Materials and methods
Postmenopausal participants were divided into non-osteoporotic (N = 21) and osteoporotic (N = 25) groups based on Bone mineral density (BMD) T scores in accordance with the World Health Organization osteoporosis characterization. BMD measurement was performed for the entire individuals via Dual-energy x-ray absorptiometry (DEXA). None of these volunteers had received chemotherapy/radiation treatment before blood sampling. Alcohol consumption, steroid use, hospitalization, previous fractures, kidney diseases, or cancer were also considered exclusion criteria.
From osteoporotic and non-osteoporotic postmenopausal women, 20 ml of peripheral blood was obtained sterilely. PBMCs were isolated from samples using Ficoll (lymphosep) 1.077 g/ml (Biosera Inc., East Sussex, UK) and gradient centrifugation 25 min, 450g technique. Magnetic-activated cell sorting (MACS) technique was also used to isolate T cells with a negative selection protocol using the Pan T Cell Isolation Kit (Order no. 130-096-535; Miltenyi Biotec, San Diego), as recommended by the manufacturer.
T-cell samples were centrifuged for 10 min at 1500g. Then, the obtained supernatant was centrifuged for 15 min at 17,000g, after which the supernatant was spun again for 1 h at 160,000g by an ultracentrifuge. Using the western blotting technique, the exosomes in the obtained pellet were identified by exosomal markers, including CD81, CD63, and CD9. Also, the morphology and size of the exosomes were evaluated by scanning electron microscope (SEM).
Isolation of Human osteoblasts (HOBs) was done from femoral heads of patients undergoing hip replacement surgery, as described previously . Briefly, isolated bone samples were refined from the soft tissue and broken down into small fragments. Digestion was performed three times in a mixture of 2.0 mg/ml collagenase P (Roche Diagnostics, Germany) and 0.7 mg/ml collagenase II (Biochrom Germany), both from clostridium histolyticum, dissolved in phosphate‐buffered saline (PBS; PAA Laboratories, Austria) with 30 min of gentle agitation at 37 °C. Then, bone fragments culture was performed at 37°C in a water-saturated atmosphere with 5% CO2 in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 IU/ml penicillin (Invitrogen Germany). After 3–4 weeks, the cells were trypsinized, transferred to plates, and cultured using the same α-MEM medium as explained above. These cells were treated with T cells exosomes driven from non-osteoporotic and osteoporotic volunteers.
Real-time quantitative PCR
Then, bone fragments culture was performed at 37 °C in a water-saturated atmosphere with 5% CO2 in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 IU/ml penicillin (Invitrogen Germany). After 3–4 weeks, the cells were trypsinized, transferred to plates, and cultured using the same α-MEM medium as explained above. These cells were treated with T cells exosomes driven from non-osteoporotic and osteoporotic volunteers.
ALP activity was colorimetrically assessed by employing para nitrophenyl phosphate as the substrate (ALP kit 104-LL; Sigma), described previously by Kura et al. . Briefly, cells were seeded in a density of 100 cells/well in 96-well plate format and incubated in a complete culture medium containing obtained exosomes from ordinary or osteoporotic postmenopausal women. Wells without exosomes and enjoying merely culture medium were considered as controls. After 14 days of incubation, cells were washed twice with PBS and subsequently solubilized with 1% Triton-X (BDH Laboratory Supplies, Poole, UK) (50 μL/well) for 20 min. Then, 50 μL of 1.5 mM 2-amino-2- methyl-1-propanol (pH 10.3) (Sigma) and four mM para nitrophenyl phosphate disodium (Sigma) mixture was added to each well and incubated at 37 °C for 30 min. The reaction was terminated by adding 150 μL of 1 M NaOH. ALP activity determination was performed by measuring the optical density at 405 nm (A405) via a spectrophotometer (Titertek Multiskan Plus, Helsinki, Finland). The standard curve of the Sigma Units was used to calculate the experimental samples' International Units (IU/L).
Data were presented as mean ± standard deviation (SD) of triplicate experiments. The statistical differences of this cross-sectional study were analyzed via one-way ANOVA analysis of variance, as well as a post hoc test (Dunnett’s T3 multiple comparisons test) for determining group differences in study parameters, and p < 0.05 was regarded as statistically significant. SPSS software (version 24.0 for Windows; Armonk, NY, USA) and Prism software (GraphPad Prism for Windows, version 6.01; Nashville, TN, USA) were employed to implement the fundamental statistical analyses.