Mm csf

Non-adherent bone marrow mononuclear cells isolated from WT or Pd-1h^-/- C57BL/6 mice were cultured in α-MEM supplemented with 10% FBS, 50 ng/ml mM-CSF and 50 ng/ml mRANKL (R&D Systems, Minneapolis, MN) in the presence or absence of recombinant human pro-MMP-13 for 4 d followed by TRAP staining^{3 (link)}. For bone resorption pit assays, bone marrow mononuclear cells were seeded on bovine femur slices (Immunodiagnostic Systems, United Kingdom) treated as described above. Osteoclast formation on dentin slices was confirmed by TRAP staining and bone resorption lacunae identified following hematoxylin staining^{3 (link)}. Bone resorption area was quantified using Image J^{3 (link)}.

Selection of hBMSCs was performed by selecting them according to the cells’ natural tendency to adhere to the culture dish. Primary hBMSCs were cultured in growth medium, Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco), 1% antibiotic–antimycotic solution (Invitrogen, Grand Island, NY, USA), and confluence was achieved within 7 days in 5% CO₂ at 37 °C. For osteogenesis, cells were cultured in DMEM-LG with 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma), and 50 μM ascorbic acid (Sigma) for 14 days. Mouse bone marrow-derived MSCs (mBMSCs) were isolated from the femurs and tibias of 3–5 mice between 10 and 30 weeks of age. mBMSCs were cultured in growth medium with α-MEM (Gibco) with 10% FBS, 1% antibiotic–antimycotic solution, and 2 mM l-glutamine (Invitrogen, Carlsbad, CA, USA) in 5% CO₂ at 37 °C^{45 (link)}. To induce osteogenic differentiation, cells were cultured in growth medium with 10 mM β-glycerophosphate and 50 μM ascorbic acid for 8 days. For osteoclastogenesis, mouse bone marrow monocyte cells (mBMMs) were cultured for 3–4 days in α-MEM containing 15 ng/ml mRANKL (R&D systems, MN, USA) and 40 ng/ml mMCSF (R&D systems)^{46 (link)}.