In vitro osteoclast differentiation was accomplished as previously described 38 (link). Briefly, BMM cells from femurs of 6 to 8 weeks old mice were cultured in full α-MEM with M-CSF (10 ng/mL, R&D systems, USA) for 1 d and then were differentiated into osteoclasts using RANKL (50 ng/mL, R&D systems, USA) and M-CSF (30 ng/mL) for 5 d. 50 μg/mL exosomes were added to 2 × 106 recipient cells (TRAP+ monocytes) on the fourth day. For in vivo treatment, 50 μg exosomes were intravenously injected into 6-week-old female BALB/c nude mice every other day for 3 weeks. In the control group, PBS was used.
For the culture of osteoblast progenitor cells, calvariae from newborn mice were dissected aseptically and treated with 0.1% collagenase and 0.2% dispase. The cells were maintained in the minimum essential medium (MEM) alpha containing 10% FBS. For osteoblast differentiation, primary osteoblasts were cultured in α-MEM containing 10% FBS and 10 nM dexamethasone (Sigma), 50 µg/mL of ascorbic acid, and 5 mM β-glycerophosphate for 6 days. For in vitro treatment, 50 μg/mL of exosomes were added to 1 × 104 recipient cells on the first day. Alkaline phosphatase (ALP) staining was carried out using a Vector Blue Substrate Kit (SK-5300; Vector Laboratories).
For the culture of osteoblast progenitor cells, calvariae from newborn mice were dissected aseptically and treated with 0.1% collagenase and 0.2% dispase. The cells were maintained in the minimum essential medium (MEM) alpha containing 10% FBS. For osteoblast differentiation, primary osteoblasts were cultured in α-MEM containing 10% FBS and 10 nM dexamethasone (Sigma), 50 µg/mL of ascorbic acid, and 5 mM β-glycerophosphate for 6 days. For in vitro treatment, 50 μg/mL of exosomes were added to 1 × 104 recipient cells on the first day. Alkaline phosphatase (ALP) staining was carried out using a Vector Blue Substrate Kit (SK-5300; Vector Laboratories).
Free full text: Click here
