Alizarin red

Osteoblastogenesis induction and alizarin red staining were performed as described previously [65 (link)]. BMSCs with a density of 5 ​× ​10⁴ ​cells/cm² were seeded on the surfaces of sterilized Ti, TNrs, and Fe₃O₄-TNrs or into the wells without any sheets, treated with 1 ​mT 15 ​Hz SEMF for 1 ​h per day. BMSCs were cultured with the osteogenic differentiation induction medium (Cyagen Biosciences, CA, USA). After 21 days of culture, the cells were fixed with 4% paraformaldehyde for 20 ​min, and stained with alizarin red (Cyagen Biosciences, CA, USA) for 5 ​min, washed with PBS three times, then the cells could be placed under the microscope (EVOS FL Auto, Life Technologies, USA) to observe and photograph.

Tibiaes and femurs were excised from rats following anaesthesia. MSCs were flushed with DMEM/F12 and isolated from the tibiae and femur marrow of 8-week old male SD rats (15 (link)). bone marrow-derived MSCs were cultured with DMEM/F12 containing 1% glutamine, 2% FBS, 100 U/ml penicillin and 100 U/ml streptomycin in incubator (37°C and 5% CO₂). As cells reached 80–90% confluence, MSCs were passaged every 3–4 days by trypsinization (Beijing Solarbio Science & Technology Co., Ltd.) and cells from the 3rd to 8th passage were used for experiments. Cells (5×10⁵) in a plate were cultured with adipogenic or osteogenic induction media (Cyagen Biosciences, Guangzhou, China) every 3 days. After 2 weeks, cells reached 90% confluence and were stained with oil red O or alizarin red (Cyagen Biosciences) in a culture plate. MSCs exhibited osteogenic and adipogenic differentiation. Biological cell surface markers of MSCs, including CD29, CD44 (both allophycocyanin-labeled), CD90, CD45 and CD34 (all phycoerythrin-labeled), were detected by flow cytometry (BD FACSCalibur; BD Biosciences, San Jose, CA, USA).

For ex vivo experiment, BM-MSCs were isolated, cultured to 80% confluence, and then incubated with osteogenic differentiation medium (Cyagen Biosciences, USA; Cat^# MUBMX-90021). Half of the induction medium was replaced every other day. For in vitro experiment, BM-MSCs were isolated from 6-month-old SAMP8 mice, cultured to 80% confluence, and then incubated with osteogenic differentiation medium containing 1 × 10¹⁰ particles/mL hESC-sEVs or vehicle (PBS). Half of the induction medium was replaced by an equivalent volume of fresh osteogenic medium supplemented with 1 × 10¹⁰ particles/mL hESC-sEVs or with PBS every other day. The expression levels of osteogenic-related genes were assayed by RT-qPCR on day 7 after the osteogenic induction. To assess osteogenic differentiation, the cells were stained with 1% Alizarin Red (Cyagen Biosciences) on day 21 after the osteogenic differentiation. Calcified nodules were eluted with 10% cetylpyridinium chloride (CPC), and the absorbance was calculated at 562 nm.

DPSCs were
seeded in 6-well plates at a density of 2 × 10⁵ per
well and then cocultured with OIM, which include different concentrations
(0, 0.1, 1, and 10 μg/mL) of GOQDs for 14 days after cell attachment.
The medium was changed every 2 days. The cells were fixed by 4% paraformaldehyde
for 30 min and then were dyed by Alizarin red (Cyagen Biosciences)
for 30 min. The photographs of calcium nodules were taken by an inverted
fluorescence microscope (Zeiss). For semi-quantitative interpretation,
10% cetylpyridinium chloride (Sigma) was added into each well, and
the OD value was detected at a wavelength of 562 nm.