# Effects of Epigallocatechin-3-O-Gallate on Bone Health
## 3 Biochemical Effects of EGCG
### Effects on RUNX2 Protein Level  
_Runx2_, a member of the runt-domain gene family, regulates the differentiation of osteoblasts from multipotent mesenchymal stem cells [48] and plays a fundamental role in osteoblast maturation and homeostasis [78; 79; 80; 81]. RUNX2 enhances osteoblast differentiation at an early stage but suppresses osteoblast maturation at late stages [82]. The RUNX2 transcription factor operates by binding to the osteoblast-specific cis-acting element 2 (_Ose2_), located in the promoter region of all major osteoblast-related genes, where it regulates their expression. Its level and/or activity are dictated by a number of different external cues, while multiple signalling pathways that affect osteoblast function merge to and are integrated by RUNX2 [78]. The ectopic expression of RUNX2 in mesenchymal cell lines leads to the upregulation of osteoblast-specific genes like _Oct_, _Alp_, collagenase-3 (_Col3_), matrix metalloproteinase-13 (_Mmp-13_), bone sialoprotein, and collagen type Ia1 [78; 83]. The Type I isoform of RUNX2 seems to be mainly involved in the intramembranous bone formation, while the Type II isoform has an exclusive role in the endochondral bone formation [84]. During the intramembranous bone formation, the Type I isoform is widely expressed in osteoprogenitor cells and in active osteoblasts, whereas the Type II isoform expression is stringently restricted to cells lining mineralised bones [84]. RUNX2 also plays a crucial role in chondrocyte maturation by the direct regulation of Indian Hedgehog (Ihh) expression. Ihh is a signalling molecule that regulates cartilage development and bone growth. RUNX2 induces the expression of Sp7 (specificity protein 7). Sp7 (also known as Osterix) is a transcription factor essential for osteoblast differentiation and bone formation. Additionally, the cooperation among RUNX2, Sp7, and canonical Wnt signalling, a pathway important for cell proliferation and differentiation, is essential for the differentiation of preosteoblasts into immature osteoblasts [80; 81]. It also induces the proliferation of osteoprogenitors by the direct regulation of fibroblast growth factor receptors like FGFR-2 and FGFR-3 expression. These receptors are involved in signalling pathways that influence the proliferation and differentiation of osteoprogenitor cells [80].  
In many in vitro studies, the influence of EGCG on RUNX2 protein expression was extensively examined, revealing variable outcomes depending on the cell type and experimental conditions (Table 2). In SaOS-2 cells, EGCG enhanced osteoblast differentiation and mineralised nodule formation, despite a reduction in RUNX2 protein levels [48], suggesting a RUNX2-independent mechanism of osteogenesis in this context. In contrast, most studies reported an upregulation of RUNX2 expression following EGCG treatment. For example, in hBM-MSCs, EGCG increased RUNX2 and OSX expression while preventing the TNF\(\alpha\)-induced suppression of osteogenic differentiation [33]. In hBM-MSCs, EGCG reversed H\({}_{2}\)O\({}_{2}\)-induced damage and promoted _Runx2_ gene expression along with other osteogenic markers such as ALP, OSX, \(\beta\)-catenin, and cyclin D1 [40]. Similarly, in hMSCs,RUNX2 expression was elevated alongside BMP-2 and VEGF, contributing to enhanced osteogenic differentiation [32]. EGCG also upregulated _Runx2_ and other osteogenic genes in hBM-MSCs [41] and hPDLCs [29], further supporting its role in activating RUNX2-driven pathways.