Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization

The imbalance between bone resorption and bone formation in favor of resorption results in bone loss and deterioration of bone architecture. Osteoblast differentiation is a sequential event accompanying biogenesis of matrix vesicles and mineralization of collagen matrix with hydroxyapatite crystals. Considerable efforts have been made in developing naturally-occurring plant compounds, preventing bone pathologies, or enhancing bone regeneration. Coumarin aesculetin inhibits osteoporosis through hampering the ruffled border formation of mature osteoclasts. However, little is known regarding the effects of aesculetin on the impairment of matrix vesicle biogenesis. MC3T3-E1 cells were cultured in differentiation media with 1–10 μM aesculetin for up to 21 days. Aesculetin boosted the bone morphogenetic protein-2 expression, and alkaline phosphatase activation of differentiating MC3T3-E1 cells. The presence of aesculetin strengthened the expression of collagen type 1 and osteoprotegerin and transcription of Runt-related transcription factor 2 in differentiating osteoblasts for 9 days. When ≥1–5 μM aesculetin was added to differentiating cells for 15–18 days, the induction of non-collagenous proteins of bone sialoprotein II, osteopontin, osteocalcin, and osteonectin was markedly enhanced, facilitating the formation of hydroxyapatite crystals and mineralized collagen matrix. The induction of annexin V and PHOSPHO 1 was further augmented in ≥5 μM aesculetin-treated differentiating osteoblasts for 21 days. In addition, the levels of tissue-nonspecific alkaline phosphatase and collagen type 1 were further enhanced within the extracellular space and on matrix vesicles of mature osteoblasts treated with aesculetin, indicating matrix vesicle-mediated bone mineralization. Finally, aesculetin markedly accelerated the production of thrombospondin-1 and tenascin C in mature osteoblasts, leading to their adhesion to preformed collagen matrix. Therefore, aesculetin enhanced osteoblast differentiation, and matrix vesicle biogenesis and mineralization. These findings suggest that aesculetin may be a potential osteo-inductive agent preventing bone pathologies or enhancing bone regeneration.

Effects of Sapindus mukorossi Seed Oil on Proliferation, Osteogenetic/Odontogenetic Differentiation and Matrix Vesicle Secretion of Human Dental Pulp Mesenchymal Stem Cells

Stem cells have attracted great interest in the development of tissue engineering. However, the self-regeneration and multi-differentiation capabilities of stem cells are easily impaired during cell transplantation. Recent studies have demonstrated that Sapindus mukorossi (S. mukorossi) seed oil has various positive biological effects. However, it is not yet clear whether S. mukorossi seed oil can increase the growth and differentiation of dental pulp mesenchymal stem cells (DPSCs). The aim of this study is to investigate the effects of S. mukorossi seed oil on the proliferation and differentiation of DPSCs. DPSCs with and without S. mukorossi seed oil, respectively, were evaluated and compared. The viabilities of the cells were assessed by MTT tests. The osteogenetic and odontogenetic capacities of the DPSCs were tested using Alizarin red S staining and alkaline phosphatase (ALP) activity assays. In addition, real-time PCR was performed to examine the gene expression of ALP, BMP-2 and DMP-1. Finally, extracellular matrix vesicle secretion was detected via scanning electron microscopy. No significant difference was observed in the viabilities of the DPSCs with and without S. mukorossi seed oil, respectively. However, under osteogenic and odontogenic induction, S. mukorossi seed oil increased the secretion of mineralized nodules and the ALP activity of the DPSCs (p < 0.05). The ALP gene expression of the differentiation-induced DPSCs was also enhanced. Finally, a greater secretion of extracellular matrix vesicles was detected in the DPSCs following odontogenic induction complemented with S. mukorossi seed oil. Overall, the present results show that S. mukorossi seed oil promotes the osteogenic/odontogenic differentiation and matrix vesicle secretion of DPSCs.