Exosomes derived from umbilical cord mesenchymal stem cells in mechanical environment show improved osteochondral activity via upregulation of LncRNA H19

Abstract

Background: Exosomes derived from stem cells have been demonstrated to be good candidates for the treatment of osteochondral injury. Our previous studies have demonstrated that mechanical stimulation could be crucial for the secretion of exosomes derived from umbilical cord mesenchymal stem cells (U-MSCs). Therefore, we explore whether mechanical stimulation caused by a rotary cell culture system (RCCS) has a beneficial effect on exosome yield and biological function.

Methods: U-MSCs were subjected to an RCCS at different rotational speeds and exosomes were characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting. small-interfering RNAs of Rab27a (siRNA-Rab27a) was used to reduce exosome production. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of mechanically sensitive long non-coding RNA H19 (LncRNA H19). The effects of exosomes on chondrocyte proliferation were examined using cell counting kit-8 (CCK-8), toluidine blue staining and a series of related genes. Annexin V-FITC and PI (V-FITC/PI) flow cytometry was used to detect the effect of exosomes on the inhibition of chondrocyte apoptosis. Macroscopic evaluation, MRI quantification and immunohistochemical staining were conducted to investigate the in vivo effects of exosomal LncRNA H19 through SD rat cartilage defect models.

Results: RCCS significantly promoted exosome production at 36 rpm/min within 196 h. Mechanical stimulation was able to increase the expression level of exosomes. The exosomal LncRNA H19 was found to promote chondrocyte proliferation and matrix synthesis and inhibit apoptosis in vitro. Chondral regeneration activity was lost in LncRNA H19-defective exosomes. The injection of exosomal LncRNA H19 in vivo resulted in improved macroscopic assessment, MRI quantification and histological analysis. Moreover, exosomal LncRNA H19 was able to relieve pain levels during the early stages of cartilage repair in an animal experiment.

Conclusion: Our findings confirmed that mechanical stimulation can enhance exosome yield as well as biological function for the repair of cartilage defects. The underlying mechanism may be related to the high expression of LncRNA H19 in exosomes. The translational potential of this article: This study provides a theoretical support of optimizing exosome production. It advances the yield of mesenchymal stem cell exosome and facilitate the clinical application to repair of osteochondral damage.

Keywords: Chondrocytes; Exosomes; LncRNA H19; Mechanical stimulation; Mesenchymal stem cells.

Figure 1

Characterisation of U-MSCs and exosomes. (A) Morphological observation of U-MSCs (×100). (B) U-MSCs exhibited multi-differentiation capacity for osteogenesis, adipogenesis and chondrogenesis. (C) Flow cytometric analysis of umbilical cord mesenchymal positive markers, such as CD105, CD73 and CD90, and negative markers, such as CD31, CD34, CD45 and HLA-DR. (D) The concentration and size distribution of exosomes by Nanosight. (E) Morphology of exosomes under transmission electron microscopy, scale bar: 200nm. (F) Western blot analysis of exosome surface markers (TSG101, CD63, CD81 and Calnexin).

Figure 2

High-yield exosome production from RCCS. (A) Yield of exosomes isolated by RCCS at different rotational speeds (n = 12). Exosome yield = the number of exosome measured by Nanosight/the number of cells. (B) Particle purity of exosomes at different rotational speeds ( = 12). Particle purity = the number of particles/exosomal protein (μg). (C) Yield of exosomes isolated by RCCS at 36 rpm/min from different culture times (n = 4). (D) Average diameter of particles isolated by RCCS at 36 rpm/min from different culture times (n = 5). (E) Morphology of exosomes under transmission electron microscopy after 48∗5 h, scale bar: 200nm. Plots show yield for each method and the mean ± SD of all measurements. ∗p < 0.05, ∗∗p < 0.01, n = 3.

Figure 3

Effect of Rab27a inhibition on U-MSCs exosomes secretion and yield. (A-B) The protein and mRNA level of Rab27a was decreased in the Rab27a-siRNAsgroup. (B) The expression of exosomal marker (TSG101, CD63 and CD81) in U-MSCs by western blot. (C) The expression of exosomal marker (TSG101, CD63 and CD81) in exosome derived from U-MSCs by western blot. (D) The yield of exosome at static and optimal speed with or without interference. Plots show yield for each method and the mean ± SD of all measurements (∗p < 0.05; ∗∗p < 0.01, n = 3). (E) Therefore, siRNA1 was used for the following interference assay. We cultured U-MSCs transfected with siRNA1 at two RCCS speeds: one completely static (0 rpm/min) and the other at the optimal rotational speed (36 rpm/min). Without mechanical stimulation (0 rpm/min), the exosome yield in the siRNA group was reduced approximately three fold (p<0.01). However, the yield only dropped 1.5-fold in the mechanical environment (p<0.01, Fig. 3E). These results demonstrated that mechanical stimulation can attenuate the reduction in exosome production caused by siRNA interference.

Figure 4

Exosomal LncRNA H19 in mechanical environment enhances proliferation and inhibits apoptosis of chondrocytes. (A) The expression level of LncRNA H19 in exosomes under mechanical environment detected by qRT-PCR. (B) The expression level of LncRNA H19 in U-MSCs treated with different siRNAs. (C) The proliferation was assessed by CCK-8 assay. (D–E) The protein and mRNA level of genes associated with proliferation (PCNA, Cyclin D1) and matrix synthesis (Col II, Sox 9, MMP 13, ADMST5). (F) Toluidine blue staining and the quantification of chondrocytes treated with different kinds of exosomes. (G) The quantification of toluidine blue staining. (H) Chondrocytes were pretreated with si-Exos and S-Exos followed by IL-1β (10 ng/mL) challenge for 24 h. Apoptosis was assessed by flow cytometry. (I) Apoptotic rate of chondrocytes. (J–K) The protein and mRNA level of genes associated with anti-apoptosis (Bcl-2, Bax). Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, n = 3; ^#p < 0.05, ^##p < 0.01 compared to each other group, n = 3.

Figure 5

Effect of exosomes on repair of cartilage defect. (A) Representative macroscopic, MRI, T2 mapping images of the regenerated tissues. (B) ICRS macroscopic scores. (C) T2 mapping scores. (D) Staining results of HE, TB, Saf-O and immunohistochemical staining for type II collagens. (E) Wakitani scores for the histological sections. (F) Percentage of collagen II stain area. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, n = 3. (G) Time-dependent nociceptive responses after injection of different exosomes. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 compared to the si-Exos group; ^#p < 0.05, ^##p < 0.01 compared to the control group, n = 3.

Figure 6

Schematic presentation involved in the proliferation and anti-apoptosis of chondrocytes by exosomal LncRNA H19 derived from U-MSCs in mechanical environment. Mechanical stimulation caused by RCCS could enhance exosome biological function for repair of cartilage defect. The underlying mechanism may be through high expression of LncRNA H19 in exosomes.

Figs1Anterior view of the RCCS system.

Figs2

Lateral view of the RCCS system.