BMP9 overexpressing adipose-derived mesenchymal stem cells promote cartilage repair in osteoarthritis-affected knee joint via the Notch1/Jagged1 signaling pathway

Abstract

Osteoarthritis (OS) is a common disease in orthopedics. Although OS is known as an inflammation mediated by inflammatory cytokines; however, the mechanism is poorly understood. In the present study, the role of bone morphogenetic protein-9 (BMP9) was investigated in chondrogenic differentiation of adipose-derived mesenchymal stem cells (ADMSCs). ADMSCs were transfected with BMP9. BMP9 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Type II collagen and aggrecan expression was detected by western blotting and RT-qPCR. Mouse models of knee OS were established. Hematoxylin-eosin staining and toluidine blue staining were performed to observe changes in the OS-affected knee joint. After intra-articular injection of ADMSCs transfected with BMP9, intra-articular expression of type II collagen and aggrecan was detected by western blot analysis and RT-qPCR. After the Notch signaling pathway was inhibited in ADMSCs, ADMSCs were injected into the articular cavity. The expression of Notch signaling pathway-related proteins Notch1 and Jagged1 was detected by western blot analysis and RT-qPCR. BMP9 promoted chondrogenic differentiation of ADMSCs. After injection of BMP9 overexpressing ADMSCs into the articular space, type II collagen and aggrecan expression was increased. When the Notch signaling pathway of ADMSCs was inhibited, the ability of BMP9 overexpressing ADMSCs to repair the cartilage in the OS-affected knee joint was attenuated. These results demonstrate that upregulating BMP9 protein expression may promote the chondrogenic differentiation of ADMSCs. Intra-articular injection of ADMSCs contributes to cartilage repair in OS-affected knee joints through the Notch1/Jagged1 signaling pathway.

Keywords: BMP9; Notch1/Jagged1 signaling pathway; adipose-derived mesenchymal stem cells; osteoarthritis.

Figure 1.

Chondrogenic induction of ADMSCs. ADMSCs induced cartilage cells, western blot assay and RT-qPCR detected the collagen II and Aggrecan expression. (A) Western blot assay. (B) RT-qPCR detection. Compared with control group, *P<0.05. ADMSC, adipose-derived mesenchymal stem cells.

Figure 2.

BMP9 promoted chondrogenic differentiation of ADMSCs. (A) Fluorescence signal intensity 24, 48 and 72 h after pVSV-G-BMP9 lentivirus-transfected ADMSCs (magnification, ×500), (B) Determination of the transfection of BMP9, RT-qPCR was performed to detect intracellular BMP9 expression. (C and D) Detection of expression of type II collagen and aggrecan after BMP9 transfected into ADMSCs by western blot assay and RT-qPCR, respectively. Compared with Induced group, *P<0.05. ADMSC, adipose-derived mesenchymal stem cells; BMP9, bone morphogenetic protein-9.

Figure 3.

BMP9 regulated the Notch/Jagged1 signaling pathway promotes the chondrogenic differentiation of ADMSCs. (A) Western blot assay for detecting the Notch/Jagged1 signaling pathway related protein expression, and determining the relationship between BMP9 and Notch pathway. (B) RT-qPCR detection. Compared with Induced group, *P<0.05. BMP9, bone morphogenetic protein-9.

Figure 4.

ADMSCs promoted cartilage repair in OS affected joints in mice. After OS model was established, ADMSCs were injected into joint cavity, and the changes of joint were observed by H&E staining and toluidine blue staining. Western blot assay and RT-qPCR were used to detect the collagen II and Aggrecan expression. (A) H&E staining (magnification, ×400). (B) Toluidine blue staining (magnification, ×400). (C) Western blot assay. (D) RT-qPCR detection. Compared with sham group, *P<0.05; Compared with OS group, ^#P<0.05; Compared with MSC group, ^$P<0.05; Compared with BMP9 group, ^@P<0.05. BMP9, bone morphogenetic protein-9; OS, osteoarthritis; MSC, mesenchymal stem cells; ADMSC, adipose-derived MSC.

Figure 4.

ADMSCs promoted cartilage repair in OS affected joints in mice. After OS model was established, ADMSCs were injected into joint cavity, and the changes of joint were observed by H&E staining and toluidine blue staining. Western blot assay and RT-qPCR were used to detect the collagen II and Aggrecan expression. (A) H&E staining (magnification, ×400). (B) Toluidine blue staining (magnification, ×400). (C) Western blot assay. (D) RT-qPCR detection. Compared with sham group, *P<0.05; Compared with OS group, ^#P<0.05; Compared with MSC group, ^$P<0.05; Compared with BMP9 group, ^@P<0.05. BMP9, bone morphogenetic protein-9; OS, osteoarthritis; MSC, mesenchymal stem cells; ADMSC, adipose-derived MSC.

Figure 5.

BMP9 overexpressing ADMSCs promote OS healing in mice by regulating the Notch/Jagged1 signaling pathway. After OS model was established, ADMSCs were injected into joint cavity, western blot assay and RT-qPCR were utilized to detect Notch signaling pathway related protein expression. (A) Western blot assay. (B) RT-qPCR detection. Compared with sham group, *P<0.05; Compared with OS group, ^#P<0.05; compared with BMP9 group, ^$P<0.05. BMP9, bone morphogenetic protein-9; OS, osteoarthritis; ADMSC, adipose-derived mesenchymal stem cells.