[A 3D hydrogel loaded with exosomes derived from bone marrow stem cells promotes cartilage repair in rats by modulating immunological microenvironment]

Abstract

Objective: To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage.

Methods: The supernatant of cultured bone marrow stem cells was subjected to ultracentrifugation separate and extract the exosomes, which were characterized by transmission electron microscopy, particle size analysis and Western blotting of the surface markers. The changes in rheology and electron microscopic features of GelMA hydrogel were examined after loading the exosomes. We assessed exosome release from the hydrogel was detected by BCA protein detection method, and labeled the exosomes with PKH26 red fluorescent dye to observe their phagocytosis by RAW264.7 cells. The effects of the exosomes alone, unloaded hydrogel, and exosome-loaded hydrogel on the polarization of RAW264.7 cells were detected by q-PCR and immunofluorescence assay. We further tested the effect of the exosome-loaded hydrogel on cartilage repair in a Transwell co-culture cell model of RAW264.7 cells and chondrocytes in a rat model of knee cartilage injury using q-PCR and immunofluorescence assay and HE and Masson staining.

Results: GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells (P < 0.05). In the Transwell co-culture model, the exosome-loaded GelMA hydrogel significantly promoted the repair of injured chondrocytes by regulating RAW264.7 cell transformation from M1 to M2 (P < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage.

Conclusion: GelMA hydrogel loaded with bone marrow stem cell-derived exosomes can significantly promote the repair of cartilage damage in rats by improving the immune microenvironment.

Keywords: GleMA hydrogel; cartilage damage; exosomes; immunoregulation.

图 1

骨髓干细胞及其外泌体的鉴定 Identification of bone marrow stem cells (BMSCs) and their exosomes. A: Typical spindle- like morphology of the BMSCs. B: Alizarin Red staining and Oil-Red-O staining of the BMSCs. C: TEM of the obtained nanoparticles. D: Particle size analysis of the nanoparticles by qNano^® system. E: Western blotting of exosome markers CD9 and TSG101.

图 2

负载外泌体的GelMA水凝胶的特性 Characteristics of GelMA hydrogel loaded with exosomes. A: NMR characterization of methacrylated gelatin. B: Rheological analysis of GelMA-Exo hydrogel. C: SEM image of GelMA-Exo hydrogel. D: Daily release curve of exosomes (n=3). E: Cumulative release profile of exosomes for 14 days (n=3).

图 3

每组生物相容性评估 Biocompatibility evaluation in each group. A: Live/Dead analysis in each group after 1 day of cell culture (Scale bar=200 μm). B: CCK8 assay of chondrocytes in each group (^**P < 0.01, ^***P < 0.001, n=5). C: The cytoskeleton images showing adhesion of the chondrocytes after 3 days of culture in each group (Scale bar=100 μm).

图 4

GelMA-Exo明显促进RAW264.7细胞的极化 GelMA- Exo significantly promotes the polarization of RAW264.7 cells. A: Exosomes loaded in the hydrogel are taken up by RAW264.7 cells (Scale bar=50 μm). B: q-PCR of RAW264.7 cells after 3 and 7 days of culture in each group (n=3). C: Immunofluorescence detection of RAW264.7 cells after 7 days of culture in each group (Scale bar=50 μm). D: Quantitative analysis of fluorescence intensity of iNOS and ARG- 1 (n=3). ANOVA followed by Tukey's test was used for statistical analysis (^**P < 0.01, ^***P < 0.001).

图 5

GelMA-Exo明显促进IL-1β损伤的软骨细胞的修复通过改善免疫微环境 GelMA-Exo significantly promotes repair of chondrocytes with IL-1β-induced injury by improving the immune microenvironment. A: q-PCR of the chondrocytes after 7 days of culture in each group (n=3). B: Immunofluorescence detection of the chondrocytes after 7 days of culture in each group (Scale bar=100 μm). C: Quantitative analysis of fluorescence intensity of COL-2 and MMP-13 (n=3). ANOVA followed by Tukey' s test was used for statistical analysis (^*P < 0.05, ^**P < 0.01, ^***P < 0.001).

图 6

GelMA-Exo明显促进大鼠膝关节软骨损伤的修复 GelMA-Exo significantly promotes repair of knee articular cartilage injury in rats (HE and Masson staining).