Ti sheets (99.9% purity) were cut into square pieces (1 mm × 10 mm × 10 mm) for the in vitro studies. The specimens were cleaned ultrasonically in deionized water for 30 min, sterilized at high temperature and high pressure, and both sides were exposed to a UV lamp for half an hour. The specimens were divided into four groups: (1) control group (BMSCs + titanium); (2) 10 µg/ml BMSCs-exos + BMSCs + titanium; (3) 25 µg/ml BMSCs-exos + BMSCs + titanium; and (4) 50 µg/ml BMSCs-exos + BMSCs + titanium. For the experiments, the specimens were all cultured in a 24-well plate at 37 °C and 5% CO2 in a constant-temperature incubator.
BMSC culture and identification
After fixation, the rabbits were injected 1.5% pentobarbital at 2 ml/kg into the ear vein to anesthetic. The concave part of the inner side of the femur was used for the puncture site. Sterile gloves were worn, the puncture point was determined, and a number 12 bone marrow puncture needle was used to puncture a point from the skin to the periosteum. A syringe pump was slowly connected to the bone marrow tissue, and the bone marrow was extracted, than the rabbits were euthanized by intravenous injection of 100 mg pentobarbital at the ear edge. The bone marrow was then quickly injected into a sterile anticoagulant tube containing heparin (Sanli, Jiangsu, China) and was suspended in complete culture medium (Dulbecco’s modified Eagle’s medium( (DMEM), Gibco) containing 10% foetal bovine serum (FBS) (Gibco) and stored at 37 °C and 5% CO2 in a constant-temperature incubator for culturing. The first medium change was performed after 48 h. Then, the medium was changed every 2–3 days. Approximately 10 days later, the cells grew to 80–90% confluency and were passaged. Unless otherwise specified, the cells were cultured with normal medium (DMEM containing 10% FBS) and seeded on the specimens at a density of 3 × 104 cells/well.
Flow cytometry was used to identify the surface markers of third generation rabbit BMSCs. There is no specific identification marker for BMSCs, but because BMSCs are non-hematopoietic stem cells, they do not express the surface markers of hematopoietic stem cells such as CD-34, CD-14 and CD45, but express their own marker, CD-44. We detected CD34-PE (NeoBioscience GTX5741), CD14-PE (Abcam ab186689), CD45-PE (NeoBioscience GTX5741) and CD44-FITC (Novus nbp2-22530apc) by flow cytometry. Osteogenic differentiation of the BMSCs was induced with osteogenic medium (normal culture medium containing 10 mM β-glycerophosphate (Sigma), 50 µg/ml ascorbic acid (Sigma), and 10 nM dexamethasone (Sigma)) when the third generation cells grew to 90% confluency. BMSCs were concomitantly induced with adipogenic medium (1 µM dexamethasone (Sigma), 1 mM pyruvate (Sigma), 2 mM L-glutamine (Sigma), 0.5 M isobutyl methylxanthine (Sigma) and 10 µM insulin (Sigma)). Both the osteogenic and adipogenic experiments were used to identify the multidirectional differentiation potential of the BMSCs.
BMSC culture and identification
In this experiment, a differential ultracentrifuge (Optima, L-100XP) was used to extract the rabbit BMSC-exos. For exosome extraction from the 3rd generation BMSCs, cells grown to approximately 80% confluency were cultured with DMEM containing 10% non-exosome FBS (the bottom 10% was removed from the FBS after centrifugation at 120,000 g for 14 h at 4 °C). After 24 h, the cell supernatant was collected, and 50 ml of the pre-extracted cell supernatant was centrifuged at 300 g for 10 min and 2000 g at 4 °C. The following steps were carried out: centrifugation for 10 min, discard the pellet, centrifugation at 10,000 g for 30 min, discard the pellet, centrifugation at 100,000 g for 70 min, discard the supernatant, resuspension in 30 µl of Phosphate Buffer solution(PBS), centrifugation at 100,000 g for 70 min, and resuspension in 100 µl of PBS. Finally, the sample was transferred to an EP tube and stored at -80 °C until use.
Analysis of BMSC-exo morphology by field emission scanning electron microscopy(FE-SEM)
The morphology of the BMSC-exos was identified by FE-SEM. The obtained exosome sample was resuspended in 100 µl PBS, and 10 µl drops were pipetted on a 2 mm sample copper net for 1 min. The floating liquid was absorbed with filter paper, and the sample was counterstained at room temperature for 5 min with 3% phosphotungstic acid. Filter paper was used again to absorb the floating liquid, and the sample was washed 1–2 times with double-deionized water and dried at room temperature. FE-SEM imaging was performed at 80 kV-120 kV, and 20 exosomes were selected randomly to measure their diameter. Exosome morphology was identified.
Western blot analysis Western blot analysis was performed to identify the BMSC surface markers/antigens CD63, Hsp70 and Alix. Western blot was did with Wes instrument (WES, WS2494), and the following parameters were set: the separation glue was extracted for 200 s, the concentrated glue was absorbed for 15 s, the sample uptake time was 9 s, the electrophoresis time was 25 min, the electrophoresis constant pressure was 375 V, the time of gel clearance was 230 s with 3 washes of 150 s each, the sealing time was 5 min, the incubation time of the primary antibody was 30 min, and the incubation time of the secondary antibody was 30 min to expose the luminescent solution (HDR). Then, the “start” button was clicked to begin the operation.
BMSC-exos effects on BMSC adhesion and stretching on specimens BMSCs were seeded on the specimens and allowed to settle for 0.5, 1, and 4 h. At each time point, the cells on the specimens were carefully moved to a new 24-well plate, rinsed three times with PBS, and fixed in 500 µl 4% paraformaldehyde (PFA) for 15 min per well. The fixative was aspirated, and the cells were rinsed 3 times with PBS for 3 min each time and stained with DAPI (Sigma) for 10 min. BMSCs fixed at time points 1, 4, and 12 h were stained with fluorescein isothiocyanate(FITC) - phalloidin (Sigma) and counterstained with DAPI. Fluorescence images were taken by a fluorescence microscope (Olympus, FSX100). The cell number and distribution were assessed in five randomly chosen fields captured at 40 × magnification and 400 × magnification. The number of cells was quantified by Image J 1.8.0 software (Rawak Software, Germany, Version 1.8.0).
BMSC-exos for BMSC proliferation on specimens The effect of BMSC-exos on the proliferation of BMSCs on the specimens was assessed using a Cell-Counting Kit 8 (CCK-8, Boster, Wuhan, China). BMSCs were seeded on the specimens and allowed to culture for 24 h, 48 h, and 72 h using medium with BMSC-exos. The medium was changed on the second day. At each time point, the cells on the specimens were carefully moved to a new 24-well plate and rinsed three times with PBS. CCK-8 solution and normal medium were prepared as working solutions at 1:9, and 300 µg of the mixture solution was added to each well. The optical density (OD) of the solution was measured at 450 nm with a microplate reader (Thermo, Shanghai, China).
ALP activity assays The ALP activity of the specimens was assessed qualitatively using a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) ALP colour development kit (Beyotime, Shanghai, China). BMSCs were seeded on the specimens at a density of 8 × 104 cells/well. Three days later, the normal medium was changed to BMSC-exo osteogenesis induction medium, and the cells were cultured for 7 days and 14 days. The medium was changed every three days. At each time point, the cells on the specimens were carefully moved to a new 24-well plate and rinsed three times with PBS. Then, the cells were fixed with 4% PFA for 15 min and stained with the working solution for 24 h. After washing 3 times with PBS for 3 min each time, titanium tablets were added dropwise. Two hundred microlitres of the ALP working solution was added to ensure that the working fluid covered the surface of the sample, and the sample was incubated for 24 h at room temperature in the dark. The staining solution was aspirated, and the sample was washed with PBS 2 times to stop the colour development and observe the staining. The stained area was quantified by Image J 1.8.0 software.
Enzyme-linked immunosorbent assay (ELISA) An ELISA was used to determine the secretion of collagen I on the specimen. BMSCs was cultured in the same way former for 7 days and 14 days. At each time point, the cells were moved on 24-well plate seemly. Then, 150 µl of RIPA lysate buffer was added and incubated on ice for 30 min, and the cells were scraped from the titanium sheet with a cell scraper into a sterile EP tube. Then, a rabbit COL-I kit (TSZ, RG01236) was used according to the assay procedure for the calculation of collagen I secretion.
Mineralization assay Mineralization on the specimens was evaluated by Alizarin Red S (Solarbio) staining. BMSCs was cultured in the same way former for 7 days and 14 days. The cells on the specimens were moved seemly on the 24-well plate at each time point. Then, the cells were fixed with 4% PFA and stained with Alizarin Red S for 30 min at room temperature. Then, the cells were rinsed three times with PBS for 5 min each time. The images of the specimens were captured (40 × lens) using an inverted light microscope (Olympus, FSX100).
The quantitative data are expressed as the mean ± standard deviation (SD). All quantified results were calculated using ImageJ Software 8.0. All data analysis was carried out using GraphPad Prism 8 software (GraphPad Software, USA, Version 8.0.0). Unless otherwise stated, the p values were calculated using Student’s t tests, and the error bars indicate the SD. The significance levels were subdivided into * (p value < 0.05), ** (p value < 0.01), *** (p value < 0.001) and **** (p value < 0.0001). For correlation analysis, Pearson’s value was calculated. Statistical analyses were performed by one-way analysis of variance and the Student-Newman-Keuls-q (SNK-q) test using SPSS v14.0 statistical software when comparing the concentration and time correlation.