Hypoxia Aggravates Alendronate-Induced Cytotoxicity and Extracellular Matrix Disruption in Gingival Fibroblasts: A Comparative In Vitro Study of Three Bisphosphonates

Background This study aimed to evaluate how oxygen tension influences the effects of three bisphosphonates—Alendronate (ALN), Zoledronate (ZA), and Ibandronate (IB)—on human gingival fibroblasts (HGnFs), focusing on cytotoxicity, wound healing, and extracellular matrix (ECM) regulation. We hypothesized that hypoxia exacerbates bisphosphonate-induced dysfunction, particularly with ALN, and that hyperbaric oxygen (HBO) could partially mitigate these effects. Methods HGnFs were cultured under normoxia, hypoxia (1% O₂), or HBO (2.4 ATA) conditions and exposed to ALN, ZA, or IB at clinically relevant concentrations. Cell viability was measured using the CCK-8 assay. Wound closure was assessed via scratch assays quantified with ImageJ. Western blotting analyzed intracellular and extracellular levels of fibronectin, collagen I, and HIF-1α in cell lysates and conditioned media. Results Hypoxia significantly reduced viability and migration in all bisphosphonate-treated groups, with ALN showing the most pronounced cytotoxicity. Under hypoxia, ALN at 50 µM almost completely halted migration by 24 h and severely impaired it at 48 h, representing the strongest inhibitory effect among all bisphosphonates tested. HBO partially restored wound healing, particularly in ZA- and high-ALN-treated cells, but did not fully reverse migration deficits. Hypoxia increased intracellular fibronectin and HIF-1α while reducing extracellular fibronectin and collagen I, indicating ECM disruption. HBO enhanced fibronectin secretion but had limited effect on collagen I. Conclusions Despite its oral administration and perceived lower potency, ALN exerts the most severe inhibitory effects on fibroblast migration and ECM integrity under hypoxic conditions. Hypoxia exacerbates bisphosphonate-induced dysfunction, and HBO provides only partial protection, primarily through increased fibronectin secretion. These findings highlight the potential risk of soft tissue healing complications even with routine oral bisphosphonate use in hypoxic environments, such as in elderly or systemically compromised patients, and suggest HBO as a possible adjunctive therapy.