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Bone tissue engineering has shown a promising way in order to renew bone defects without conflicting with previously known side effects. Three main building blocks including seeding cells, scaffold, and signaling molecules are required for this process. The human amniotic membrane (hAM) is the innermost of the placental membranes. As well as providing a source of stem cells and growth factors, hAM has several features which make it an appropriate stem cell containing scaffold in order to be used in orthopedic surgery. The present investigation aimed to demonstrate the effect of bone morphogenetic protein-9 (BMP-9) alongside phenamil and simvastatin on osteogenic induction of hAM with its sessile epithelial cells (hAECs). For this purpose, hAM was cultured using six different osteogenic media for 14 days. The basic osteogenic media was chosen as the first group and other media were made by addition of BMP-9, phenamil, simvastatin, BMP-9 alongside phenamil, and BMP-9 alongside simvastatin to the basic osteogenic media. Finally, viability, mineralization, calcium and phosphate content, lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activity were evaluated. Among all study groups, simvastatin-containing groups showed a significantly lower level of viability. Although, all media could induce osteogenic features, the hAECs which were cultured in media containing BMP-9 and phenamil could demonstrate a wider area of mineralization and a significantly higher level of calcium, phosphate, LDH, and ALP activity. These findings indicated that the application of phenamil alongside BMP-9 could synergistically show in situ osteogenic induction in hAECs.


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