Enhancement of acellular cartilage matrix scaffold by Wharton's jelly mesenchymal stem cell-derived exosomes to promote osteochondral regeneration

Abstract

Articular cartilage defect repair is a problem that has long plagued clinicians. Although mesenchymal stem cells (MSCs) have the potential to regenerate articular cartilage, they also have many limitations. Recent studies have found that MSC-derived exosomes (MSC-Exos) play an important role in tissue regeneration. The purpose of this study was to verify whether MSC-Exos can enhance the reparative effect of the acellular cartilage extracellular matrix (ACECM) scaffold and to explore the underlying mechanism. The results of in vitro experiments show that human umbilical cord Wharton's jelly MSC-Exos (hWJMSC-Exos) can promote the migration and proliferation of bone marrow-derived MSCs (BMSCs) and the proliferation of chondrocytes. We also found that hWJMSC-Exos can promote the polarization of macrophages toward the M2 phenotype. The results of a rabbit knee osteochondral defect repair model confirmed that hWJMSC-Exos can enhance the effect of the ACECM scaffold and promote osteochondral regeneration. We demonstrated that hWJMSC-Exos can regulate the microenvironment of the articular cavity using a rat knee joint osteochondral defect model. This effect was mainly manifested in promoting the polarization of macrophages toward the M2 phenotype and inhibiting the inflammatory response, which may be a promoting factor for osteochondral regeneration. In addition, microRNA (miRNA) sequencing confirmed that hWJMSC-Exos contain many miRNAs that can promote the regeneration of hyaline cartilage. We further clarified the role of hWJMSC-Exos in osteochondral regeneration through target gene prediction and pathway enrichment analysis. In summary, this study confirms that hWJMSC-Exos can enhance the effect of the ACECM scaffold and promote osteochondral regeneration.

Keywords: Articular cartilage; Exosomes; Mesenchymal stem cells; Regeneration; Tissue engineering.

Graphical abstract

Fig. 1

Schematic illustration of the whole study.

Fig. 2

(a) Extracted exosomes by differential ultracentrifugation. (b) Osteochondral defects model. Scale bar = 5 mm. (c) The ACECM scaffold was implanted into the defect site. (d) Groups used in the osteochondral defects repair experiments.

Fig. 3

(a) Wharton's jelly stripped from the umbilical cord. (b) P3-generation hWJMSCs morphology under an optical inverted microscope. (c) Osteogenic differentiation of hWJMSCs stained with alizarin red ( × 100). (d) hWJMSCs differentiated into fat and stained with oil red O ( × 200). (e) hWJMSCs differentiated into cartilage and stained with alcian blue ( × 40). (f) Flow cytometry showed that hWJMSCs highly expressed CD73, CD90, and CD105 (both ≥95%) but did not express CD34 and CD45 (both ≤2%). (g) TEM shows that Exos have the shape of a disc with a diameter of approximately 100 nm (Exos shown by the red arrowhead). (h) NTA shows the particle size distribution of Exos. (i) WB results for the Exo markers CD9, TSG101, and CD63 and the negative marker Calnexin. CL is a positive control (cell lysate). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

(a) Macroscopic view of the ACECM scaffold. (b) SEM results of the cross-section of the ACECM scaffold. (c) SEM results for a longitudinal section of the ACECM scaffold. (d) SEM results for BMSCs planted on the ACECM scaffold for 4 days (the red arrowhead indicates the clustered cells). (e–g) Confocal laser observation of live/dead-stained BMSCs planted on the ACECM scaffold for 1, 4, and 7 days (green indicates live cells, and red indicates dead cells). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

(a) Effects of different concentrations of Exos on the migration of BMSCs in vitro (purple shows the cells). Scale bar = 500 μm. (b) Effects of Exos on the migration of BMSCs in vitro (Transwell method). (c) Effects of Exos on the proliferation of BMSCs in vitro (CCK-8 method). (d) Effects of Exos on the proliferation of chondrocytes in vitro (CCK-8 method). (e) The effect of Exos on macrophage polarization in vitro (RT-PCR). (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

(a) Macroscopic results for new cartilage. The red circles show the locations of the defects. Scale bar = 5 mm. (b) ICRS cartilage repair macroscopic score. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

(a) Micro-MRI findings. The red arrowheads show the locations of the defects. (b) MRI WORMS score results. (c) Micro-CT findings, the red rectangles show the subchondral bone at the defect sites. (d–e) Micro-CT BVF analysis and Tb.Th analysis results. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

(a) HE staining results. The black arrow indicates the defect boundary. The left side of the arrow is normal cartilage, and the right side of the arrow is repaired tissue. Scale bar = 100 μm. (b) Safranin O-fast green staining result. Scale bar = 100 μm. (c) Type II collagen immunohistochemical staining results. Scale bar = 100 μm. (d) Histomorphology score of the repaired tissue (cartilage). (e) Histomorphology score of the repaired tissue (subchondral bone). (f) Young's modulus of the repaired tissue. (g) The collagen content of the repaired tissue. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

(a) Immunohistochemical staining (IL-10, IL-1, TNF-α) for synovium and the osteochondral defects in rats at 10 and 20 days after surgery, the red rectangles show the repaired tissue, scale bar = 200 μm. (b–d) Semi-quantitative results of immunohistochemical staining for IL-10, IL-1 and TNF-α. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

(a) DAPI and CD206 immunofluorescence staining of the synovium in rats at 10 days and 20 days after surgery, blue fluorescence are the nucleus, red fluorescence are the CD206 positive cells, scale bar = 100 μm. (b) DAPI, CD68和CD86 immunofluorescence staining of the synovium in rats at 10 days and 20 days after surgery, blue fluorescence are the nucleus, green fluorescence are the CD68 positive cells, red fluorescence are the CD86 positive cells, scale bar = 100 μm. (c) The rate of CD206 positive cells in the synovium. (d) The rate of CD68 positive cells in the synovium. (e) The proportion of CD86 positive cells to total macrophages. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

(a) The immunotissue fluorescence staining of the osteochondral defects of rats at 10 days and 20 days after surgery, blue fluorescence are the nucleus, green fluorescence are CD73 positive cells, red fluorescence are CD105 positive cells, scale bar = 100 μm. (b) The enlarged images of the area show by the green rectangles in Fig. 11a. (c) Statistical analysis results of CD73 and CD105 double positive cells. (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 12

(a) The top 50 high-abundance miRNAs according to the total read count of hWJMSC-Exo miRNA sequencing. The abscissa represents each miRNA, and the ordinate represents the count values of these miRNAs; (b–e) The target genes of Top50 miRNAs were enriched in KEGG analysis, GO biological process analysis, GO cellular component analysis and GO molecular function analysis (the first 20 results were plotted). The abscissa represents the number of genes annotated in the pathway, the ordinate represents the pathway, the color of the column represents the q value, and q < 0.05 was significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 13

The possible mechanism of MSC-Exos promoting osteochondral regeneration. (a) Promoting macrophage polarization toward the M2 phenotype. (b) Promoting MSC migration. (c) Promoting MSC proliferation. (d) Promoting chondrocyte proliferation. (e) Promoting the secretion of cartilage matrix. (f) inhibiting chondrocyte hypertrophy.