Abstract
ObjectiveHuman chorion membrane extracts (CME) are known to exhibit osteogenic effects when used for treating human osteoblast-like cells (MG63 cells), but the active compound in CME remains unknown. The aim of this study was to identify the presence of exosomes in CME and to determine the osteogenic effect of CME exosomes on MG63 cells.MethodsExosomes were isolated from human placenta CME using the ExoQuick-TC solution and were characterized. The activity and deposition of alkaline phosphatase (ALP) on MG63 cells cultured with or without exosomes in osteogenic induction medium (OIM) were determined. Human amniotic membrane extracts (AME) were used as controls as they had not affected the osteogenic differentiation of MG63 cells in our previous study.ResultsTransmission electron microscopy (TEM) revealed that exosomes isolated from CME and AME (CME-Exo and AME-Exo, respectively) had a cup-shaped structure. NanoSight™ particle tracking analysis (NTA) confirmed that the size of these exosomes was 100–150 nm. In vitro osteogenic experiments demonstrated that the exosomes from CME, but not those from AME, presented increased alkaline phosphatase (ALP) activity and resulted in the mineralization of MG63 cells in a dose-dependent manner.ConclusionExosomes were identified in CME and AME from the human placenta. Further, the exosomes from CME were found to be capable of promoting osteogenic differentiation, suggesting that exosomes are a key component of CME that stimulate the osteogenesis of human osteoblast-like cells. CME exosomes can be developed as promising therapeutic candidates for bone regeneration.
Highlights
The purpose of this study was to investigate the osteogenic effect of human chorion membrane exosomes (CME-Exo) using human osteoblast-like cells
The exosomes isolated from human amniotic membrane extracts (AME) and chorion membrane extracts (CME) using the ExoQuick-TCTM solution were first characterized based on size
NanoSightTM particle tracking analysis (NTA) revealed that the average size of the AME and CME exosomes was in the range of 100–150 nm (Fig. 1c)
Summary
The purpose of this study was to investigate the osteogenic effect of human chorion membrane exosomes (CME-Exo) using human osteoblast-like cells. Exosomes have not yet been isolated from the chorionic membrane of the human placenta. Our previous studies had demonstrated that chorion membrane extracts (CME) from human placenta have an excellent osteogenic efficacy, especially with respect to the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoblast-like cells [14, 15]. We hypothesized that CME-Exo play an important role in mediating the osteogenic effect of CME in human osteoblast-like cells. Treatment of MG63 cells with CME-Exo induced osteogenesis, ALP activity was critically increased from 3 to 7 days in the early stage of osteogenesis, and the mineral deposition was observed from 14 to 20 days in the late stage of osteogenesis [14, 18]. We investigated the ALP activity and mineralization of CME-Exotreated MG63 cells during osteogenesis in vitro
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