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. 2021 Mar 9:2021:6624874.
doi: 10.1155/2021/6624874. eCollection 2021.

Exosomes from Kartogenin-Pretreated Infrapatellar Fat Pad Mesenchymal Stem Cells Enhance Chondrocyte Anabolism and Articular Cartilage Regeneration

Jiahua Shao  1 , Jun Zhu  1 , Yi Chen  1 , Qiwei Fu  1 , Lexiang Li  1 , Zheru Ding  1 , Jun Wu  1 , Yaguang Han  1 , Haobo Li  1 , Qirong Qian  1 , Yiqin Zhou  1
Affiliations

Exosomes from Kartogenin-Pretreated Infrapatellar Fat Pad Mesenchymal Stem Cells Enhance Chondrocyte Anabolism and Articular Cartilage Regeneration

Jiahua Shao et al. Stem Cells Int. .

Abstract

Objective: To evaluate the effect of Kartogenin-pretreated exosomes derived from infrapatellar fat pad mesenchymal stem cells on chondrocyte in vitro and articular cartilage regeneration in vivo.

Methods: Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were isolated from rabbits to harvest exosomes. After identification of mesenchymal stem cells and exosomes, rabbit chondrocytes were divided into three groups for further treatment: the EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells), KGN-EXO group (chondrocytes treated with exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN), and control group. After processing and proliferation, phenotypic changes of chondrocytes were measured. In the in vivo study, 4 groups of rabbits with articular cartilage injury were treated with KGN-EXO, EXO, IPFP-MSCs, and control. Macroscopic evaluation and histological evaluation were made to figure out the different effects of the 4 groups on cartilage regeneration in vivo.

Results: The proliferation rate of chondrocytes in the EXO or KGN-EXO group was significantly higher than that in the control group (P < 0.05). The qRT-PCR results showed that the expression of Sox-9, Aggrecan, and Col II was the highest in the KGN-EXO group compared with the EXO group and the control group (P < 0.05). The results of Western blot were consistent with the results of qRT-PCR. In vivo, the cartilage defects in the KGN-EXO group showed better gross appearance and improved histological score than those in IPFP-MSC groups, EXO groups, and control groups (P < 0.05). At 12 weeks, the defect site in the KGN-EXO group was almost completely repaired with a flat and smooth surface, while a large amount of hyaline cartilage-like structures and no obvious cracks were observed.

Conclusion: Our study demonstrates that the exosomes isolated from infrapatellar fat pad mesenchymal stem cells pretreated with KGN have potent ability to induce chondrogenic differentiation of stem cells, effectively promoting the proliferation and the expression of chondrogenic proteins and genes of chondrocytes. The KGN-EXO can also promote the repair of articular cartilage defects more effectively, which can be used as a potential therapeutic method in the future.

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Conflict of interest statement

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
Isolation and identification of IPFP-MSC. (a) The P3 generation IPFP-MSC. (b) The results of flow cytometry. Blue line: negative control; red line: IPFP-MSCs. (c) Three-line differentiation experiments.
Figure 2
Figure 2
Isolation and identification of exosomes. (a) TEM results. The red arrow indicates exosomes. (b) Identification of exosome surface proteins. Western blotting showed that these exosomes were positive for the exosome-specific surface proteins CD9 and TSG101. (c) NTA test results. The size of the particles within the precipitate were predominantly 40–100 nm in diameter. Red line: range of particle concentration at different diameter sizes; black line: average concentration of particles at different diameter sizes.
Figure 3
Figure 3
The proliferation of chondrocytes after treatment with exosomes. The proliferation of chondrocytes in the EXO group and the KGN-EXO group increased significantly compared with that in the control group. ∗Significant difference between the EXO group and the control group (P < 0.05). #Significant difference between the KGN-EXO group and the control group (P < 0.05). d: days.
Figure 4
Figure 4
The Western blotting results showed that the expression levels of Sox-9, Aggrecan, and Col II, increased significantly after treatment with EXO and KGN-EXO when compared with those in the control group. Moreover, the KGN-EXO treatment was more effective than the EXO treatment alone (P < 0.05). Note that different letters above bars indicate significant differences (P < 0.05) while matched letter means no significant difference in each comparison among the KGN-EXO/EXO/control groups.
Figure 5
Figure 5
qRT-PCR results showed that the expression levels of Sox-9, Aggrecan, and Col II were significantly increased after treatment with KGN-EXO and EXO. For all three genes, there were statistical differences between the KGN-EXO group and the control group (P < 0.05) and statistical differences between the KGN-EXO group and the EXO group (P < 0.05). Note that different letters above bars indicate significant differences (P < 0.05) while matched letters mean no significant difference in each comparison among the KGN-EXO/EXO/control groups.
Figure 6
Figure 6
In vivo cartilage repair at 4 and 12 weeks after surgery. (a) The gross appearance, Saf-O/Fast Green and HE staining at 4 weeks. (b) The gross appearance, Saf-O/Fast Green and HE staining at 12 weeks. (c) The ICRS and histological scores at 12 weeks.
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