Bisphosphonates (BPs) are first-line therapies for prevention and treatment of osteoporosis, with well-established efficacy in reducing both vertebral and nonvertebral fracture risk (Deardorff et al., 2022, Bock, 2008). Commonly prescribed BPs, such as alendronate (ALN), risedronate (RIS), and zoledronic acid (ZA), exert effects on several bone cells (Patntirapong et al., 2012, Patntirapong et al., 2021a, Patntirapong et al., 2021b, Lilakhunakon et al., 2021, Koch et al., 2011, Malavasi et al., 2016). Although the primary targets of BPs are osteoclasts (bone-resorbing cells), they can also directly expose to and affect osteoblasts (bone-forming cells). The impact of BPs on osteoblasts is complex and can depend on several factors including drug types, dosages, treatment duration, and cell phenotypes.

Several studies have demonstrated that BPs exert direct effects on osteoblasts and pre-osteoblasts. In vitro studies have shown that ALN and ZA at moderate to high concentrations inhibit osteoblast proliferation, differentiation and function (Patntirapong et al., 2012, Patntirapong et al., 2021a, Patntirapong et al., 2021b). RIS at high doses suppresses proliferation and viability but enhances type I collagen synthesis, alkaline phosphatase (ALP) activity, and matrix mineralization (Malavasi et al., 2016). Moreover, timing and duration of BP exposure significantly influence osteoblast mineralization capacity (Patntirapong, 2022). In vivo studies have further demonstrated that BPs may delay bone healing under certain conditions (Koyama et al., 2020, Kates and Ackert-Bicknell, 2016).

Although the prescription of BPs are not limited to a certain age group, they are most commonly used in older adults whose bone-forming cells may already be experiencing aging-related changes (Deardorff et al., 2022, Vandenbroucke et al., 2017). Under a controlled cell culture, cells with increasing passages lose their proliferative capacity (Peterson et al., 2004). Osteoblasts at higher passages often exhibit a reduced capacity to differentiate and form bone matrix (Chung et al., 1999). Stimuli, such as BP, that can alter activities in low-passage cells, might be less effective in high-passage cells. The possibility that cells interacting with and being affected by the drugs may differ with passage number. However, in vitro study investigating BP effects on osteoblasts utilize early-passage (typically below passage 22, P22) or highly proliferative cells (Patntirapong et al., 2021a), which may not adequately reflect the biological responses of aging osteoblasts. This discrepancy underlines the need for an in vitro model that is more closely mimic the aging skeletal microenvironment. Utilizing high-passage osteoblast cells may serve as more relevant model for aging bone tissue.

Although BP is a known factor influencing changes in osteoblast activities, only a few studies examine the effect of passage number on osteoblasts. Furthermore, there is still lack of study investigating the interaction between cell passage and BP treatment, particularly in pre-osteoblasts. To evaluate whether further passaging alters cellular responses to BPs, this study investigated key factors: cell passages, BP treatment, or interaction between BP treatment and cell passage, on the responses of pre-osteoblasts. MC3T3-E1 pre-osteoblast cell line was used. These cells are well-established in vitro model that demonstrates proliferative and functional decline beyond P40, with further decline at very high passages beyond P60 (Peterson et al., 2004, Chung et al., 1999). Higher passage MC3T3 display senescent markers. Cells stain positive to β-galactosidase and express higher p21 gene compared to lower passage (P17 vs P7) (Xu et al., 2022).

In this study, P42 and P62 represented two different stages of prolonged in vitro passaging. According to previous studies, MC3T3 cells at P40–60 are considered high-passage, while those above P60 represent late or very high passage (Peterson et al., 2004, Chung et al., 1999). Cells at P42 and P62 were treated with ALN. Then, cell growth, adhesion, migration, cell spreading, cytoskeletal organization, and gene expressions were examined. This finding could provide the effects of cell passage, BP treatment, and their interaction on pre-osteoblasts, addressing a frequently overlooked variable in in vitro bone research.