(-)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Abstract

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (-)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

Keywords: (−)-epigallocatechin-3-gallate (EGCG); antioxidant; human bone marrow mesenchymal stem cells (BMSCs); mineralization; osteogenesis.

Figure 1

Effects of (−)-epigallocatechin-3-gallate (EGCG) on human bone marrow stem cells (hBMSCs) in 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTS). There was no significant change in MTS assay and cell cycle after EGCG treatment at 1 and 10 µmol/L for 24 and 48 h. With treatment of EGCG, the viability of hBMSCs was not affected by EGCG at both 1 and 10 µmol/L (both p > 0.05).

Figure 2

The mRNA expression of osteogenic marker genes. The mRNA expression of Runx2 (A), bone morphogenetic protein 2 (BMP2) (B), ALP (C), osteonectin (D) and osteocalcin (E) increased significantly after EGCG treatment for 24 and 48 h, at concentrations of both 1 and 10 µmol/L. The expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin were quantified by real-time PCR. There were significant changes in all genes after treatment for 48 h but not 24 h, except in the case of Runx2. In Runx2, mRNA expression increased 57% (p < 0.01) and 85% (p < 0.05) with 1 and 10 µmol/L, respectively, at 24 h and 169% (p < 0.01) and 203% (p < 0.01) with 1 and 10 µmol/L, respectively, at 48 h. In BMP2, mRNA expression increased 459% (p < 0.01) and 502% (p < 0.01) with 1 and 10 µmol/L, respectively. ALP mRNA expression was enhanced 239% (p < 0.01) and 210% (p < 0.01) at concentrations of 1 and 10 µmol/L of EGCG, respectively. The mRNA expression in osteonectin was amplified 239% (p < 0.01) and 383% (p < 0.01) after EGCG treatment at concentrations of 1 and 10 µmol/L, respectively. The mRNA expression in osteocalcin was amplified 86% (p < 0.01) and 134% (p < 0.01) after EGCG treatment at concentrations of 1 and 10 µmol/L, respectively. ^# p < 0.05, ^## p < 0.01.

Figure 2

The mRNA expression of osteogenic marker genes. The mRNA expression of Runx2 (A), bone morphogenetic protein 2 (BMP2) (B), ALP (C), osteonectin (D) and osteocalcin (E) increased significantly after EGCG treatment for 24 and 48 h, at concentrations of both 1 and 10 µmol/L. The expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin were quantified by real-time PCR. There were significant changes in all genes after treatment for 48 h but not 24 h, except in the case of Runx2. In Runx2, mRNA expression increased 57% (p < 0.01) and 85% (p < 0.05) with 1 and 10 µmol/L, respectively, at 24 h and 169% (p < 0.01) and 203% (p < 0.01) with 1 and 10 µmol/L, respectively, at 48 h. In BMP2, mRNA expression increased 459% (p < 0.01) and 502% (p < 0.01) with 1 and 10 µmol/L, respectively. ALP mRNA expression was enhanced 239% (p < 0.01) and 210% (p < 0.01) at concentrations of 1 and 10 µmol/L of EGCG, respectively. The mRNA expression in osteonectin was amplified 239% (p < 0.01) and 383% (p < 0.01) after EGCG treatment at concentrations of 1 and 10 µmol/L, respectively. The mRNA expression in osteocalcin was amplified 86% (p < 0.01) and 134% (p < 0.01) after EGCG treatment at concentrations of 1 and 10 µmol/L, respectively. ^# p < 0.05, ^## p < 0.01.

Figure 3

Concentration and time response of ALP activity upregulation by EGCG. In comparison to control cultures, the ALP activities of EGCG (1 and 10 µmol/L)-treated cultures increased by 11% and 30% (p < 0.05) on the 4th day, 30% (p < 0.01) and 52% (p < 0.01) on the 7th day, and 20% (p < 0.05) and 37% (p < 0.05) on the 14th day, respectively. ^# p < 0.05, ^## p < 0.01.

Figure 4

Effects of EGCG on hBMSC mineralization (A)–(B). Different concentrations of EGCG, 1 and 10 µmol/L, increased mineralization by 43% (p < 0.01) and 76% (p < 0.01), with respect to the control, respectively. With antioxidant ability evaluated as playing an important role in enhancing mineralization of EGCG in hBMSCs, EGCG was combined with air with O₂ to deplete its antioxidant ability. After EGCG was combined with air, it was still able to increase mineralization by 37% (p < 0.01) and 75% (p < 0.01), with respect to the control, respectively. There was no difference between fresh EGCG and EGCG in air, which indicated that antioxidant ability did not play an important role in enhancing mineralization. ^## p < 0.01 compared with CON.