DISSCUSSIONS:

1. (-)-EPIGALLOCATECHIN-3-GALLATE (EGCG) EHNAHCES OSTEOGENESIS IN A BONE MARROW MESENCHYMAL STEM CELL LINE Chung-Hwan Chen1,3; Mei-Ling Ho2,3 ; Je-Ken Chang1,3 ; Shao-Hung Hung,3 ; Gwo-Jaw Wang1,3 Departments of Orthopaedics1, and Physiology2, School of Medicine, Orthopaedic Research Center3 Kaohsiung Medical University, Kaohsiung, TAIWAN INTRODUCTION: Green tea is one of the most popular beverages in the world. Among the catechins, (-)-epigalloncatechin -3-gallate (EGCG) has received by far the most attention(1). Previous studies verified the beneficial effects of catechins in decreasing serum lipid, reducing blood pressure, antitumorigenic and antibacterial effects. Recent surveys have reported to reduce the risk of having a hip fracture with higher bone mineral density (BMD) by habitual tea drinkers (2). However, the effective components and the action mechanisms of tea on bone remodeling remain unclear. In this study, we attempt to elucidate the osteogenic effects of a green tea catechin, EGCG, on a stem cell line by screening the mRNA expressions of some osteogenic markers Furthermore, the effects of EGCG on the osteocalcin protein production, ALP activity and mineralization are also evaluated. Besides, we evaluate the effect of EGCG on cell proliferation and apoptosis. MATERIALS AND METHODS: The cloned pluripotent mesenchymal cells, D1, were maintained in Dulbecco Modified Eagle Medium (DMEM) (3). D1 cells were treated with EGCG at concentration of 1 µmol/L or 10 µmol/L for 48 hours respectively. The mRNA expressions of Cbfa1/Runx2, osterix, osteocalcin (OC) and alkaline phosphatase (ALP) were examined by RT-PCR. Protein production of OC was evaluated by ELISA after treatment for 4 and 7 days. ALP activity will be assayed by chemiluminescent method after EGCG treatment for 4, 7 and 14 days. The degree of mineralization effected by EGCG was determined by von Kossa stain at the 3rd and 4th week. Cell proliferation was assayed by thymidine incorporation after treated for 24 hours. Cells were stained with tryphan blue for cell count after treated for 24 hours. Apoptotic effect of EGCG was evaluated by TUNEL stain after treated for 24 hours. RESULTS: First the mRNA expression of osterix, ALP, and OC have shown a markedly increase with the addition of different concentrations (1 µmol/L or 10 µmol/L) of EGCG, 66 % ( P<0.05) and 137% (P<0.01) for osterix, 44% ( P<0.05) and 109% (P<0.01) for ALP, and 40% ( P<0.05) and 44% ( P<0.05) for OC, respectively. (Fig.1) Second, with respect to the control and the increase of EGCG concentrations, ALP activities increased by 202% and 278% (P<0.01) on the 4th day, 161% and 183% (P<0.01) on the 7th day, and 134% and 148% (P<0.01) on the 14th day (Fig.2). Third, OC production was increase the addition of different concentrations (1 µmol/L or 10 µmol/L) of EGCG, 96% and 135% on the 4th day and 71% and 590% on the 7th day(Fig.4). Fourth, On the 3rd week, different concentration of EGCG, 1 and 10 µmol/L, increased mineralization by 5% and 29%, with respect to the control, respectively. The differences extend on the 4th week to 25% and 44%, with respect to the control, by addition of EGCG, 1 and 10 µmol/L (Fig.4). Fifth, a 24-hour treatment of EGCG decreased thymidine incorporation by 12% (1 µmol/L) and 24% (10 µmol/L) (P<0.05) (Fig.5). Sixth, cell count after EGCG treated for 24 hours decreased by 30% (1 µmol/L) and 30% (10 µmol/L) (P<0.05) (Fig.6). Seventh, no obvious apoptotic effect of EGCG on D1-cell cultures after treated for 24 hours. Fig 3. EGCG increases Mineralization • DISSCUSSIONS: • Our results illustrated that the effective concentration of EGCG is at the range of 1 to 10 µmol/L. Previous report indicated that one cup of green tea drinking could accumulate the circulating level of EGCG to 1 µmol/L. Our results indicated that EGCG acts on bone marrow mesenchymal cells by modulating the differentiation through augmenting the gene expressions of Cbfa1/Runx2, osterix, ALP and OC. Our results further demonstrated that EGCG can increase the potential of terminal osteoblastic differentiation by increasing OC protein production, ALP activity, which eventually leads to mineralization confirmed by von Kossa stain. EGCG’s stimulatory effects on OC protein production, ALP activity and mineralization in D1-cell cultures further confirmed its post-transcriptional influences on osteogenesis. However, a 24-hour treatment of EGCG inhibited thymidine incorporation of D1-cells and decreased cell count. No obvious apoptotic effect of EGCG on D1-cell under the concentration of 1 µmol/L and 10 µmol/L. These results demonstrated that long-term treatment of EGCG increases the expressions of osteogenic genes and OC protein production, elevates ALP activity and eventually stimulates mineralization, in spite of its inhibitory effect on proliferation. The osteogenic effect of EGCG may rely on inducing osteogenic differentiation, not via increasing proliferation. Elucidations on the detail mechanisms of EGCG in mesenchymal stem cells and osteoblasts, both in mice and in humans, require further investigation. • REFERENCES: • 1. Liao S, Kao YH & Hiipakka RA, Vitam Horm: 1-94, 2001. • 2. Wang GJ, Cui Q & Balian G, Clin Orthop: 295-310, 2000. • 3. Hegarty VM et.al., Am J Clin Nutr: 1003-1007, 2000. Fig 1. mRNA * : P<0.05 **: P<0.01 Fig 2 ALP activity