Characterization of ASC-Exos
The TEM and NTA revealed spherical vesicles with a particle size distribution of 50 to 150 nm. Western-blotting analysis showed that these vesicles had strong surface expression of exosomal markers, including CD9, CD63, and TSG-101 (Figure 1).
Inflammation analysis
Cell proliferation
Dexamethasone treatment significantly decreased the proliferation intensity of rat raw cells to 0.82-, 0.89-, and 0.88-fold at 12, 24, and 48 hours, respectively, compared with the control group.
To our surprise, additional treatment with ASC-Exos did not further decrease the proliferation intensity, but overrode the detrimental effect of dexamethasone on the proliferation of raw cells, with the GCs + ASC-Exos group showing no significant differences in proliferation intensity compared with the control group (Figure 2A).
Cell migration
Dexamethasone treatment significantly decreased the migration of rat raw cells to 0.69-fold at 24 hours, compared with the control group.
Consistent with the cell proliferation results, additional treatment with ASC-Exos did not further decrease cell migration, but overrode the detrimental effect of dexamethasone on the migration of raw cells, with the GCs + ASC-Exos group showing no significant differences in migration compared with the control group (Figure 2B, 2C).
Secretion of inflammatory-related cytokines
Pro-inflammatory cytokines
Dexamethasone treatment significantly downregulated the secretion of TNF-α, IL-1α, and IL-1β from rat raw cells to 0.75-, 0.85-, and 0.49-fold, respectively, compared with the control group.
In contrast to the cell proliferation and migration results, additional treatment with ASC-Exos further significantly downregulated the secretion of TNF-α, IL-1α, and IL-1β to 0.54-, 0.76-, and 0.33-fold, respectively, compared with the control group.
Significant differences were also detected between the GCs group and the GCs + ASC-Exos group, indicating that additional treatment with ASC-Exos might exert a stronger anti-inflammatory effect than dexamethasone alone (Figure 3A, 3B, 3C).
Anti-inflammatory cytokines
Dexamethasone treatment significantly upregulated the secretion of IL-4 to 1.39-fold, and significantly downregulated the secretion of IL-10 to 0.81-fold, respectively, compared with the control group.
Additional treatment with ASC-Exos further significantly upregulated the secretion of IL-4 and IL-10 to 3.68- and 2.31-fold, respectively, compared with the control group.
Consistent with the pro-inflammatory cytokine results, significant differences were also detected between the GCs group and the GCs + ASC-Exos group, indicating that additional treatment with ASC-Exos might exert a stronger anti-inflammatory effect than dexamethasone alone (Figure 3D, 3E).
Cytoprotective analysis
Cell proliferation
Dexamethasone treatment significantly decreased the proliferation intensity of rat tenocytes to 0.74-, 0.91-, and 0.70-fold at 12, 24, and 48 hours, respectively, compared with the control group.
Additional treatment with ASC-Exos not only overrode the detrimental effect of dexamethasone on the proliferation of rat tenocytes, but further significantly promoted their proliferation intensity to 1.17-, 1.22-, and 1.13-fold at 12, 24, and 48 hours, respectively, compared with the control group (Figure 4A).
Cell migration
Dexamethasone treatment significantly decreased the migration of rat tenocytes to 0.55-fold at 24 hours, compared with the control group.
Additional treatment with ASC-Exos not only overrode the detrimental effect of dexamethasone on the migration of rat tenocytes, but further significantly increased their migration to 1.13-fold at 24 hours, compared with the control group (Figure 4B, 4C).
Cell senescence
Dexamethasone treatment significantly increased the senescence intensity of rat tenocytes to 1.92-fold at 24 hours, compared with the control group.
Additional treatment with ASC-Exos overrode the detrimental effect of dexamethasone on the senescence intensity of rat tenocytes, as shown by the lack of a significant difference in the senescence intensity between the GCs + ASC-Exos group and the control group (Figure 5A, 5B).
Cell apoptosis
Dexamethasone treatment significantly increased the apoptosis percentage of rat tenocytes to 1.34-fold at 24 hours, compared with the control group.
Additional treatment with ASC-Exos overrode the detrimental effect of dexamethasone on the apoptosis percentage of rat tenocytes, as shown by the lack of a significant difference in the apoptosis percentage between the GCs + ASC-Exos group and the control group (Figure 5C, 5D).
Transcription of ROS
Dexamethasone treatment significantly upregulated the transcription of ROS of rat tenocytes to 2.40-fold of rat tenocytes at 72 hours, compared with the control group.
Additional treatment with ASC-Exos not only overrode the effect of dexamethasone on the ROS transcription by rat tenocytes, but further significantly decreased their transcription to 0.41-fold at 72 hours, compared with the control group (Figure 6A).
Transcription of degradative enzymes and their inhibitors
Dexamethasone treatment significantly upregulated the transcription of MMP-2, MMP-9, and MMP-13 by rat tenocytes to 1.79-, 2.33-, and 3.62-fold, respectively, compared with the control group. Additional treatment with ASC-Exos overrode the effect of dexamethasone on the transcription of degradative enzymes of rat tenocytes, as shown by the lack of significant difference between the GCs + ASC-Exos group and the control group.
Dexamethasone treatment had little effect on the transcription of degradative enzyme inhibitors by rat tenocytes, as shown by the lack of significant difference between the GCs group and the control group regarding the transcription of TIMP-1 and TIMP-3. Additional treatment with ASC-Exos upregulated the transcription of TIMP-1 and TIMP-3 of rat tenocytes to 3.95- and 2.72-fold at 72 hours, respectively, compared with the control group (Figure 6B–6F).
Transcription of tenocytic matrix molecules
Dexamethasone treatment significantly downregulated the transcription of decorin and biglycan by rat tenocytes to 0.65- and 0.53-fold at 72 hours, respectively, compared with the control group. Additional treatment with ASC-Exos not only overrode the effect of dexamethasone on the decorin and biglycan transcription by rat tenocytes, but further significantly increased their transcription to 2.41- and 1.40-fold at 72 hours, respectively, compared with the control group (Figure 7A, 7B).
Dexamethasone treatment significantly downregulated the transcription of type I collagen by rat tenocytes to 0.27-fold at 72 hours compared with the control group. Transcription of type III collagen was also downregulated in the GCs group to 0.78-fold compared with the control group, but this difference did not reach statistical significance. Additional treatment with ASC-Exos not only overrode the effect of dexamethasone on the transcription of type I and type III collagen by rat tenocytes, but further significantly increased their transcription to 4.90- and 3.23-fold at 72 hours, respectively, compared with the control group (Figure 7C, 7D).
Dexamethasone treatment also significantly downregulated the type I/III transcription ratio to 0.34-fold compared with the control group. Additional treatment with ASC-Exos not only overrode the effect of dexamethasone on the type I/III transcription ratio of rat tenocytes, but further significantly increased their transcription ratio to 1.52-fold at 72 hours compared with the control group (Figure 7E).
Macroscopic evaluation of the supra-infraspinatus-humerus complex
The torn supraspinatus tendon was connected to the humerus through fibrous tissue, and the connection between the infraspinatus tendon and the humerus remained complete. There was no evidence of infection.
Histological analysis
Injured supraspinatus tendon
Generally, increased cellularity, vascularity, thinning, separation, and disorganization of collagen fibers, and fatty infiltration were observed in all three groups. These signs were most significant in the GCs group (Figure 8).
Quantitatively, the GCs group exhibited significantly higher fatty infiltration, and severe collagen degeneration (significantly lower type I collagen and higher type III collagen density) than both the control and GCs + ASC-Exos group. The immunohistochemical variables did not significantly differ between the control and GCs + ASC-Exos groups (Figure 9A, 9B, 9C).
Semi-quantitative analysis results showed that the histological properties in the GCs group were significantly worse than those in the control and GCs + ASC-Exos groups. There were no significant differences between the control and GCs + ASC-Exos groups (Figure 9D).
Intact infraspinatus tendon
Generally, slightly increased cellularity, vascularity, thinning, separation, and disorganization of collagen fibers were observed in all three groups. These signs were relatively most significant in the GCs group. There was no evidence of fatty infiltration in any of the three groups (Figure 10).
Quantitatively, the GCs group exhibited severe collagen degeneration (significantly lower type I collagen and higher type III collagen density) than both the control and GCs + ASC-Exos groups. There were no significant differences between the control and GCs + ASC-Exos groups with regard to collagen degeneration (Figure 11A, 11B).
Semi-quantitative analysis results showed that the histological properties in the GCs group were significantly worse than those in the control and GCs + ASC-Exos groups. There were no significant differences between the control and GCs+ ASC-Exos groups (Figure 11C).
Biomechanical analysis
Injured supraspinatus tendon
The cross-sectional area of the supraspinatus tendon did not significantly differ between the three groups (Figure 12A).
The GCs injection significantly decreased the ultimate load to failure and the ultimate stress to failure to 0.66- and 0.71-fold, respectively, compared with the control group (Figure 12B, 12C). Concomitant injection of ASC-Exos and GCs overrode the GCs-induced detrimental effects on the biomechanical properties of the injured supraspinatus tendon, as shown by the lack of significant differences between the GCs + ASC-Exos group and the control group in the ultimate load to failure and the ultimate stress to failure (Figure 12B, 12C).
All specimens failed at the middle of the tendon.
Intact infraspinatus tendon
The cross-sectional area of the intact infraspinatus tendon did not significantly differ between the three groups (Figure 13A).
The GCs injection significantly decreased the ultimate load to failure and the ultimate stress to failure to 0.64- and 0.74-fold, respectively, compared with the control group (Figure 13B, 13C). Concomitant injection of ASC-Exos and GCs overrode the GCs-induced detrimental effects on the biomechanical properties of the intact infraspinatus tendon, as shown by the lack of significant differences between the GCs + ASC-Exos group and the control group in the ultimate load to failure and the ultimate stress to failure (Figure 13B, 13C).
All specimens failed at the tendon-to-bone interface.