2.5. Alcian blue staining

Chondrogenic differentiation of hMSCs was accomplished by a modification of the protocol outlined by Johnstone et al. [38]. In brief, aliquots of 250,000 DPSCs suspended in 0.5 mL medium were distributed to 15 mL conical polypropylene centrifuge tubes (VWR, West Chester, PA). The cells were centrifuged for 5 min at 600 g and pelleted at the bottom of the tube, and cultured in serum-free chondrogenic medium (Millipore). Tubes were placed in an incubator with caps loosened to permit gas exchange. The sedimented cells formed a spherical mass at the bottom of the tube within 24 h. Medium was replaced three times per week. Cell pellets were harvested by rinsing in D-PBS followed by fixation for 1 h in 4% formaldehyde in D-PBS, made fresh. Samples were then transferred into 70% ethanol, dehydrated in ethanol and xylene series, and paraffin-embedded. Sections of 5 μm were cut through the center of each pellet. Sections were stained with Alcian blue stain and images were captured with a light microscope.