BMP-7 modified exosomes derived from synovial mesenchymal stem cells attenuate osteoarthritis by M2 polarization of macrophages

Abstract

Background: Although the exosomes derived from mesenchymal stem cells (MSCs) display a therapeutic effect on inflammatory diseases, its application on OA has great limitations due to lack of specificity and targeting. The current study aimed to elucidate the potential therapeutic role of bone morphogenetic proteins-7(BMP-7) modified synovial mesenchymal stem cells-derived exosomes (SMSCs-exo) on OA and mechanism.

Methods: For in vitro experiments, LPS-treated macrophages RAW264.7 were treated with SMSCs-exo (exo) or BMP-7 modified SMSCs-exos (BMP-7-exo). The levels of inflammatory factors were assessed by ELISA. Also, the proportion of iNOS and CD206 positive cells were quantified by flow cytometry. Chondrocytes and RAW264.7 were co-culture to evaluate the effects of macrophage polarization on chondrocytes cellular behaviors. This effect on KOA was verified by an experiment in vivo. HE staining and Safranin fast green staining were used to observe the damage of articular cartilage. Immunohistochemistry was used to determine the expression of collagen II and aggrecan in articular cartilage, as well as the expression of iNOS and CD206 in synovial tissues.

Results: Our in vitro results showed that BMP-7-exo treatment promoted LPS-induced proliferation of macrophages and chondrocytes, and showed a better ability to reduce inflammation by promoting macrophages M2 polarization. After co-culture with LPS treated macrophages, the proliferation rate and migration of chondrocytes were significantly decreased, while the apoptosis was significantly increased. The macrophages treated with BMP-7-exo and exo partially reversed these changes. The chondrocytes in BMP-7-exo group had higher proliferation rate and migration, as well as lower apoptosis compared with the exo group. Also, the in vivo results showed BMP-7-exo treatment improved the pathological changes of KOA and promoted synovial macrophages M2 polarization.

Conclusions: Our results demonstrated that BMP-7-exo attenuated KOA inflammation and cartilage injury by synovial macrophages M2 polarization, suggesting that BMP-7-exo carry much therapeutic potential for OA.

Keywords: BMP-7; Exosomes; Inflammation; Macrophage polarization; Osteoarthritis; Targeted drug delivery.

Fig. 1

Identification and characterization of SMSCs-exos. A: Representative TEM image (scale bar = 200 nm). B: The particle size of SMSCs-derived exosomes was assessed by NTA. C: The typical surface markers of MSCs-exos including CD63, CD81 and CD9 was detected by Western blot.

Fig. 2

The expression of BMP-7 in the transfected SMSCs and SMSCs-exos. Western blot was used to detect the expression of BMP-7 in the transfected SMSCs(A) and SMSCs-exos(B). **p < 0.01.

Fig. 3

BMP-7-exo promoted LPS-induced proliferation of macrophages and chondrocytes. A. The proliferation of RAW264.7 was detected by CCK-8 assay. B. The proliferation of chondrocytes was detected by CCK-8 assay. *P < 0.05, **p < 0.01.

Fig. 4

BMP-7-modified SMSCs-exos induces M2 polarization of macrophages RAW264.7. A and B: The proportion of iNOS and CD206 positive cells in macrophages RAW264.7 were detected by flow cytometry. C: The ratio of iNOS-positive cells/CD206-positive cells. D: The levels of inflammatory related factors including IL-β, IL-6, TNF-α, IL-10 and TGF-β in macrophages RAW264.7 were detected by ELISA. *P < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

Macrophage M2 polarization promotes proliferation and migration of chondrocytes. A. The migration of chondrocytes was detected by transwell assay (scale bar = 50 μm) B. The number of migration cells. C. The proliferation of chondrocytes was detected by CCK-8 assay. *P < 0.05, **p < 0.01.

Fig. 6

Macrophage M2 polarization promotes chondrocytes apoptosis. A. The representative diagram of apoptosis detected by flow cytometry. B. The apoptosis ratio of chondrocytes. The apoptosis ratio is the sum of the upper right quadrant and the lower right quadrant. **p < 0.01.

Fig. 7

BMP-7 modified SMSCs-exos alleviate histological injury of KOA. A. HE staining. Safranin-O Fast green staining. C. Histological score. **p < 0.01. D. The expression of aggrecan were detected by immunohistochemistry (scale bar = 200 μm). E. The expression of collagen II was detected by immunohistochemistry (scale bar = 200 μm).

Fig. 8

BMP-7-modified SMSCs-exos reduces KOA inflammation by induced synovial macrophages M2 polarization in synovial tissues. A. The expression of iNOS and CD206 in synovial tissues were detected by immunohistochemistry (scale bar = 50 μm). B. The proportion of iNOS positive cells in synovial tissues. C. The proportion of CD206 positive cells in synovial tissues. D. The levels of inflammatory related factors including IL-β, IL-6, TNF-α, IL-10 and TGF-β in synovial tissues were detected by ELISA. *P < 0.05, **p < 0.01.