Repair of osteoarthritis in animal model with exosomes derived from BMSCs transfected by the siRNA -Piezo1 through CT navigation

Abstract

To investigate the effect of the exosomes from bone marrow mesenchymal stem cells (BMSCs) transfected with silence plasmid of Piezol small interference RNA (siRNA)on osteoarthritis (OA) animal model. Twenty male SD rats with specific pathogen free (SPF) were selected, ranging in age from 5.46 to 6.96 months, with a mean of (6.21± 0.75) months;and ranging in weight from 385.76 g to 428.66 g, with a mean of (407.21±21.45) g. BMSCs were extracted. The siRNA silencing plasmid of piezo1 was constructed by siRNA technology. After lentivirus was transfected into BMSCs, the exosomes were extracted. At the cellular level, BMSCs were divided into blank plasmid group and siRNA silencing plasmid group according to whether siRNA-Piezo1 was transfected or not. The osteogenic induction ability of siRNA-Piezo1 on BMSCs was detected by RT-PCR and Western blot. At the animal model level, the OA model was established by surgical resection of cruciate ligament of knee joint.According to different treatment schemes, SD rats were divided into 4 groups:blank control group, model group, BMSCs group and exosome group. SD rats in the blank control group were not treated. In the model group, the cruciate ligaments of rats were excised and OA animal model was established. In BMSCs group, BMSCs were injected into knee joint under CT guidance after OA model establishment, and the cell volume was 5×105/ml, loading amount of 2 ml, twice a week for 4 weeks. In the exosome group, 100 μg exosomes from siRNA BMSCs were added twice a week for 4 weeks after OA model establishment. The cartilage of the animal model was detected by hematoxylin eosin (HE) staining and safranin solid green staining, and quantified by the modified Minkin score and the score of the international society for osteoarthritis research (OARSI). Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the relative mRNA expression level of aggrecan type II collagen in cartilage. The lentivirus transfection efficiency was(92.11±4.22)%. RT-PCR showed that the relative expression of Piezo1 mRNA in blank plasmid group was 1.07±0.06, which was significantly different from that of 0.31±0.01 in siRNA silencing plasmid group (t=2.907, P<0.05). The results of HE staining and safranine solid green staining showed that there was cartilage structure and smooth cartilage surface in the knee joint of SD rats in the blank control group. The knee joint structure in the model group had been completely destroyed, the knee joint cartilage structure in the BMSCs group was not clear, and there were subchondral bone components in the OA rats in the exosomes group. There was significant difference between the modified Minkin score of HE staining and the OARSI score of safranin solid green staining (F=15.903, 19.005;P<0.05). Among them, the repair effect of exosome group was significantly better than that of BMSCs group and model group (P<0.05). RT-PCR results showed that the relative expression of aggrecan mRNA in BMSCs group was significantly higher than that in model group (P< 0.05), and the relative expression of aggrecan mRNA in exosome group was higher than that in BMSCs group and model group (P<0.05). The relative expression of CollagenⅡmRNA in BMSCs group was higher than that in model group (P<0.05), and the relative expression of CollagenⅡmRNA in exosomes group was higher than that in BMSCs group and model group (P<0.05). Piezo1 siRNA silencing vector can promote the differentiation of BMSCs into chondrocytes and effectively inhibit the progression of OA, so as to delay the disease of OA.