Allogeneic adipose-derived mesenchymal stem cells promote the expression of chondrocyte redifferentiation markers and retard the progression of knee osteoarthritis in rabbits

Abstract

Osteoarthritis (OA) is a progressively degenerative disease of joints. In vitro culture of chondrocytes results in dedifferentiation, which is characterized by the development of fibroblast phenotypes, reduced ability to produce cartilage extracellular matrix (ECM) and increase the expression of molecular markers Col1a1, Col10a1 and Runx2. Redifferentiation of chondrocytes is indicated by increased expression of the molecular markers Col2a1, Aggrecan and Sox9. In the current study, we investigated the effects of allogeneic rabbit adipose-derived mesenchymal stem cells (ADSCs) on articular chondrocytes, and explored the therapeutic effect of ADSCs on damaged articular cartilage at different stages in a rabbit OA model. In vitro, the proliferation and migration of primary articular chondrocytes were enhanced by cocultured rabbit ADSCs, and the expression of redifferentiation markers in chondrocytes cocultured with ADSCs was increased at both the mRNA and protein levels, while the expression of dedifferentiation markers was decreased. In vivo, the rabbit model of OA was established by anterior cruciate ligament transection (ACLT) with complete medial meniscectomy (MMx). Two weeks after surgery, ADSCs were used for OA rabbit treatment. Intra-articular injection of ADSCs gradually alleviated articular cartilage destruction, decreased Osteoarthritis Research Society International (OARSI) and Mankin scores, and reduced MMP13 expression at different stages in the rabbit model of OA. During the experiment, allogeneic ADSCs did not cause any adverse events. The current study demonstrates the effects and molecular mechanisms of ADSCs on articular chondrocytes and provides a favorable reference for clinical OA treatment with mesenchymal stem cells (MSCs) derived from adipose tissue.

Keywords: ADSCs; articular cartilage; articular chondrocyte; molecular marker; osteoarthritis.

Figure 1

Morphological properties and identification of rabbit ADSCs. (A) Light microscopy images showing the characteristic fibroblast-like morphology of passage 3 ADSCs at 50×, 100×, and 200× magnification. Scale bars in (A1-A3) represent 400, 200 and 100 μm respectively. (B) The multipotency of ADSCs demonstrated by adipocytic differentiation (oil red O staining), osteogenic differentiation (alizarin red staining), and chondrogenic differentiation (toluidine blue staining). Scale bars represent 200 μm. (C) Photomicrographs of dual-immunofluorescence labeling of ADSCs for CD44 and CD90. Scale bars represent 100 μm.

Figure 2

Morphological properties and identification of rabbit primary articular chondrocytes. A. Light microscopy images of rabbit primary articular chondrocytes. B. Images of toluidine blue staining. C. Images of Col2a1 labeling. Scale bars represent 200 μm.

Figure 3

Effects of ADSCs on proliferation and migration of cocultured chondrocytes. A. EdU incorporation showing that ADSCs enhance the proliferation of cocultured chondrocytes. B. Quantification of proliferation of cocultured chondrocytes. Data represent the mean ± SEM of three independent experiments. C. Chondrocyte migration assays showing migrated chondrocytes stained by crystal violet. Scale bars represent 200 μm. **P < 0.01.

Figure 4

RT-qPCR and western blot analyses of molecular marker expression of chondrocyte redifferentiation for primary articular chondrocytes cocultured with ADSCs. A-C. Increased relative mRNA expression of Aggrecan, Col2a1 and Sox9 in chondrocytes cocultured with ADSCs. D, E. Increased relative protein expression of Aggrecan, Col2a1 and Sox9 in chondrocytes cocultured with ADSCs. F-H. Decreased relative mRNA expression of Col1a1, Col10a1 and Runx2 in chondrocytes cocultured with ADSCs. I, J. Decreased relative protein expression of Col1a1, Col10a1 and Runx2 in chondrocytes cocultured with ADSCs. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Macroscopic examination and histological assessment of intra-articular injection of ADSCs in OA rabbits at six and ten weeks post-surgery. A. Macroscopic examination of the FC and HE and SOFG staining six weeks post-surgery. B, C. OARSI and Mankin score assessment of the cartilage damage at six weeks post-surgery. D. Macroscopic examination and HE and SOFG staining of the FC at ten weeks post-surgery. E, F. OARSI and Mankin score assessment of the cartilage damage at 6 weeks post-surgery. FC: femoral condyle; HE: hematoxylin and eosin; SOFG: Saffron O and fast green. Scale bars represent 200 μm. **P < 0.01, ***P < 0.001.

Figure 6

The expression of MMP13 in articular cartilage after intra-articular injection of ADSCs in OA rabbits established by surgery. (A) Immunofluorescence analysis of MMP13 expression in the articular cartilage of the medial femoral condyle six weeks post-surgery. (B, C) Quantification of MMP13-positive cells and the mean optical density of MMP13 based on the staining results of (A). (D) Immunofluorescence analysis of MMP13 expression in articular cartilage of the medial femoral condyle ten weeks post-surgery. (E, F) Quantification of MMP13-positive cells and the mean optical density of MMP13 based on the staining results of (D). Scale bars represent 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001.