Abstract
Human amniotic membrane extracts contain numerous growth factors and bioactive substances. However, osteogenic effects of amnion and chorion membrane extracts (AME and CME, respectively) on osteoblasts are unclear. In this study, we explored the ability of AME and CME to promote the osteogenic differentiation of osteoblast-like MG-63 cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without exogenous AME and CME. CME enhanced the osteogenic differentiation of MG-63 cells compared with AME, as indicated by increased mineralization; alkaline phosphatase activity; and mRNA expression of osteogenic marker genes encoding integrin-binding sialoprotein (IBSP), RUNX2, OSTERIX, and osteocalcin (OCN). Interestingly, AME and CME contained different combinations of osteogenesis-related growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGFβ-1), and epidermal growth factor (EGF), which differentially regulated the osteogenic differentiation of MG-63 cells. bFGF and TGFβ-1 present in CME positively regulated the osteogenic differentiation of MG-63 cells, whereas EGF present in AME negatively regulated the differentiation of MG-63 cells. Moreover, exogenous treatment of EGF antagonized CME-induced mineralization of extracellular matrix on MG-63 cells. We compared the osteogenic efficacy of CME with that of BMP2, bFGF, and TGFβ-1 alone or their combinations. We observed that CME greatly enhanced osteogenesis by providing a conductive environment for the differentiation of MG-63 cells. Together, our results indicated that human AME and CME exerted differential effects on osteogenesis because of the presence of different compositions of growth factors. In addition, our results highlighted a new possible strategy of using CME as a biocompatible therapeutic material for bone regeneration.
Highlights
Osteogenesis is a sequential process involving the proliferation and differentiation of osteogenic cells, activation of osteogenesis-specific genes, and maturation and mineralization of extracellular matrix (ECM) [1, 2]
While cells cultured in only osteogenic induction medium (OIM) showed initiation of osteogenic differentiation compared with cells cultured in only growth medium (GM), cells cultured in OIM containing CME showed more obvious evidence of osteogenesis than only OIM condition (Fig 1A, left square image)
To determine whether growth factors present in CME enhanced the osteogenesis of MG-63 cells, we examined the inhibitory effect of suramin sodium, which is known to block the interaction of various growth factors such as basic fibroblast growth factor (bFGF), PDGF, epidermal growth factor (EGF), and transforming growth factor beta-1 (TGFβ-1) with their respective surface receptors [23,24,25],on MG-63 cells cultured in OIM supplemented with CME
Summary
Osteogenesis is a sequential process involving the proliferation and differentiation of osteogenic cells, activation of osteogenesis-specific genes, and maturation and mineralization of extracellular matrix (ECM) [1, 2]. Osteoblast differentiation is tightly controlled by specific extracellular regulatory proteins such as growth factors, hormones, vitamins, and cytokines [5]. Growth factors and cytokines modulate cell fate, including proliferation, differentiation, survival, and migration. We previously demonstrated the potential of human amniotic membrane extracts to promote the osteogenic differentiation of osteoblast-like MG-63 cells [12]. The extracts prepared from amnion/ chorion membrane have showed greater effect on osteogenesis of MG-63 cells than the extracts from a single layer of the amnion membrane. This may be because of the different growth factors present in AM and CM
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