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Objective To compare the differences between exosomes derived from three-dimensional (3D) cultured human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and conventional (2D) cultured hUC-MSCs in promoting osteoblast differentiation, and to explore the application possibility of exosomes derived from 3D-cultured hUC-MSCs in treatment related to promoting bone formation. Methods hUC-MSCs were divided into 2D group and 3D group, and cultured respectively.The morphological characteristics of the both groups were observed under optical microscope, and the viability of 3D-cultured hUC-MSCs was detected by calcein AM/PI double staining of living and dead cells. Transcriptome sequencing was performed to screen the differentially expressed genes between 2D group and 3D group, and GO enrichment analysis was conducted. Exosomes of 2D group and 3D group were extracted and divided into 2D-Exo group and 3D-Exo group. Characterization of exosomes was conducted by transmission electron microscope, nanoparticle tracking analysis and Western blotting. The exosomes excreted by 2D group and 3D group were applied to C57BL/6J mouse calvarial osteoblasts, respectively, and osteogenic differentiation was induced in vitro. The effects of exosomes cultured in 2D and 3D on osteogenic differentiation were identified by alizarin red staining, alkaline phosphatase staining and RT-qPCR. Results Comparing to 2D group, cells in 3D group were equal in size and grew into spherical shape. 3D-cultured hUC-MSCs showed higher rate of viability verified by calcein AM/PI double staining of living and dead cells.Transcriptome sequencing results showed that the up-regulated genes in 3D group were enriched in bone mineralization, cartilage development, composition of extracellular matrix, osteoblast differentiation, angiogenesis, cell proliferation and gene expression.The down-regulated genes were enriched in negative regulation of cell proliferation, cell migration, and apoptotic process, etc.Transmission electron microscope observation showed that the exosome diameter in both 2D-Exo group and 3D-Exo group were around 100 nm and exhibited typical cup shape features, and expressed exosome related marker proteins CD9, CD63 and CD81.Staining results of osteoblasts in calvaria of newborn rats showed that the number of calcium nodules stained with alizarin red and the intensity of alkaline phosphatase staining increased significantly in 3D-Exo group than in 2D-Exo group. Results of RT-qPCR showed that the relative expression levels of osteogenic differentiation related genes Bglap, Runx2, Alp, Col1a1 and Spp1 mRNA increased significantly in 3D-Exo group than in 2D-Exo group with statistically significant differences (P<0.05). Conclusion Exosomes from 3D-cultured hUC-MSCs can up-regulate the expressions of osteogenic genes Bglap, Runx2, Alp, Col1a1 and Spp1,and have stronger capabilities to promote osteogenic differentiation compared with 2D-cultured exosomes.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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