To differentiate DPSC to chondrocytes, only the fourth cell passage was used. After 21 days of culture in chondrogenic induction medium, the cellular unfortunately morphological characters had changed.Figure 1Characteristics of isolated and in vitro mouse DPSC colony formation after cultured DPSC at the first passage (a and b). Cell after 48 hours after culture (c) and fibroblastic-like cells shape (d).After chondrogenic induction, the cytoplasm contracted toward the nucleus and formed round shape cells without branches (Figures 2(a) and 2(b)). Cells that have been cultured in chondrocyte differentiation medium were stained by toluidine blue to produce blue colors (Figure 2(c)). Toluidine blue staining revealed an increased production of glycosaminoglycan during induction, a phenomenon noticed only among chondrocyte cells.

Figure 2Characteristics of chondrocyte derived from mouse DPSC. After chondrogenic induction, the cytoplasm contracted toward the nucleus and formed spherical cells without branch (a and b). Glycosaminoglycans in chondrocyte tissue were stained by toluidine blue …In RT-PCR, mouse DPSC where showed to express Cd146 and Cd166 indicating that these cells belong to mesenchymal stem cells (Figures 3(a) and 3(b)). Since these cells did not express Cd31 (Figure 3(c)), it showed that they do not belong to hematopoietic stem cells. On the other hand, CollI marker was shown to be highly expressed after 14 days of induction (Figure 3(e), L1) as compared to before induction (Figure 3(e), L2) whereas activation of CollII as mature chondrocyte cells markers was observed after 21 days treatment with chondrocyte induction medium (Figure 3(f), L1).

On the other hand, before induction (Figure 3(f), L2) showed no amplification of CollII.Figure 3RT-PCR analyses of mouse DPSC in 1% (w/v) Agarose. (a) Cd146 (479bp), (b) Cd166 (630bp), (c) Cd 31(355bp), (d) Gapdh as a house keeping gene (717bp). (e, L1) The CollI marker (532bp) was after …Cell viability analyses using MTT assay during differentiation stage showed that the proliferation ability of differentiated cells is weaker as compared to the control. Statistical analyses also demonstrated significant differences (P < 0.05) between the control and chondrogenic induction group at day 7 until 21 (Figure 4). ALP activity of DPSC cultured in chondrogenic differential medium was detected using an ALP enzymological assay.

After 14 days of culturing DPSC in the chondrogenic induced medium, results showed that most of the cells became alkaline phosphatase positive as compared to control GSK-3 cells which were cultured in AMEM and 15% (v/v) FBS. Enzymatic activity of differentiated cells reached the highest valueafter21 days (Figure 5).Figure 4Cell viability by using MTT assay. The results were presented as mean �� SD.