Background: Spinal cord injury (SCI) is a destructive complication that leads to temporary or permanent paralysis of a person or animal. The increase in inflammation and the release of pro-inflammatory cytokines at the site of the lesion worsens the condition. Treatment goals should be aimed at reducing or modulating these factors. Indoleamine 2, 3- dioxygenase (IDO1) is one of the main paracrine factors of mesenchymal stem cells (MSCs). It was demonstrated that the soluble factors and extracellular vesicles (EVs) from the conditioned medium of MSCs may mimic their producer cells’ immunomodulatory properties. EVs could be administered as hydrogel-embedded entities to enhance their local persistence in the damaged tissue. Methods: In the present study, hTERT-immortalized human adipose tissue-derived mesenchymal stem cells were used, in which the ectopic expression of human IDO1 gene is stably increased. As a control group, hTERT-MSCs-GFP were applied. To isolate extracellular vesicles (EVs), 72-hour conditioned media were collected under EV-depleted culture conditions. Following the pre-clearing steps, the extraction of small EVs (sEVs) performed based on the ultrafiltration method. Freshly prepared sEVs were mixed with modified chitosan hydrogel under sterile conditions and were kept on ice. Female rats (N=30, ~230 g) were used and divided into five groups including Sham, SCI, Hydrogel, hTERT-MSCs-GFP-EVs, and hTERT-MSCs-IDO1-EVs. Laminectomy of the T10 was performed based on the previously described method. The drake aneurysm clip is placed on the spinal cord for a minute. 60 µl of hydrogel or hydrogels containing 100 µg sEVs from control GFP and IDO1 groups were locally injected immediately after SCI. 72 h after surgery, the rats were euthanized and central and epicenter parts of the lesion were removed and placed in RIPA solution. The levels of interleukin (IL)-6, IL-10, and tumor necrosis factor alpha (TNF-α) were measured in the supernatant of spinal cord homogenates using ELISA kits. Results: Comparison of the concentration of different cytokines among the experimental groups reveals that the changes in the level of TNF-α was significant (P<0.05) for IDO1 (43.11±8.21) vs. control groups (54.21±6.21). Moreover, the ratio of IL-6/IL-10 (0.14±0.05) and TNF-α/IL-10 (0.23±0.08) was decreased for MSCs-IDO1-EVs, indicating their anti-inflammatory effects in comparison to other treatment groups, although it was not statistically significant. Conclusion: MSCs-EVs enriched from the conditioned medium of the cells equipped with the ectopic expression of IDO1, carry immunomodulatory content which may be effective in treatment of inflammatory diseases.

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