[Experimental study on osteogenic differentiation of adipose-derived stem cells treated with different concentrations...

Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adipose-derived stem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches.

Five New Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adipogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 x 10(5) cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 x 10(-9) (group C), 1 x 10(-8) (group D), 1 x 10(-7) (group E), 1 x 10(-6) (group F), and 1 x 10(-5) mol/L (group G) dexamethasone, respectively. The cell proliferation and the mRNA expressions of osteocalcin (OC) and core binding factor alpha1 (Cbfalpha1) were detected by MTT and RT-PCR, respectively. The activity of alkaline phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by alizarin red staining.

ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value declined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P < 0.05). The mRNA expressions of OC and Cbfal reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then declined gradually. There was no significant difference in the mRNA expressions of OC and Cbfalpha1, the activity of ALP, and the percentage of mineral area between groups D and E (P > 0.05), but significant differences were found between groups D and E and other groups (P < 0.05). After 14 days, the cells of group G died, and the result of alizarin red staining was positive in groups B, C, D, E, and F.

When the concentration of dexamethasone in osteogenic medium is 1 x 10(-8) mol/L, it could not only reduce the inhibitive effect on cells proliferation, but also induce osteogenic differentiation of ADSCs more efficiently.