Stemxvivo chondrogenic base media

In order to perform osteogenic differentiation, third-passage cells were cultured in media consisting of StemXVivo Chondrogenic Base Media supplemented with 10% of StemXVivo Human Osteogenic Supplement (R&D Systems) and 1% of P/S/A. After 11 days of culture, cells were fixed in 4% PFA for 15 minutes at room temperature. Following three times washing with HBSS cells were stained with 1% Alizarin Red S solution in water for 10 minutes to visualize mineralized matrix. Moreover, we performed RT-PCR to evaluate quantitatively effectiveness of differentiation process. Investigated genes involved are RUNX-2 (runt-related transcription factor 2), BMP-2 (bone morphogenetic protein 2), and COLL-1 (collagen type I). Sequences of primes are listed in Table 2.

DPSCs and PDLSCs were treated with 3.5 µM SF for 3 days; then, the cells were collected as 5 × 10⁵ cells in 15-ml polypropylene tubes. Cells were centrifuged, and the media were replaced with the StemXVivo Chondrogenic Base Media supplemented with StemXVivo Chondrogenic Supplement (R&D Systems, Minneapolis, Minnesota, United States) and 1% antibiotic/antimycotic. Control cells were cultured in the normal growth medium. Every 3 days, half of the medium was replaced by a new medium. All cultures were grown for 21 days; then, the pellets were collected and frozen by OCT compound (Thermo Fisher), cryosectioned and stained by Collagen Type II immunofluorescent staining. Photographs were captured using a phase-contrast microscope at ×20 magnification. Chondrogenic quantification was done using ImageJ software (NIH).