Background: Recent studies showed that bone marrow-derived mesenchymal stem cells (BMSCs) had risk of ectopic bone formation. In this study, we aimed to investigate the effect of growth and differentiation factor 6 (GDF-6) on the tenogenic differentiation of BMSCs in vitro, and then combined with small intestine submucous (SIS) to promote tendon regeneration in vivo.
Methods: The BMSCs were isolated from the green fluorescent protein (GFP) rats, and were characterized by multi-differentiation assays following our previous study protocol. BMSCs cultured with different concentrations of GDF-6, without growth factors served as control. After 2 weeks, mRNA expression and protein expression of tendon specific markers were examined by qRT-PCR and Western blotting to define an optimal concentration of GDF-6. Mann-Whitney U-test was used to compare the difference in relative mRNA expression among all groups; P ≤ 0.05 was regarded as statistically significant. The GDF-6 treated BMSCs combined with SIS were implanted in nude mice and SD rat acute patellar tendon injury model, the BMSCs combined with SIS served as control. After 12 and 4 weeks in nude mice and tendon injury model, the samples were collected for histology.
Results: After the BMSCs were treated with different concentration of GDF-6 for 2 weeks, the fold changes of the specific markers (Tenomodulin and Scleraxis) mRNA expression were significantly higher in GDF-6 (20 ng/ml) group (P ≤ 0.05), which was also confirmed by Western blotting result. The BMSCs became parallel in orientation after GDF-6 (20 ng/ml) treatment, but the BMSCs in control group were randomly oriented. The GDF-6 (20 ng/ml) treated BMSCs were combined with SIS, and were implanted in nude mice for 12 weeks, the histology showed neo-tendon formation. In the SD rat patellar tendon window injury model, the histology also indicated the GDF-6 (20 ng/ml) treated BMSCs combined with SIS could promote tendon regeneration.
Conclusions: GDF-6 has tenogenic effect on the tenogenic differentiation of BMSCs, and GDF-6 (20 ng/ml) has better tenogenic effect compared to other concentrations. The GDF-6 (20 ng/ml) treated BMSCs combined with SIS can form neo-tendons and promote tendon regeneration.