[Experimental study on chondrogenic differentiation of adipose-derived stem cells co-cultured with chondrocytes]

Abstract

Objective: To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs.

Methods: ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 x 10(4)/mL and 1 mL ADSCs at concentration 2 x 10(4)/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR.

Results: ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 +/- 0.07, 2.13 +/- 0.08, and 1.08 +/- 0.08) were significantly higher than those in the control group (0.04 +/- 0.03, 0.13 +/- 0.04, and 0.10 +/- 0.02) (P < 0.05).

Conclusion: ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.