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. 2019 Mar 12;10(1):86.
doi: 10.1186/s13287-019-1193-1.

Intraarticular senescent chondrocytes impair the cartilage regeneration capacity of mesenchymal stem cells

Xu Cao  1 , Pan Luo  1 , Junjie Huang  1 , Chi Liang  1 , Jinshen He  1 , Zili Wang  1 , Dongyong Shan  2 , Cheng Peng  3 , Song Wu  4
Affiliations

Intraarticular senescent chondrocytes impair the cartilage regeneration capacity of mesenchymal stem cells

Xu Cao et al. Stem Cell Res Ther. .

Abstract

Background: Senescent cells exert a significant influence over their surrounding cellular environment. Senescent chondrocytes (SnChos) were found to be accumulated in degenerated cartilage present in joints affected by osteoarthritis. The influence of SnChos on exogenously transplanted stem cells has yet to be reported.

Methods: In this study, we evaluated the interactions between SnChos and bone marrow mesenchymal stem cells (BMSCs) when co-cultured as well as in the intra-articular senescent microenvironment (IASM). The effect of IASM on cartilage regeneration was also assessed.

Results: It was found that a small fraction of SnChos induced BMSC cellular senescence and apoptosis. SnChos also inhibited proliferation, facilitated stemness, and suppressed chondrogenic differentiation of BMSCs. BMSCs induced the apoptosis of SnChos, reduced the proportion of SnChos, stimulated SnChos proliferation, and revealed a bidirectional effect on SnChos inflammaging. IASM significantly suppressed the survival, proliferation, and appropriate differentiation of grafted BMSCs in vivo, all of which impaired cartilage regeneration. Anti-senescence agent ABT-263 was able to partly rescue the cells from the negative effects of SnChos.

Conclusions: The SnChos and BMSCs interacted with each other at cellular senescence, apoptosis, proliferation, differentiation, and cell functions. This interaction impaired the cartilage repair of MSCs. Anti-senescence agent provided a possible solution for this impairment.

Keywords: Bone marrow mesenchymal stem cells (BMSCs); Cartilage repair; Intra-articular senescent microenvironment (IASM); Osteoarthritis (OA); Senescent chondrocytes (SnChos).

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Conflict of interest statement

Ethics approval

All experiments involving animals were performed in accordance with guidelines approved by the IRB of Third Xiangya Hospital, Central South University (NO:2015-S056) and coincided with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
The confirmation of senescence induction and proliferation of cells in co-culture. a The staining for SA-β-Gal (green) in non-IR Chos (left) and SnChos (right) 10 days after IR exposure. The ratio of SA-β-Gal-positive cells was calculated (right, n = 3). b The expression level of p16Ink4a and p21Cip1 in cartilage from non-IR rat joints and post-IR rat joints (n = 3). c, d The EdU staining (green) in proliferating BMSCs (c) and SnChos (d) in co-culture at 7 and 21 days. Nucleus were stained by Hoechst 33342 (blue). Bars = 100 μm. The ratio of EdU-positive cells was calculated (right, n = 3). *P < 0.05, **P < 0.01
Fig. 2
Fig. 2
The apoptosis of cells in co-culture. a, b The immunocytochemistry staining for caspase-3 (brown) in apoptotic BMSCs (a) and SnChos (b, arrows) in co-culture at 7 and 21 days. Bars = 100 μm. The ratio of caspase-3-positive cells was calculated (right, n = 3). c, d The expression level of Bax and Bcl-2 in BMSCs (c) and SnChos (d) in co-culture at 7 and 21 days (n = 3). The BMSCs (7d) or SnChos (7d) were used as control. *P < 0.05, **P < 0.01002E
Fig. 3
Fig. 3
The senescence of cells in co-culture. a, b The staining for SA-β-Gal (green) in senescent BMSCs (a) and SnChos (b) in co-culture at 7 and 21 days. Bars = 100 μm. The ratio of SA-β-Gal positive cells was calculated (right, n = 3). c, d The expression level of p16Ink4a and p21Cip1 in BMSCs (c) and SnChos (d) in co-culture at 7 days and 21 days (n = 3). The MSCs (7d) or SnChos (7d) were used as control. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
The inflammation, differentiation, and chondrogenesis. a, b The expression level of stemness and differentiation markers in BMSCs (a) and inflammatory factors in SnChos (b) in co-culture at 7 and 21 days (n = 3). The MSCs (7 days) or SnChos (7 days) were used as control. c, d The generation of cartilage matrix protein Col II and Agg by BMSCs (c) and SnChos (d) in co-culture at 21 days (n = 3). The protein levels were normalized to GAPDH. *P < 0.05, **P < 0.01
Fig. 5
Fig. 5
The effect of IASM on viability, apoptosis, senescence, and proliferation of MSCs in rat joint. a, b, c, d The Calcein-AM (green)/PI (red) (a), Caspase-3 (brown) (b), SA-β-Gal (green) (c), and EdU (green) (d) staining of MSCs in gel scaffold in cartilage defect 1 week after transplantation. Nucleus were stained by Hoechst 33342 (blue). Bars = 50 μm. Positive cells were calculated respectively (right, n = 3). *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
The effect of IASM on BMSCs cartilage regeneration. a Safranin O/fast green staining of cartilage repair 6 weeks after BMSCs implantation. The residual immature area (RIA, white dashed line) and residual gel area (RGA, yellow dashed line) were calculated (right, n = 3). Bars = 400 μm. b–d The immunohistochemistry staining for Col II (b), Agg (c), and Col I (d) of cartilage repair 6 weeks after BMSCs implantation. Bars = 400 μm. e Safranin O/fast green staining of cartilage repair 3 months after BMSCs implantation. Bars = 400 μm. The Pineda score of cartilage regeneration was evaluated (right, n = 3). *P < 0.05, **P < 0.01
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