Osteogenic differentiation medium

Newborn TLR9^−/− or WT mice (2–3 days after birth) were used to harvest calvaria. After sacrifice, the calvaria were dissected and digested five times in α-MEM containing 0.1% collagenase (Roche) and 0.2% dispase (Roche). Cells collected from the last four digestions were pooled together and cultured in α-MEM containing 10% FBS, 100 U·mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin. Cells showing confluence of 60%–80% were replated at a density of 1 × 10⁵ per well in 12-well plates, and the culture medium was changed to osteogenic differentiation medium (Cyagen) to induce the differentiation of osteoblasts. After 7 days of differentiation, the cells were either fixed for ALP staining or lysed for RNA extraction or the ALP assay (Jiancheng Bioengineering, Nanjing). For the collection of osteoblast culture supernatants, cells were cultured in regular 10% FBS α-MEM for an additional 2 days before collecting the supernatants. All experiments were performed in duplicate, and the number of biological replicates is indicated by the number of dots in each bar plot.

To induce osteoblast differentiation, a total of 1 × 10⁵ PDLSCs were seeded into each well of 6-well plates. Upon reaching a density of 70%, the PDLSCs were grown in osteogenic differentiation medium (Cyagen, United States) for 21 days. In some osteogenic induction assays, the PDLSCs were treated with recombinant human IL-1β protein (R&D, United States) at the indicated concentration every 2 days. After 21 days, the cells were fixed with 4% PFA in PBS and stained with 2% Alizarin red (pH = 4.2).

The hBMSCs were inoculated in 24-well plates. When cell confluence reached 80%, the medium was changed to osteogenic differentiation medium (Cyagen, USA). The medium was changed every 3 days and when the medium was changed, exosomes were added to the medium. Alkaline phosphatase (ALP) staining and Alizarin Red S staining were done to evaluate the level of osteogenic differentiation. After the hBMSCs were cultured in osteogenic differentiation medium for 7 days, ALP staining was conducted according to the manufacturer’s instructions (Beyotime, China). The cells were incubated with 10 mM p-nitrophenyl phosphate (Meilunbio, China) as the substrate at 37 °C for 15 min. Afterward, ALP activity was quantified at 420 nm by a microplate reader [34 (link)]. After cultured in osteogenic differentiation medium for 3 weeks, the hBMSCs were fixed, stained, and cleared according to the instructions of the Alizarin Red S staining kit (Solarbio, China). Based on the previously reported methods, the stained mineralization nodules were dissolved with 10% cetylpyridinium chloride at 37 °C for 30 min [35 (link)]. The solution was added to a 96-well plate, and a microplate reader was used detect the OD value at 562 nm for quantitative analysis.

HUMSCs were assessed using Osteogenic-Differentiation Medium, Adipogenic-Differentiation Medium, and Complete Chondrogenic Medium (Cyagen Biosciences, Santa Clara, CA, USA). At the end of the incubation period, the differentiation of HUCMSCs was detected by three kinds of staining. Alizarin red solution (Solarbio, Beijing, China), oil red-O staining (Solarbio, Beijing, China), and alcian blue staining (Solarbio, Beijing, China) were used to detect osteogenic differentiation, lipogenic differentiation, and chondrogenic differentiation according to manufacturer’s instructions, respectively.

For the in vitro adipogenic and osteogenic differentiation assays, BMSCs were treated with adipogenic differentiation medium (Cyagen) or osteogenic differentiation medium (Cyagen) follow the operating instruction in 24‐well plates for 7 or 14 days. The differentiation medium was refreshed every 2 days. Oil Red O staining, Western blot and qRT‐PCR were used to measure the adipogenic differentiation efficacy. Alizarin red (AR) staining, alkaline phosphatase (ALP) staining, Western blotting and qRT‐PCR were used to measure the osteogenic differentiation efficacy.