protein was extracted using M-PER protein extraction reagent (Thermo, USA). Each protein extraction was separated by 7.5% SDS-PAGE to detect the GPR177 (Millipore), VPS35 (Abcam), ATP6V0 (Abcam), Wnt3a (Millipore), and β-actin (Millipore). The secreted Wnt3a in the collected medium was quantified by ELISA (USCN Life Science, Wuhan, PR China).
RNAi treatment for GPR177, VPS35, and V-ATPases
MSCs were transfected with siRNA for GPR177, VPS35, and ATP6V0 (Santa Cruz), respectively on days 1, 4, and 7 by using the same protocol above described. Silencing was detected by western blotting after the treatments. The secreted Wnt3a in the collected medium was quantified by ELISA (USCN).
Data are given as mean ± standard devision (SD) of the results from three or four different samples in each item of the experiment. The cells from each sample were separately evaluated. Differences between two groups were measured by the Student’s t-test. A p value less than 0.05 was defined statistically significant difference.