Moreover, the role of autophagy in osteogenic differentiation in either human or animal MSC of any origin, as well as its dependence on AMPK/Akt/mTOR signaling, has not been investigated so far. The present study combines pharmacological inhibition and genetic knockdown approach to investigate the role of AMPK, mTOR, Akt,

autophagy and their interplay in osteogenic differentiation of hDP-MSC. Our data indicate a coordinated involvement of AMPK/Akt/mTOR signaling in this process, relying on time-dependent induction of AMPK/mTOR-dependent autophagy and activation of Akt/mTOR signaling Navitoclax price axis. Extracted teeth were collected at the School of Dentistry, University of Belgrade, in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Ethical approval was obtained from the ethics committee of the School of Dentistry, University of Belgrade. All participants provided written informed consent. The dental pulps isolated from deciduous tooth were kept in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; PAA Laboratories, Linz, Austria) and delivered to the laboratory for the isolation AZD2281 concentration of hDP-MSC in less than 2 h. After centrifugation and supernatant removal, extracted pulp

Adenosine triphosphate tissues were digested in a solution of 3 mg/ml collagenase type I (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS; PAA Laboratories, Linz, Austria) supplemented with 20% FBS for 45 min at 37 °C. Afterwards, PBS containing 2% FBS was added to cell suspensions, which were then pelleted by centrifugation and enumerated for viable cells by trypan blue dye exclusion test. HDP-MSC were isolated

based on their ability to adhere to culture plates, as described previously [17]. Namely, the cells obtained from one tooth were seeded into 25 cm2 plastic tissue culture flasks (Sarstedt, Numbrecht, Germany) (1 × 104 cells/cm2) and cultured in a DMEM growth medium containing 15% FCS, 200 μM l-ascorbic acid-2-phosphate (Sigma-Aldrich, St. Louis, MO), 100 units⁄ml penicillin/streptomycin (PAA Laboratories, Linz, Austria) at 37 °C in a humidified atmosphere containing 5% CO2. After 3 days, non-adherent cells were removed and fresh medium was added to allow further growth. Fresh medium was replaced every 2–3 days and cells were left to grow to subconfluency (80–90% of surface occupancy). These adherent cells were defined as passage zero cells, while later passages were named accordingly. For passaging, the adherent cells were washed twice with Ca2 +/Mg2 +-free PBS and detached with 0.25% trypsin-EDTA solution (PAA Laboratories, Linz, Austria) for 5–10 min at 37 °C.