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.
Highlights
Bone is an extremely heterogeneous tissue built up of cells and mineralized extracellular matrix [1].It undergoes an unceasing process of remodeling that involves removal of old bone by osteoclasts and formation of new bone by osteoblasts
We examined the effects of betulin on the differentiation and mineralization of osteoblasts of two human cell lines both in the presence of an osteogenic medium and without an osteogenic supplement such as ascorbic acid [32,33]
To avoid the cytotoxicity of the compound towards osteoblasts, the effect of BET on the viability of hFOB 1.19 and Saos-2 cells was determined by the LDH assay
Summary
Bone is an extremely heterogeneous tissue built up of cells and mineralized extracellular matrix [1]. It undergoes an unceasing process of remodeling that involves removal of old bone by osteoclasts and formation of new bone by osteoblasts. In physiological conditions, these events are balanced and provide tissue homeostasis [2]. Various diseases including rheumatoid arthritis, chronic inflammatory periodontal diseases, multiple myeloma, bone metastatic malignant cancers, or drugs adversely affect bone health and can contribute to the development of osteoporosis.
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