Aesculetin Accelerates Osteoblast Differentiation and Matrix-Vesicle-Mediated Mineralization

Abstract

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.

Keywords: aesculetin; collagen mineralization; hydroxyapatite; matrix vesicles; non-collagenous proteins; osteoblast differentiation.

Figure 1

Chemical structure of aesculetin (A), cytotoxicity of MC3T3-E1 cells by 1–20 μM aesculetin (B), upregulation of bone morphogenetic protein-2 (BMP-2) expression (C) and alkaline phosphatase (ALP) activity (D) and staining (E) by aesculetin. MC3T3-E1 cells were cultured for 3 days and 21 days with 1–20 µM aesculetin in differentiation media. Cell viability was measured by MTT assay (B). Bar graphs for viability (mean ± SEM, n = 3) was expressed as percent cell survival compared to untreated cells. Further, MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin for three days (BMP-2) and seven days (ALP). Whole cell lysates were subject to SDS-PAGE and Western blot with a specific antibody against BMP-2 (C). β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative results of blots obtained from a densitometer. The ALP activity (D, mean ± SEM, n = 6) was measured at λ = 405 nm. The ALP staining was visualized under light microscopy (E, 4 separate experiments). Scale bar = 40 μm. Respective values not sharing a small letter are different at p < 0.05.

Figure 2

Upregulation of Runt-related transcription factor 2 (Runx2) transcription (A), and expression of collagen type 1 (B) and osteoprotegerin (OPG, C) by aesculetin. MC3T3-E1 cells were cultured for nine days with 1–10 µM aesculetin in differentiation media. The transcription of Runx2 was measured by real-time polymerase chain reaction assay, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used for the internal controls (A). For the expression of collagen type 1 and OPG, whole cell lysates were subject to SDS-PAGE and Western blot with a specific antibody against collagen type 1 or OPG (B,C). β-Actin was used as an internal control. The bar graphs represent quantitative results of blots obtained from a densitometer. Respective values (mean ± SEM, n = 3) not sharing a small letter are different at p < 0.05.

Figure 3

Temporal induction of bone sialoprotein (BSP) II (A) and elevation of induction of BSP II (B), osteopontin (C), and osteocalcin (D) by aesculetin in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin for up to 21 days. For the measurement of expression of BSP II, osteopontin and osteocalcin of aesculetin, MC3T3-E1 cells were differentiated for 15 days. Whole cell lysates were subject to SDS-PAGE and western blot with a specific antibody against BSP II, osteopontin or osteocalcin. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative results of blots obtained from a densitometer. Respective values not sharing a small letter are different at p < 0.05.

Figure 4

Temporal induction of osteonectin (A), induction elevation of osteonectin (B) and formation of calcium nodules (C) by aesculetin in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin for up to 21 days. To examine the osteonectin expression of aesculetin, MC3T3-E1 cells were differentiated for 15 days. Whole cell lysates were subject to SDS-PAGE and Western blot with a specific antibody against osteonectin. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative results of blots obtained from a densitometer. Respective values not sharing a small letter are different at p < 0.05. Matrix mineralization was measured by Alizarin red S staining (C). Microphotographs were representative of 21 day-grown osteoblasts on the wells. Heavy reddish staining of Alizarin red S is proportional to the area of mineralized matrix in osteoblastic MC3T3-E1 cells. Scale bar = 40 μm. The calcium nodules were visualized under light microscopy (5 separate experiments).

Figure 5

Induction of thrombospondin-1 and tenascin C by aesculetin (A,B), temporal induction of annexin V and PHOSPHO 1 (C), and elevation of annexin V and PHOSPHO 1 with aesculetin (D). MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin up to 21 days. To measure the effects of aesculetin on expression of annexin V, PHOSPHO 1, thrombospondin-1, and tenascin C, cells were differentiated for 18 days (A,B,D). Whole cell lysates were subject to SDS-PAGE and Western blot with a specific antibody against annexin V, PHOSPHO 1, thrombospondin-1 or tenascin C. β-Actin was used as an internal control. The bar graphs (mean ± SEM, n = 3) represent quantitative results of blots obtained from a densitometer. Respective values in bar graphs not sharing a small letter are significantly different at p < 0.05.

Figure 6

Immunofluorocytochemical analysis showing formation of tissue-nonspecific alkaline phosphatase (TNSALP) by aesculetin. MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin for 21 days. The matrix vesicle formation was confirmed by FITC-green staining of TNSALP on the top of differentiated MC3T3-E1 cells (n = 3). Nuclear staining of these cells was carried out with 4′, 6-diamidino-2-phenylindole (DAPI, blue), and visualized under light microscopy. Scale bar = 50 μm.

Figure 7

Immunofluorocytochemical analysis showing production of collagen type 1 by aesculetin. MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 μM aesculetin for 21 days. The collagen type 1 production was confirmed by Cy3-red staining of collagen 1 on the top of differentiated MC3T3-E1 cells (n = 3). Nuclear staining of these cells was carried out with 4′, 6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm.

Figure 8

Schematic diagram showing the effects of aesculetin on osteoblastogenesis and collagen mineralization. The arrows indicate activation by aesculetin. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; BSP, bone sialoprotein; OPG, osteoprotegerin; Runx2, Runt-related transcription factor 2; TNSALP, tissue-nonspecific alkaline phosphatase.