12-16 In cell-based treatment of tissue defects, one strategy is to transplant fully-differentiated cells into the injured site. For this reason, the subject of the optimization of MSC chondrogenic differentiation is of particular importance.17 Some investigations have indicated that the differentiation of MSCs into cartilage cells mostly occurs following the activation of certain signaling pathways, particularly the Wnt (wingless type) pathway. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 (GSK-3) enzyme. The inhibition of this molecule initiates the signaling pathway.18-20 On the other hand,
some investigators have reported that a small molecule referred to as BIO (6-bromoindirubin-3–oxim), Inhibitors,research,lifescience,medical derived from Tyrian purple indirubins, Inhibitors,research,lifescience,medical possesses a GSK-3-selective inhibitory function. It acts by binding on a groove between ATP and GSK-3ß, resulting in the activation of the Wnt signaling pathway.21 A number of investigations have so far been conducted using the Wnt-activating property of BIO. These studies have reported some interesting effects of this small molecule. Some have found that the addition of BIO
into the cell culture medium results in culture protection against apoptotic changes.22,23 Inhibitors,research,lifescience,medical Others have concluded that the presence of BIO in the culture medium enhances the growth capacity of the cultured cells.24,25 Finally, a few studies have reported that BIO supplementation leads to the maintenance of pluripotency in embryonic stem cell culture.26-28 There is no report regarding the effect of Inhibitors,research,lifescience,medical BIO on MSC in vitro chondrogenesis. The objective of
the present investigation was to examine whether or not the addition of BIO into the culture medium could improve cartilage differentiation of marrow-derived MSCs. Materials and Methods Animals MSCs from 10 NMRI male mice (4-8 weeks old) were studied in the current experimental study. Prior to the experiment, approval for animal use was obtained from the Ethics Committee of Royan Institute. Bone Marrow Cell Culture The mice were killed by cervical dislocation, and their tibia and femur were removed Inhibitors,research,lifescience,medical and transferred to the cell culture lab. Within laminar cabinet, the bone marrow was flushed out of the medullary canal using an insulin needle inserted into the clipped end of the long bones. The marrow was mixed with 5 ml AV-951 DMEM (Dulbecco’s Modified Eagle Medium, Gibco, Germany) containing 15% FBS (fetal sellectchem bovine serum, Gibco, Germany) and 100 IU penicillin and 100 µg/ml streptomycin (Gibco, Germany) and centrifuged at 400 g for 3 min. The cell pellet was suspended in DMEM, cultivated at 10 6 -cells/ml in 75-cm culture flasks, and incubated in an atmosphere of 5% CO2 at 37ºC. The medium was replaced twice weekly until the culture reached confluency. At this time, the cultures were subcultured with a 1:3 ratio into new culture flasks. Passaged-3 cells were used at the following experiments.