Guan P, Cui R, Wang Q, Sun Y
Department of Spine Surgery, Third Affiliated Hospital of Sun Yat- sen University, Guangzhou 510630, China.
Department of Organ Transplantation, Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Apr 20;42(4):528-537. doi: 10.12122/j.issn.1673-4254.2022.04.08.
To assess the efficacy of GelMA hydrogel loaded with bone marrow stem cell-derived exosomes for repairing injured rat knee articular cartilage.
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.
GelMA hydrogel loaded with exosomes significantly promoted M2-type polarization of RAW264.7 cells ( < 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 ( < 0.05). HE and Masson staining showed that the exosome-loaded hydrogel obviously promoted cartilage repair in the rat models damage.
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.
评估负载骨髓干细胞来源外泌体的甲基丙烯酰化明胶（GelMA）水凝胶修复大鼠膝关节损伤软骨的疗效。
对培养的骨髓干细胞的上清液进行超速离心以分离并提取外泌体，通过透射电子显微镜、粒径分析和表面标志物的蛋白质免疫印迹对其进行表征。在负载外泌体后检测GelMA水凝胶的流变学和电子显微镜特征变化。通过BCA蛋白质检测法检测水凝胶中外泌体的释放情况，并用PKH26红色荧光染料标记外泌体以观察RAW264.7细胞对其的吞噬作用。通过实时定量聚合酶链反应（q-PCR）和免疫荧光测定法检测单独的外泌体、未负载水凝胶以及负载外泌体的水凝胶对RAW264.7细胞极化的影响。我们进一步使用q-PCR、免疫荧光测定法以及苏木精-伊红（HE）和马松（Masson）染色，在大鼠膝关节软骨损伤模型的RAW264.7细胞与软骨细胞的Transwell共培养细胞模型中测试负载外泌体的水凝胶对软骨修复的作用。
负载外泌体的GelMA水凝胶显著促进RAW264.7细胞向M2型极化（<0.05）。在Transwell共培养模型中，负载外泌体的GelMA水凝胶通过调节RAW264.7细胞从M1型向M2型转化，显著促进损伤软骨细胞的修复（<0.05）。HE和Masson染色显示，负载外泌体的水凝胶明显促进大鼠损伤模型中的软骨修复。
负载骨髓干细胞来源外泌体的GelMA水凝胶可通过改善免疫微环境显著促进大鼠软骨损伤的修复。
