Objective: To study the effect of transforming growth factor beta1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction.
Methods: Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditions to induce them to differentiate into chondrocytes. Toluidine blue staining and immunofluorescence were used to identify those induced chondrocytes. TGF-beta1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups (TGF-beta1 + IGF-1 group, 10 ng/ml and 50 ng/ml respectively; TGF-beta1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group (without growth factor). In TGF-beta1 + IGF-1 group, toluidine blue staining and immunofluorescence staining were carried out at 14 days and 21 days. The effect of TGF-beta1 and IGF-1 on the expression of collagen II gene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressions of collagen II were compared between two culture patterns.
Results: In TGF-beta1 +-IGF-1 group, the histological examination and immunofluorescence showed that those inducted chondrocytes could express collagen II at 14 days. The gel electrophoresis results showed that the fragment of collagen II gene was seen in TGF-beta1 +IGF-1 group and TGF-beta1 group and that no fragment of collagen II gene was seen in IGF-1 group and control group. The expression of collagen II gene was stronger in TGF-beta1+ IGF-1 group than in TGF-beta1 group, showing significant difference (P<0. 05). Cells expressed more collagen II under pellet culture than under monolayer culture.
Conclusion: IGF-1 could enhance the effect of TGF-beta1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.