Safranin o

Synchondroses in paraffin sections made from the demineralized cranial base were stained with safranin O. Briefly, 4% PFA/PBS-fixed mouse heads were decalcified in 10% EDTA/0.1 M Tris (final pH 7.0) for one week at room temperature with agitation and embedded in paraffin. Paraffin sections (4 μm) were cut with a microtome blade (PATH BLADE + PRO, Matsunami) on a sliding microtome (Yamato Kohki, REM-710). After deparaffinization, sections were stained with Weigert’s iron haematoxylin solution, washed with 0.5% HCl in 70% ethanol, and stained sequentially in 0.05% fast green (Polysciences) and 0.1% safranin O (Polysciences). Slides were then evaluated by microscopy (BX53, Olympus).

Dulbecco’s minimum essential medium (DMEM), fetal bovine serum, stock antibiotic/antimycotic mixture were obtained from Invitrogen (Carlsbad, CA). ⁴⁵Calcium was obtained from Perkin-Elmer (Boston, MA). Antibody specific to MMP-13 (Lifespan Biosciences, Seattle, WA), ADAMTS5 (Santa Cruz Biotechnology, Dallas, TX), Cox-2 (Santa Cruz Biotechnology, Dallas, TX), and CCL-5 (Bioss, Woburn, MA) were obtained from the commercial sources indicated. Safranin-O, fast green, picrosirius red, and alcian blue were obtained from Polysciences (Warrington, PA). PC and PC-E were prepared according to the method described [32 (link)]. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Poly(dimethylsiloxane) (PDMS) pre-polymer (Sylgard 184) and a curing agent were purchased from Dow Corning (Midland, MI, USA). Tris-HCl was purchased from Biosesang (Seongnam, Korea). Dopamine hydrochloride and Alizarin Red S were obtained from Sigma-Aldrich (St. Louis, MO, USA). MSCs, HUVECs, MSC growth medium (MSCM), endothelial cell growth medium (ECM), endothelial cell growth supplements (ECGS), MSC growth supplements (MSCGS), fetal bovine serum (FBS), penicillin/streptomycin (P/S) solution, trypsin-EDTA (T/E) solution, Dulbecco’s phosphate-buffered saline (DPBS), MSC chondrogenic differentiation medium (MCDM), MSC chondrogenic differentiation supplement (MCDS), MSC osteogenic differentiation medium (MODM), and MSC osteogenic differentiation supplement A and B (MODS-A and MODS-B) were purchased from ScienCell (Carlsbad, CA, USA). Transforming growth factor-β3 (TGF-β3) and live/dead viability/cytotoxicity kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Safranin O was obtained from Polyscience (Warrington, PA, USA). Norland optical adhesive63 (NOA63) was purchased from Norland Products (Jamesburg, NJ, USA). Polystyrene (PS) sheets and poly(ethylene terephthalate) (PET) film were obtained from Goodfellow (Pittsburgh, PA, USA).