Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases

Abstract

Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.

Electroless Palladium-Coated Polymer Scaffolds for Electrical Stimulation of Osteoblast-Like Saos-2 Cells

“…Once it was demonstrated that daily ES of foams increased the proliferation of osteoblast at least up to 14 days, we decided to analyze the expression of six different genes related with osteoblast differentiation. Osteoblast differentiation is usually divided in two stages: an early stage, when the extracellular matrix begins to mature, and a late stage, when the extracellular matrix mineralizes [ 41 , 42 , 43 ]. The length of each period is not clear and varies among reports, but the extend of the early stage is considered by most authors to be between 7 and 14 days, and at approximatively 14 days the late stage begins [ 41 , 42 , 43 ].…”

“…ALPL gene encodes for alkaline phosphatase protein and, generally, it is accepted that this gene is expressed through proliferation and early extracellular matrix maturation, that is, between 7–14 days [ 41 , 43 ]. However, some authors have also reported ALPL expression in late stages [ 44 ].…”

“…COL1 gene encodes for type I collagen and, according to several authors [ 41 , 42 ], its maximum expression is during proliferation and decreases during extracellular matrix maturation. However, some other authors consider COL1 as a late stage expression gene [ 43 ]. In our work, the expected pattern was clear on cells grown on glass, electrically stimulated or not, where a decrease at 14 days was observed, but not on Pd-coated PU foams where an increased level of COL1 expression was still present at day 14.…”

Role of MAPK/JNK signaling pathway on the regulation of biological behaviors of MC3T3‑E1 osteoblasts under titanium ion exposure

“…JnK signaling has been shown to be extensively activated by oxidative stress, where it serves as a regulator of several life-cycle processes, including cell meiosis, mitosis, differentiation and energy metabolism (30). in particular, JnK activity is required for the initiation of early osteogenic differentiation in mesenchymal stem cells (MScs) (31,32). By contrast, attenuation of JnK activity using JnK inhibitors was found to result in increased late stage osteogenic differentiation (33).…”

MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI

“…Mesenchymal stem cells (MSCs) extracted from bone marrow can form osteoblasts through osteogenic differentiation, which was first discovered by Friedenstein et al in the late 1960s-early 1970s [1]. Osteoblasts participate in the formation of bone and work together with osteoclasts, which is crucial for the repair of fractures, bone development and maintenance [2]. Abnormal differentiation of these two cells may cause several orthopedic diseases, such as osteoporosis, osteoarthritis and fractures, subsequently affect patients' physical health and life expectancy [3].…”