Intra-Articular Injection of miR-29a-3p of BMSCs Promotes Cartilage Self-Repairing and Alleviates Pain in the Rat Osteoarthritis

Abstract

Background: Stem cells intra-articular injection stagey indicated a potential therapeutic effect on improving the pathological progress of osteoarthritis (OA). However, the long-term effect of stem cells intra-articular injection on the cartilage regeneration remains unclear. miR-29a-3p is predicted to be a critical target for inhibiting insulin-like growth factor-1 expression and may aggravate the progression of OA.

Methods: In this study, we investigated the therapeutic efficacy of intra-articular injection of bone marrow mesenchymal stem cells (BMSCs) transfected with miR-29a-3p inhibitor in OA.

Results: miR-29a-3p inhibitor transfection did not influence cell viability of BMSCs, while the chondrogenic differentiation potential of BMSCs was significantly improved. Interestingly, intra-articular injection of BMSCs with miR-29a-3p inhibition significantly prevented articular cartilage degeneration by up-regulating the expression of Sox 9, Col-2a1, aggrecan and down-regulating the expression of matrix metalloproteinase, as well as relieved pain in OA.

Conclusion: The double effects on cartilage protection and pain relief indicated a great potential of intra-articular injection of miR-29a-3p inhibitor-transfected BMSCs for the treatment of OA.

Keywords: BMSCs; Cartilage; Osteoarthritis; Pain; miR-29a-3p.

Fig. 1

The transfection of miR-29a-3p inhibitor significantly inhibited miR-29a-3p and enhanced IGF-1 in BMSCs. A Predicted binding sites of miR-29a-3p on 3′-UTR of IGF-1. B Relative miR-29a-3p expression in BMSCs after transfection of miR-29a-3p inhibitor. C Relative gene expression of IGF-1 in BMSCs transfected with miR-29a-3p inhibitor. D–E Protein expression level of IGF-1 in BMSCs when transfected with miR-29a-3p inhibitor detected by western blot. F Luciferase activity with various reporters was detected in the presence or absence of miR‐29a-3p mimic in HEK293 cells. **p < 0.01, ***p < 0.001, n = 6

Fig. 2

Cell viability of BMSCs transfected with miR-29a-3p inhibitor. A The cell proliferation assay was studied with chondrocytes for 1, 4, and 7 days by CCK-8 assay. B Representative fluorescence microscopy images after Calcein-AM/PI staining of chondrocytes incubated for 3 days. Green fluorescence represented live cells, and red fluorescence indicated dead cells. C THE survival rates of BMSCs under different conditions. n = 6

Fig. 3

Chondrogenic differentiation of BMSCs was stimulated with downregulation of miR-29a-3p. A–C RT-qPCR analysis of chondrogenic genes Sox 9, Col-2a1, and aggrecan of BMSCs transfected with miR-29a-3p inhibitor lentivirus. (D–E) Safranine O and alcain blue of BMSCs transfected with miR-29a-3p inhibitor lentivirus. *p < 0.05, **p < 0.01, n = 6

Fig. 4

A, B H&E and safranin O staining at 6 and 12 weeks after the intra-articular injection. C, D OARSI scores and Markin scores were statistically analyzed in each group. *p < 0.05, **p < 0.01, ***p < 0.001, n = 6

Fig. 5

RT-qPCR analysis of cartilage-related genes of A Sox 9, B Col-2a1, C aggrecan, and D MMP-13 in OA rat model after intra-articular injection. *p < 0.05, **p < 0.01, n = 6

Fig. 6

A Representative immunofluorescence images of Sox 9, Col-2a1, aggrecan, and MMP-13 in cartilage at 6 weeks and 12 weeks post-injection. B–E Quantitative statistics of Sox 9, Col-2a1, aggrecan, and MMP-13 expression. *p < 0.05, **p < 0.01, ***p < 0.001, n = 6

Fig. 7

Paw withdrawal threshold (PWT) was measured to test mechanical allodynia *p < 0.05, ***p < 0.001, n = 6