## 4 Discussion  
This study explored the impacts of compressive force and ZA on osteoblast proliferation and differentiation. BPs are robustly related to osteonecrosis in the jaw, but the pathogenesis of MRONJ remains unclear. Recent studies have suggested that BPs might directly interfere with osteoblasts. As reported by [PERSON] et al., high doses of ZA inhibit both osteoclast and osteoblast functions and bone remodeling in vivo [10]. A previous in vitro study also identified inhibitory effects of ZA on cell viability, cell proliferation, and osteoblast differentiation of mesenchymal stem cell [11]. Furthermore, other reports have mentioned that higher doses of ZA decrease osteoblast cell viability in a dose-dependent manner [12; 13]. In the present study, higher micromolar dose (10 or 100 \(\upmu\)M) of ZA de  
Figure 4: Matrix mineralization is assessed by Alizarin Red S (ARS) staining (**A**). After ARS staining, dye is extracted and quantified (**B**). Data represent mean \(\pm\) SD. * \(p\) < 0.05.  
creased cell proliferation on days 1-7. Higher concentrations of ZA exert direct cytotoxic effects on cells, and a lower micromolar dose (1 uM) of ZA also affects cell proliferation with prolonged exposure. Changes in expression levels of osteoblast markers are typical events during osteoblast differentiation. ALP and Runx2 are closely related to osteoblast differentiation and matrix mineralization [14]. Col I expression is one of the most widely appreciated early markers for osteoblast differentiation [15]. Furthermore, osteoblast differentiation and bone formation are closely related to blood vessel formation, and VEGF play a definitive role in bone repair, since angiogenesis and osteogenesis are highly coupled [16]. Both intramembranous and endochondral ossification depend on the VEGF signaling pathway, and blocking VEGF receptors decreases blood vessel formation and bone regeneration [17]. In addition, VEGF-A mRNA expression is increased during terminal differentiation with other mRNA expressions, such as bone sialoprotein and osteoclastin [18; 19]. We obtained evidence of the anti-osteoblast differentiation properties of 1 uM of ZA according to the results of real-time PCR. Furthermore, the combination of 1 uM of ZA and compressive force robustly inhibited osteoblast differentiation (Figure 3). Matrix mineralization is one of the bone formation events initiated by osteoblasts and is the key process in bone formation. In this study, we performed ARS staining to observe matrix mineralization in vitro. As reported by [PERSON] et al., BPs including ZA may increase or decrease osteogenesis in a concentration-dependent manner [20]. Furthermore, previous reports have also mentioned that nanomolar doses of ZA induced no differences in matrix mineralization compared with the control, while micromolar levels of ZA produced decreases in mineralization [21]. The present study found that mineralized nodule formation was decreased with the addition of 1 uM of ZA, and the combination of 1 uM ZA with compressive force markedly decreased the matrix mineralization of cells; these results indicated that the combination of ZA and compressive force negatively affects osteoblast differentiation. In the fields of dentistry and oral and maxillofacial surgery, mechanical stress (including compressive force) is closely related to the pathogenesis of various diseases, such as periodontal disease [22; 23], peri-implant bone loss [24] and temporomandibular joint disorder [9]. Alveolar bone experiences continuous mechanical stress such as occlusal forces from tooth contact. Furthermore, the oral mucosa and alveolar bone of dedutulous patients also receive mechanical stress from the denture base. [PERSON] et al. reported that a compressive force of 1.0 g/cm\({}^{2}\) increased expression levels of osteogenesis-related transcript factors, mRNAs encoding Runx2, Osterix, Dlx5 and Msnx2 [25].