Efficacy and mechanisms of human umbilical cord mesenchymal stem cells-derived exosomes in repair of tendon injury in rats / 中华创伤杂志

ABSTRACT

Objective:To investigate the effect and mechanism of exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSC) in repair of tendon injury in rats.Methods:The hUC-MSC were cultured and the surface markers were identified by flow cytometry. The cells were induced to differentiate into osteoblasts, chondroblasts and adipocytes using a specific media. Meanwhile, the exosomes were isolated from the cell supernatant using exosome separation columns, and were identified by transmission electron microscopy, PKH67 staining and Western blot. A total of 40 Wistar rats were used to establish the Achilles tendon injury model by surgical resection. The rats were divided into hUC-MSC group (Group A) (with 100 μg exosome injected at the injured site) and control group (Group B) (with 250 μl normal saline injected at the injured site) according to the random number table, with 20 rats per group. The expressions of transforming growth factor β (TGF-β), bone morphogenetic protein (BMP-2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF-2), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the tendon tissues of both groups were detected using q-PCR, Western blot and immunofluorescence assay at 4 weeks following injection. The expression of collagen III in the injured tissues of both groups was detected by immunohiestochemistry.Results:The isolated and cultured hUC-MSC presented fusiform under an inverted microscope. After osteogenic differentiation, the cells exhibited a cube nodular structure, and the Alizarin red staining was positive. After adipogenic differentiation, the fat was observed inside the cells, which was red by oil red O staining. After chondroblast differentiation, the cells secreted a large amount of glycosaminoglycans, and a strong positive was revealed by Alisin blue staining. The hUC-MSC-derived exosomes showed round disc shape with a depressed internal structure under a transmission electron microscope, which was verified via PKH67 staining. The Western blot analysis showed high expressions of motility-related protein-1 (CD9) and lysosomal associated membrane protein 3 (CD63). The q-PCR test revealed that the mRNA expressions of TGF-β (4.887±0.767), BMP-2 (3.079±0.150), VEGF (3.108±0.508) and FGF-2 (4.211±0.522) in Group A were markedly higher than those in Group B (1.000±0.062, 0.918±0.129, 1.004±0.103, 1.010±0.169, respectively) ( P<0.01), and that the mRNA expression of IL-1β (0.697±0.037) and TNF-α (0.793±0.021) in Group A was markedly lower than those in Group B (1.004±0.089 and 1.006±0.015, respectively) ( P<0.01). The Western blot analysis revealed that the protein expressions of TGF-β (1.434±0.041), BMP-2 (1.798±0.177), VEGF (1.552±0.113) and FGF-2 (1.357±0.039) in Group A were markedly higher than those in Group B (1.002±0.032, 0.992±0.068, 1.007±0.070, 0.994±0.051) ( P<0.01), and that the protein expressions of IL-1β (0.705±0.016) and TNF-α (0.840±0.045) in Group A was markedly lower than those in Group B (1.000±0.016, 1.003±0.040) ( P<0.01). The immunofluorescence revealed that the positive expression rates of TGF-β and VEGF in Group A were not significantly different from those in Group B ( P>0.05). However, the positive expression rates of BMP-2 (2.278±0.208) and FGF-2 (4.656±0.106) in Group A were markedly higher than those in Group B (0.315±0.101, 1.661±0.110) ( P<0.05 or 0.01), and the positive expression rates of IL-1β (1.677±0.947) and TNF-α (1.520±0.088) in Group A were greatly lower than those in Group B (4.296±0.291, 2.373±0.273, respectively) ( P<0.01). In Group A, the tendon collagen fibers were arranged regularly and tightly, with relatively significant expression of collagen III; while the tendon collagen fibers in Group B were distributed loosely, accompanying broken scarlike healing. Conclusion:The hUC-MSC-derived exosomes can prompt the repair of the injured tendon tissues, which may be associated with the function in up-regulating the expressions of growth factors including TGF-β, BMP-2, VEGF and FGF-2, enhancing the expression of collagen III and inhibiting the expression of the inflammatory cytokines including IL-1β and TNF-α.