| Objective:Induced pluripotent stem cells (iPSCs) have great potential as seed cells for tissue engineering applications. Previous studies have shown that iPSCs could be induced to differentiate into bone forming cells. However, in a tissue engineering approach, seeding cells in biomaterials is required, and the effect of biomaterials on cell growth and differentiation is critical for the success of the formation of engineered tissues. In this study, we investigated the effect of akermanite, a bioactive ceramic, on the osteogenic differentiation of embryoid body (EB) cells derived from human iPSCs.Methods:(1) We first evaluated the Karyotyping analysis, Alkaline phosphatase staining, Immunostaining of human iPSCs. Furthermore, teratoma formation was also performed and stained with hematoxylin and eosin. (2) Cells were cultured in growth medium(GM), osteogenic medium(OM), medium containing extracts of akermanite with osteogenic factors and medium containing extracts of akermanite without osteogenic factors. To evaluate the osteogenic differentiation of cells in the presence of akermanite bioceramics, ALP staining and ALP activity assay after culturing for 7 days were performed, Alizarin Red S staining was performed at 21 days. After osteogenic differentiation of hEB cells at 14 and 21 days, gene expression was detected by Quantitative real-time polymerase chain reaction (Q-RT-PCR).Results:(1) We first observed the ALP expression and obtained cells express typical pluripotent stem cell markers (Oct3/4, Sox2, Nanog, SSEA-4, TRA-1-60, and RA-1-81) of iPSCs, Furthermore, teratoma formation was also observed by hematoxylin and eosin staining, All these 45 results confirmed that the teratoma is formed and contains three primary germ layers, ectoderm, endoderm and meso-derm, which indicates that the properties of the cells satisfy the criteria for human iPSCs. (2) The results showed that, in the presence of osteogenic factors (ascorbic acid, dexamethasone, and b- glycerophosphate), ionic extracts of akermanite enhanced the osteogenic differentiation of EB cells as compared with normal osteogenic medium. Alkaline phosphatase (ALP) activity and the expression of osteogenic marker genes such as osteocalcin (OCN), collagen (COL-1), RUNX2, and BMP2 are significantly increased by the stimulation of akermanite ceramic extracts at certain concentration ranges. More interesting is that the medium containing extracts of akermanite but without osteogenic factors also showed stimulatory effects on the osteogenic differentiation of EB cells as compared to normal growth medium without osteogenic factors, such as ascorbic acid, dexamethasone, and b-glycerophosphate, not at the early stage of culture, but only at the later stage of the culture period (21 days).Conclutions:These results suggest that akermanite as a bioactive material together with human iPSCs might be used for bone tissue engineering applications. |
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