Objective      The effectiveness of bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs) in combination with transforming growth factor-β1 (TGF-β1) is investigat

ed to find the optimal concentration of TGF-β1 for bone regeneration of tissue engineering.       Methods      Mouse MSC C3H10T1/2 cell line was used to construct the osteogenic differentiation cell model by the steps of transfection of recombinant adenovirus with BMP9 and stimulation with different concentrations of TGF-β1 protein(0

5

10

and 20 ng/mL). Alkaline phosphatase (ALP) activity was assayed by the modified SEAP chemoluminescence assay and histochemical staining assay. Alizarin Red S staining was used for matrix mineralization. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of collagen type Ⅰ (COL Ⅰ)

osteopontin (OPN) and osteocalcin (OCN). Viable cell counting with trypan blue staining was used to detect cell proliferation and flow cytometry to test cell cycle.       Results      It was found that BMP9 combined with 5 ng/mL TGF-β1 produced higher ALP activity and more matrix mineralization at earlier period as compared to single BMP9 treatment group. Matrix mineralization had no significant difference after 17 days’s treatment. Meanwhile

TGF-β1 alone had no difference with control group. BMP9 combined with higher concentration TGF-β1 inhibited ALP activity and matrix mineralization in a dose-dependent manner. In real-time PCR and Western blot results

BMP9 combined with 5 ng/mL TGF-β1 increased mRNA transcription and protein expression of COL Ⅰ

OPN and OCN. COL Ⅰ expression increase was observed earlier and more significant.  TGF-β1 of 20 ng/mL combined with BMP9 decreased the expression of OPN and OCN but COL Ⅰ. TGF-β1 inhibited the proliferation of MSCs during the BMP9-induced osteogenic differentiation and arrested the cells in G

0

/G

1

phase.       Conclusion      Suitable concentration of TGF-β1 can promote BMP9-induced osteogenic differentiation of MSCs. TGF-β1 and BMP9 may have synergistic effect.