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

Hypoxia Aggravates Alendronate-Induced Cytotoxicity and Extracellular Matrix Disruption in Gingival Fibroblasts: A Comparative In Vitro Study of Three Bisphosphonates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hypoxia Aggravates Alendronate-Induced Cytotoxicity and Extracellular Matrix Disruption in Gingival Fibroblasts: A Comparative In Vitro Study of Three Bisphosphonates Chia-Chen Wu, Jiiang-Huei Jeng, Chun-Chang Ting, Shu-Hung Huang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7338302/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Hypoxia Fibronectin Cytotoxicity Bisphosphonates Alendronate Wound healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Medication-related osteonecrosis of the jaw (MRONJ) is a major challenge in maxillofacial surgery [ 1 ] [ 2 ]. Bisphosphonates (BPs), which are widely prescribed for osteoporosis and bone-related disorders, contribute to MRONJ by accumulating in bone and suppressing osteoclast activity [ 1 ] [ 2 ] [ 3 ]. Clinically, MRONJ presents as exposed necrotic bone with associated soft tissue damage, particularly gingival loss, impairing oral wound healing [ 3 ] [ 4 ]. While bone cells are primary targets of BP-related complications, gingival fibroblasts (HGnFs) also play critical roles in maintaining gingival integrity and extracellular matrix (ECM) synthesis. BP-induced dysfunction in HGnFs compromises soft tissue repair, leading to persistent inflammation and impaired healing [ 5 ] [ 6 ] [ 7 ] [ 8 ] [ 9 ]. Hypoxia further disrupts ECM synthesis and wound healing, activating hypoxia-inducible factor-1α (HIF-1α), which, despite upregulating angiogenesis-related genes, fails to counteract collagen suppression [ 5 ]. Hyperbaric oxygen (HBO) therapy has been explored as a potential intervention, although its effects on BP-treated gingival fibroblasts remain unclear [ 10 ] [ 11 ]. While BP toxicity has been well documented in bone cells, its impact on fibroblasts remains underexplored, despite its critical role in forming a protective barrier over necrotic bone [ 1 ] [ 2 ] [ 12 ]. This study investigated how oxygen tension modulates the effects of alendronate, zoledronate, and iboundronate on HGnFs, with a focus on cell viability, wound healing, and ECM protein expression under normoxia, hypoxia, and HBO. We hypothesize that hypoxia exacerbates BP-induced toxicity via HIF-1α upregulation and ECM disruption, whereas HBO provides partial mitigation [ 6 ] [ 7 ] [ 13 ]. Materials and methods Cell culture and oxygen conditions Human gingival fibroblasts (HGnFs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in low-glucose DMEM (HyClone, Logan, UT, USA) supplemented with 4 mM L-glutamine, 1 g/L glucose, 110 mg/L sodium pyruvate, 100 IU/mL penicillin, 100 µg/mL streptomycin (GeneDireX, Las Vegas, NV, USA), and 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). The cells were maintained at 37°C and 5% CO₂ and subcultured at 80–90% confluence with 0.05% trypsin-EDTA. For hypoxia , the cells were incubated at 1% O₂, 5% CO₂, and balanced N₂ in a multigas incubator (MCO-50 M, PHCbi, Tokyo, Japan) to mimic hypoxic conditions relevant to MRONJ [ 5 ]. Hyperbaric oxygen (HBO) treatment was performed at 2.4 ATA in 100% O₂ for 90 minutes, followed by decompression at 1 ATA every 6 minutes [ 6 ]. Bisphosphonate Administration HGnFs were treated with alendronate (ALN; Sigma‒Aldrich, St. Louis, MO, USA) (ALN; Sigma‒Aldrich), ibandronate (IB; Sigma‒Aldrich), or zoledronate (ZA; Sigma‒Aldrich) at concentrations ranging from 1–200 µM for 1–3 days to determine effective doses [ 4 ]. The culture media containing bisphosphonates were changed daily. In dose‒response experiments, effective concentrations of ALN (50 µM), IB (50 µM), and ZA (10 µM) were used for subsequent 3-day treatments under different oxygen conditions [ 9 ] [ 10 ]. Cell viability assay HGnFs (1 × 10⁴ cells/well) were seeded in 96-well plates and treated with bisphosphonates under normoxia, hypoxia, or HBO for 3 days. Viability was assessed via the CCK-8 assay (Dojindo, Kumamoto, Japan) following the manufacturer’s instructions [ 10 ]. The absorbance at 450 nm was recorded via a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). To address concerns regarding the effects on proliferation, cell viability was assessed at 24, 48, and 72 hours to distinguish cytotoxic effects from migration-related changes in the wound healing assay. This confirmed that the observed differences in wound closure were not solely attributed to bisphosphonate-induced growth inhibition. Scratch Wound Healing Assay HGnFs (1 × 10⁵ cells/well) were cultured in 24-well plates until they reached confluence. After 3 days of bisphosphonate treatment, a 200 µL pipette tip was used to create a scratch wound. To eliminate the effects of proliferation, the cells were pretreated with 10 µg/mL mitomycin C for 2 hours before wounding to arrest DNA replication without inducing cytotoxicity. This approach ensures that wound closure is attributed primarily to migration rather than proliferation, as recommended by previous studies [ 14 ]. To standardize wound closure assessment, images were captured at 0, 24, and 48 hours post-scratch using the JuLi™ FL fluorescence cell history recorder (JuLi Stage™, NanoEntek, Seoul, Korea). Wound closure was objectively quantified via ImageJ software, which calculates the percentage of wound closure on the basis of area reduction over time. Irregular wound margins were accounted for via automated threshold adjustments and contour tracing in ImageJ [ 14 ]. Western Blotting and Conditioned Medium Preparation HGnFs (1 × 10⁶ cells/dish) were treated with bisphosphonates for 3 days, with HBO-treated cells receiving 90 minutes of HBO exposure on day 3. For protein quantification, conditioned media were collected after 3 days of treatment and centrifuged at 3,000 × g for 10 minutes at 4°C to remove cell debris. The samples were concentrated via an Amicon Ultra4 Centrifugal Filter Unit (10 kDa MWCO, Millipore, USA) at 4,000 × g for 30 minutes to increase protein detection sensitivity. Total protein quantification was performed via a BCA protein assay kit (iNtRON Biotechnology, South Korea) before the proteins were loaded onto SDS‒PAGE gels. To ensure equal loading, 20 µg of protein from cell lysates and 15 µg from conditioned media were used per lane. β-actin served as an internal control for cell lysates, while Ponceau S staining verified equal conditioned media loading before blotting. Primary antibodies against fibronectin, collagen I, and HIF-1α were used, and signal detection was performed via enhanced chemiluminescence (ECL) substrate (TOOLS, Biotools, Taiwan) and analyzed via ImageLab™ software (Bio-Rad, Hercules, CA, USA). Statistical analysis The data are presented as the means ± standard errors (SEs). One-way ANOVA was used for statistical comparisons, with significance set at P < 0.05. Results Bisphosphonate cytotoxicity in gingival fibroblasts: determining effective concentrations Human gingival fibroblasts (HGnFs) were treated with alendronate (ALN), zoledronate (ZA), or ibandronate (IB) at various concentrations (1–200 µM) for 1–3 days, and cell viability was assessed via the CCK-8 assay. ZA demonstrated significant toxicity at 50 µM within 24 hours, with further reductions observed after 48 and 72 hours. Even at 1 µM, ZA exhibited measurable cytotoxicity by day 3 (Fig. 1 A), which was consistent with its strong inhibitory effects on fibroblast proliferation and extracellular matrix (ECM) production in vitro [ 15 ]. IB showed minimal toxicity on day 1, but significant cytotoxicity was evident at 50 µM by days 2 and 3 (Fig. 1 B). Similarly, ALN induced marked cytotoxicity at 50 µM after 48–72 hours of exposure (Fig. 1 C), which aligns with previous studies showing its dose-dependent inhibitory effects on fibroblast viability [ 10 ]. To ensure clinically relevant conditions, the following effective concentrations were selected for subsequent experiments: ZA (10 µM), IB (50 µM), and ALN (50 µM) under a 3-day continuous treatment protocol. Hypoxia exacerbates bisphosphonate-induced cytotoxicity in gingival fibroblasts After 3 days of BP treatment, the HGnFs were exposed to normoxia, hypoxia (1% O₂), or hyperbaric oxygen (HBO; 2.4 ATA O₂ for 90 min). Hypoxia significantly reduced fibroblast proliferation, particularly in ALN-treated cells, compared with that in the ZA- and IB-treated groups (Fig. 2 A). These findings indicate that ALN-treated fibroblasts are more sensitive to oxygen deprivation. HBO treatment did not fully restore proliferation in BP-treated fibroblasts, suggesting that oxygen supplementation alone is insufficient to counteract BP-induced cytotoxicity. IB-treated cells exhibited comparable viability across all oxygen conditions, indicating that IB-mediated cytotoxicity is independent of oxygen levels (Fig. 2 B). Hypoxia impairs fibroblast migration and wound healing in BP-treated cells To assess wound healing, HGnFs were subjected to a scratch wound assay, with closure quantified via ImageJ -based area measurement, ensuring objective analysis. Under normoxia and HBO conditions, fibroblasts treated with low BP concentrations closed the wound within 48 hours . However, higher BP concentrations significantly impaired migration, leading to persistent wound gaps even after 48 hours (Fig. 3 A). Under hypoxic conditions, fibroblasts fail to achieve full wound closure regardless of the BP concentration, confirming that oxygen deprivation severely limits fibroblast migration. Notably, alendronate at 50 µM almost completely halted migration by 24 h (Fig. 3 B) and continued to markedly hinder fibroblast migration at 48 h (Fig. 3 C), representing the most potent inhibitory effect among all BPs tested under hypoxia. HBO partially improved wound healing, particularly in ZA- and high ALN-treated cells, but remained insufficient to fully reverse BP-induced migration deficits (Fig. 3 C). Importantly, IB-treated fibroblasts exhibited impaired migration only under hypoxia , suggesting that oxygen fluctuations affect IB effects differently than ALN or ZA do. Oxygen tension modulates fibronectin, collagen type I, and HIF-1α expression in BP-treated fibroblasts Western blot analysis was conducted to evaluate changes in ECM protein expression in response to oxygen fluctuations. Hypoxia significantly increased intracellular fibronectin expression in BP-treated fibroblasts (Fig. 4 A, B). Compared with normoxia, HBO also increased fibronectin levels, but this increase was not statistically significant . Both hypoxia and HBO inhibited type I collagen expression , suggesting that oxygen tension influences ECM integrity (Fig. 4 A, C). HIF-1α expression was strongly upregulated under hypoxia in all BP-treated groups, whereas HBO did not alter HIF-1α expression (Fig. 4 A, D). These findings indicate that hypoxia enhances cellular stress , leading to disruptions in ECM protein regulation. Hypoxia and HBO influence extracellular protein secretion in BP-treated fibroblasts To evaluate extracellular protein secretion, conditioned media were collected and analyzed. Hypoxia significantly reduced fibronectin secretion , whereas HBO increased fibronectin levels in the extracellular matrix (Fig. 5 A, B). Both hypoxia and HBO suppressed type I collagen secretion , resulting in BP-induced ECM degradation (Fig. 5 A, C). Notably, hypoxia and HBO significantly reduced extracellular HIF-1α levels , suggesting that oxygen-related stress responses are altered posttranslationally rather than at the protein level (Fig. 5 A, D). These findings underscore the interplay between oxygen tension, bisphosphonate exposure, and ECM remodeling in fibroblasts , highlighting potential limitations in HBO therapy for BP-induced cytotoxicity. Detailed physicochemical properties of the bisphosphonates are provided in Supplementary Table S1 . Discussion This study demonstrated that oxygen tension critically modulates bisphosphonate (BP)-induced toxicity in gingival fibroblasts (HGnFs). Hypoxia exacerbates BP-induced cytotoxicity and impaired wound healing, particularly in alendronate (ALN)-treated cells, whereas hyperbaric oxygen (HBO) therapy has partial but limited reparative effects. The observed reductions in cell viability, migration, and ECM protein secretion highlight the impact of oxygen availability on soft tissue repair. HGnFs play a key role in MRONJ progression, as loss of gingival integrity precedes bone exposure and necrosis [ 9 ]. Prior studies have shown that BPs disrupt fibroblast proliferation, adhesion, and ECM remodeling, contributing to MRONJ [ 15 ] [ 10 ]. Among the BPs tested, ALN exhibited the greatest cytotoxicity under hypoxia, which is consistent with its prolonged retention in bone and soft tissues [ 16 ]. While HIF-1α upregulation may mediate an adaptive response, intracellular fibronectin accumulation fails to restore ECM function due to impaired secretion, likely contributing to wound healing deficits [ 13 ]. Hypoxia markedly impaired fibroblast migration across all BP-treated groups, with alendronate at 50 µM exerting the most potent effect—almost completely halting migration by 24 h and continuing to markedly hinder closure at 48 h. This severe inhibition, objectively quantified by ImageJ-based scratch wound analysis, underscores that oxygen deprivation is a critical factor limiting fibroblast-mediated tissue repair and that ALN’s impact is disproportionately strong compared with that of other BPs. HBO partially restored migration and fibronectin secretion, particularly in ZA- and high ALN-treated cells, but its effects were insufficient to fully reverse BP-induced dysfunction[ 10 ]. These findings reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling [ 17 ]. Hypoxia markedly impaired fibroblast migration, particularly in ALN- and zoledronate-treated cells, as shown by objective scratch wound analysis via ImageJ-based measurements. These findings confirm that oxygen deprivation is a key factor limiting fibroblast-mediated tissue repair. HBO partially restored migration and fibronectin secretion, but its effects were insufficient to fully reverse BP-induced dysfunction. These results reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling. Western blot and conditioned media analyses confirmed that hypoxia reduced fibronectin secretion, whereas HBO increased extracellular fibronectin levels. Type I collagen secretion was suppressed under both hypoxia and HBO, resulting in BP-induced ECM degradation. While fibronectin and collagen I are crucial ECM components, cytokines, integrins, and matrix metalloproteinases (MMPs) also regulate fibroblast function. Upregulated MMP activity under BPs contributes to ECM remodeling but is insufficient to fully restore wound healing [ 17 ]. Similarly, BP exposure inhibits collagen synthesis and increases inflammatory cytokines, further impairing tissue regeneration [ 16 ] [ 14 ]. The suppression of type I collagen and the intracellular accumulation but extracellular deficiency of fibronectin under hypoxia highlights oxygen tension as a key regulator of ECM assembly. HBO partially enhances fibronectin secretion, supporting cell adhesion and migration, but fails to restore collagen synthesis, limiting its reparative potential [ 16 ] [ 18 ]. These findings suggest that additional molecular mediators beyond fibronectin and collagen I contribute to BP-induced dysfunction (Fig. 6 ). These results have important clinical implications for MRONJ management, particularly in hypoxic environments such as tunnels, submarines, or high-altitude workplaces, where oxygen deprivation may worsen BP-related soft tissue damage [ 19 ]. While HBO enhances fibronectin secretion and migration, its limited impact on collagen synthesis suggests that combination therapies targeting oxidative stress, collagen restoration, or BP toxicity may be necessary [ 20 ]. While this study provides mechanistic insights, the in vitro model does not fully replicate the complexity of in vivo wound healing. Future research should explore coculture models, animal studies, and advanced transcriptomic profiling to further investigate BP‒hypoxia interactions. Additionally, investigating upstream signaling pathways (e.g., the PI3K/AKT and NF-κB pathways) may provide a more comprehensive understanding of ECM remodeling in BP-treated fibroblasts [ 21 ]. Hypoxia markedly impaired fibroblast migration, particularly in ALN- and ZA-treated cells, as shown in objective scratch wound analysis using ImageJ-based measurements. This confirms that oxygen deprivation is a key factor limiting fibroblast-mediated tissue repair. Notably, despite its oral administration and perceived lower potency, alendronate exerted the most pronounced inhibitory effects on fibroblast migration and ECM integrity under hypoxic conditions, almost completely halting migration at 24 h and severely hindering closure at 48 h. HBO partially restored migration and fibronectin secretion, but its effects remained insufficient to fully reverse BP-induced dysfunction. These results reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling. Collectively, our findings suggest that in hypoxic environments—such as those occurring in elderly or systemically compromised patients—routine alendronate use may pose a higher-than-anticipated risk to oral soft tissue healing, and that HBO may offer partial but clinically meaningful mitigation. Conclusions This study demonstrates that hypoxia markedly intensifies bisphosphonate-induced cytotoxicity and extracellular matrix disruption in human gingival fibroblasts, with Alendronate producing the most severe inhibitory effects on fibroblast migration and ECM integrity. Notably, under hypoxic conditions, high-dose Alendronate nearly abolished cell migration within 24 hours and continued to impair it at 48 hours, surpassing the effects of Zoledronate and Ibandronate. Although hyperbaric oxygen therapy partially restored fibronectin secretion and modestly improved migration, it was insufficient to fully reverse bisphosphonate-induced damage. These findings underscore that even routinely prescribed oral bisphosphonates, such as Alendronate, may carry significant soft tissue healing risks in hypoxic conditions—particularly in elderly or systemically compromised patients—and suggest that HBO therapy may serve as a supportive but incomplete countermeasure. Abbreviations ALN Alendronate ZA Zoledronate IB Ibandronate BPs Bisphosphonates HGnFs Human gingival fibroblasts ECM Extracellular matrix HBO Hyperbaric oxygen HIF-1α Hypoxia-inducible factor 1-alpha CCK-8 Cell Counting Kit-8 HBO Hyperbaric oxygen at 2.4 atmospheres absolute (ATA) MRONJ Medication-related osteonecrosis of the jaw Declarations Ethics Declarations Clinical trial number Not applicable. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. The original uncropped Western blot images are not available due to the multi-marker blotting approach and the time elapsed since data collection. All Western blot experiments were independently repeated three to four times, yielding consistent and reproducible results. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the personal self-funding of the corresponding author, Edward Chengchuan Ko. No external funding was received. Authors’ contributions E.C.K. conceived and designed the study, performed experiments, analysed data, and drafted the manuscript. J. J. revised the manuscript. S.H. provided the hyperbaric oxygen chamber. Y.K., H.C., Y.K., C.T. and C.H. provided clinical correlation and interpretation. J.K. contributed clinical insights regarding bisphosphonate therapy in cancer patients. C.W. and F.W. performed the laboratory work and analysed the results. W.L., T.T. and K.H. supervised the study and reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to Miss Maruko Wanchih Huang for her efforts in building our laboratory. We also extend our gratitude to The Liberty Lab, a non-profit research institution dedicated to promoting interinstitutional and international collaborations. We welcome and sincerely appreciate any additional support or grants that may help further advance our research efforts. Authors’ information Edward Chengchuan Ko, Ph.D., DDS, MS – Corresponding author. Email: [email protected] . References Ruggiero SL, Dodson TB, Aghaloo T, Carlson ER, Ward BB, Kademani D. American Association of Oral and Maxillofacial Surgeons' Position Paper on Medication-Related Osteonecrosis of the Jaws-2022 Update. J Oral Maxillofac Surg. 2022;80(5):920–43. Campisi G, Mauceri R, Bertoldo F, Bettini G, Biasotto M, Colella G, Consolo U, Di Fede O, Favia G, Fusco V et al. Medication-Related Osteonecrosis of Jaws (MRONJ) Prevention and Diagnosis: Italian Consensus Update 2020. Int J Environ Res Public Health 2020, 17(16). Rocho FR, Bonatto V, Lameiro RF, Lameira J, Leitao A, Montanari CA. 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Cell viability was assessed via the CCK-8 assay. ZA exhibited significant cytotoxicity at 10 μM by day 3, whereas IB and ALN showed cytotoxicity at 50 μM. The data are presented as the means ± SEs. Statistical differences were analyzed via one-way ANOVA: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/8f63bc10a6ba8e7132bf73fa.png"},{"id":97899771,"identity":"c61020bd-9c96-4314-b19d-8c6623581a06","added_by":"auto","created_at":"2025-12-10 15:44:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypoxia exacerbates bisphosphonate-induced cytotoxicity in HGnFs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative images of HGnFs under normoxia, hypoxia, and hyperbaric oxygen (HBO) conditions captured with a JuLI® FL fluorescence cell history recorder at 4× magnification. (B) Cell viability assessed by a CCK-8 assay demonstrated significantly reduced survival under hypoxia. The data are expressed as the means ± SEs and were analyzed via one-way ANOVA: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with the normoxia group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/84fd597b47ee79542d025f4c.jpg"},{"id":97899810,"identity":"6814b671-4180-43ac-af7f-36cfc610f14f","added_by":"auto","created_at":"2025-12-10 15:44:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":824521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWound healing impairment in bisphosphonate-treated HGnFs under different oxygen conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Scratch wound closure in bisphosphonate-treated HGnFs was assessed under normoxia, hypoxia, and hyperbaric oxygen (HBO). Images were captured at 0, 24, and 48 hours via a JuLI® FL fluorescence cell history recorder (4× magnification).\u003c/p\u003e\n\u003cp\u003e(B) Quantification of the migration rate at 24 h revealed that hypoxia significantly impaired wound healing in all the BP-treated groups. Notably, alendronate at 50 µM almost completely halted migration by 24 h, representing the most potent inhibitory effect under hypoxia.\u003c/p\u003e\n\u003cp\u003e(C) Quantification of the migration rate at 48 h revealed that alendronate at 50 µM markedly hindered fibroblast migration, with HBO partially improving wound closure in ZA- and high-ALN-treated cells but not fully restoring migration. The data are presented as the means ± SEs and were analyzed via one-way ANOVA: *P \u0026lt; 0.05, **P \u0026lt; 0.01 compared with normoxia.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/bbbe8d746c4d9aafe0cd669e.png"},{"id":97900884,"identity":"ac5af488-7aa9-4662-9379-50aa048f5507","added_by":"auto","created_at":"2025-12-10 15:46:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of oxygen tension on intracellular ECM protein expression in bisphosphonate-treated HGnFs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of fibronectin, collagen I, and HIF-1α in HGnFs treated with bisphosphonates under normoxia, hypoxia, or HBO (n = 4).\u003c/p\u003e\n\u003cp\u003e(B) Hypoxia significantly increased intracellular fibronectin expression in BP-treated fibroblasts.\u003c/p\u003e\n\u003cp\u003e(C) Collagen I expression was suppressed under both hypoxia and HBO.\u003c/p\u003e\n\u003cp\u003e(D) Hypoxia markedly increased HIF-1α expression, whereas HBO had no significant effect. The data are the means ± SEs. Statistical differences were analyzed via one-way ANOVA: \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/42b1870c86055bd110273700.png"},{"id":97900539,"identity":"fafbce03-b41e-461f-aae9-434058699bfe","added_by":"auto","created_at":"2025-12-10 15:45:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":99175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBisphosphonate-induced changes in ECM protein secretion under different oxygen conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of fibronectin, collagen I, and HIF-1α in conditioned media from BP-treated HGnFs (n = 4).\u003c/p\u003e\n\u003cp\u003e(B) Hypoxia significantly reduced extracellular fibronectin secretion, whereas HBO increased fibronectin levels.\u003c/p\u003e\n\u003cp\u003e(C) Both hypoxia and HBO suppressed collagen I secretion.\u003c/p\u003e\n\u003cp\u003e(D) Hypoxia and HBO significantly reduced extracellular HIF-1α secretion. The data are the means ± SEs. Statistical differences were analyzed via one-way ANOVA: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared with normoxia.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/e9c57120baa257311a3c8779.png"},{"id":97900436,"identity":"be08640f-e0db-48f1-8496-090107933f36","added_by":"auto","created_at":"2025-12-10 15:45:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90305,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConceptual model of bisphosphonate-treated HGnFs under different oxygen conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRegulatory pathways influenced by bisphosphonates (BPs) affect extracellular matrix (ECM) protein expression and fibroblast function under hypoxia, normoxia, and HBO therapy. Under hypoxia, reduced O₂ activates HIF-1α, oxidative stress, and inflammatory cytokines, increasing intracellular fibronectin but reducing extracellular secretion and suppressing collagen I synthesis, leading to impaired wound healing. Under normoxia, BPs downregulate fibronectin and collagen I while maintaining partial ECM integrity. HBO increases fibronectin secretion but does not fully restore collagen I expression, suggesting partial improvement without complete ECM recovery. Question marks indicate processes potentially influenced by additional factors (e.g., cytokines and growth factors). The dashed lines represent indirect or proposed relationships.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/df85a610975e8687530c9b34.jpg"},{"id":99816755,"identity":"d52320c5-252c-49a9-ba30-9ef92102c776","added_by":"auto","created_at":"2026-01-08 14:47:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2395012,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7338302/v1/778a42e7-4387-463e-aefb-1ca352a36cd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hypoxia Aggravates Alendronate-Induced Cytotoxicity and Extracellular Matrix Disruption in Gingival Fibroblasts: A Comparative In Vitro Study of Three Bisphosphonates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMedication-related osteonecrosis of the jaw (MRONJ) is a major challenge in maxillofacial surgery [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Bisphosphonates (BPs), which are widely prescribed for osteoporosis and bone-related disorders, contribute to MRONJ by accumulating in bone and suppressing osteoclast activity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Clinically, MRONJ presents as exposed necrotic bone with associated soft tissue damage, particularly gingival loss, impairing oral wound healing [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While bone cells are primary targets of BP-related complications, gingival fibroblasts (HGnFs) also play critical roles in maintaining gingival integrity and extracellular matrix (ECM) synthesis. BP-induced dysfunction in HGnFs compromises soft tissue repair, leading to persistent inflammation and impaired healing [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHypoxia further disrupts ECM synthesis and wound healing, activating hypoxia-inducible factor-1α (HIF-1α), which, despite upregulating angiogenesis-related genes, fails to counteract collagen suppression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hyperbaric oxygen (HBO) therapy has been explored as a potential intervention, although its effects on BP-treated gingival fibroblasts remain unclear [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile BP toxicity has been well documented in bone cells, its impact on fibroblasts remains underexplored, despite its critical role in forming a protective barrier over necrotic bone [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This study investigated how oxygen tension modulates the effects of alendronate, zoledronate, and iboundronate on HGnFs, with a focus on cell viability, wound healing, and ECM protein expression under normoxia, hypoxia, and HBO. We hypothesize that hypoxia exacerbates BP-induced toxicity via HIF-1α upregulation and ECM disruption, whereas HBO provides partial mitigation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and oxygen conditions\u003c/h2\u003e\u003cp\u003eHuman gingival fibroblasts (HGnFs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in low-glucose DMEM (HyClone, Logan, UT, USA) supplemented with 4 mM L-glutamine, 1 g/L glucose, 110 mg/L sodium pyruvate, 100 IU/mL penicillin, 100 \u0026micro;g/mL streptomycin (GeneDireX, Las Vegas, NV, USA), and 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). The cells were maintained at 37\u0026deg;C and 5% CO₂ and subcultured at 80\u0026ndash;90% confluence with 0.05% trypsin-EDTA.\u003c/p\u003e\u003cp\u003eFor \u003cem\u003ehypoxia\u003c/em\u003e, the cells were incubated at 1% O₂, 5% CO₂, and balanced N₂ in a multigas incubator (MCO-50 M, PHCbi, Tokyo, Japan) to mimic hypoxic conditions relevant to MRONJ [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hyperbaric oxygen (HBO) treatment was performed at 2.4 ATA in 100% O₂ for 90 minutes, followed by decompression at 1 ATA every 6 minutes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBisphosphonate Administration\u003c/h3\u003e\n\u003cp\u003eHGnFs were treated with alendronate (ALN; Sigma‒Aldrich, St. Louis, MO, USA) (ALN; Sigma‒Aldrich), ibandronate (IB; Sigma‒Aldrich), or zoledronate (ZA; Sigma‒Aldrich) at concentrations ranging from 1\u0026ndash;200 \u0026micro;M for 1\u0026ndash;3 days to determine effective doses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The culture media containing bisphosphonates were changed daily. In dose‒response experiments, effective concentrations of ALN (50 \u0026micro;M), IB (50 \u0026micro;M), and ZA (10 \u0026micro;M) were used for subsequent 3-day treatments under different oxygen conditions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eHGnFs (1 \u0026times; 10⁴ cells/well) were seeded in 96-well plates and treated with bisphosphonates under normoxia, hypoxia, or HBO for 3 days. Viability was assessed via the CCK-8 assay (Dojindo, Kumamoto, Japan) following the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The absorbance at 450 nm was recorded via a Multiskan SkyHigh Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\u003cp\u003eTo address concerns regarding the effects on proliferation, cell viability was assessed at 24, 48, and 72 hours to distinguish cytotoxic effects from migration-related changes in the wound healing assay. This confirmed that the observed differences in wound closure were not solely attributed to bisphosphonate-induced growth inhibition.\u003c/p\u003e\n\u003ch3\u003eScratch Wound Healing Assay\u003c/h3\u003e\n\u003cp\u003eHGnFs (1 \u0026times; 10⁵ cells/well) were cultured in 24-well plates until they reached confluence. After 3 days of bisphosphonate treatment, a 200 \u0026micro;L pipette tip was used to create a scratch wound.\u003c/p\u003e\u003cp\u003eTo eliminate the effects of proliferation, the cells were pretreated with 10 \u0026micro;g/mL mitomycin C for 2 hours before wounding to arrest DNA replication without inducing cytotoxicity. This approach ensures that wound closure is attributed primarily to migration rather than proliferation, as recommended by previous studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo standardize wound closure assessment, images were captured at 0, 24, and 48 hours post-scratch using the JuLi\u0026trade; FL fluorescence cell history recorder (JuLi Stage\u0026trade;, NanoEntek, Seoul, Korea). Wound closure was objectively quantified via ImageJ software, which calculates the percentage of wound closure on the basis of area reduction over time. Irregular wound margins were accounted for via automated threshold adjustments and contour tracing in ImageJ [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWestern Blotting and Conditioned Medium Preparation\u003c/h3\u003e\n\u003cp\u003eHGnFs (1 \u0026times; 10⁶ cells/dish) were treated with bisphosphonates for 3 days, with HBO-treated cells receiving 90 minutes of HBO exposure on day 3. For protein quantification, conditioned media were collected after 3 days of treatment and centrifuged at 3,000 \u0026times; g for 10 minutes at 4\u0026deg;C to remove cell debris. The samples were concentrated via an Amicon Ultra4 Centrifugal Filter Unit (10 kDa MWCO, Millipore, USA) at 4,000 \u0026times; g for 30 minutes to increase protein detection sensitivity. Total protein quantification was performed via a BCA protein assay kit (iNtRON Biotechnology, South Korea) before the proteins were loaded onto SDS‒PAGE gels.\u003c/p\u003e\u003cp\u003eTo ensure equal loading, 20 \u0026micro;g of protein from cell lysates and 15 \u0026micro;g from conditioned media were used per lane. β-actin served as an internal control for cell lysates, while Ponceau S staining verified equal conditioned media loading before blotting.\u003c/p\u003e\u003cp\u003ePrimary antibodies against fibronectin, collagen I, and HIF-1α were used, and signal detection was performed via enhanced chemiluminescence (ECL) substrate (TOOLS, Biotools, Taiwan) and analyzed via ImageLab\u0026trade; software (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors (SEs). One-way ANOVA was used for statistical comparisons, with significance set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eBisphosphonate cytotoxicity in gingival fibroblasts: determining effective concentrations\u003c/h2\u003e\u003cp\u003eHuman gingival fibroblasts (HGnFs) were treated with alendronate (ALN), zoledronate (ZA), or ibandronate (IB) at various concentrations (1\u0026ndash;200 \u0026micro;M) for 1\u0026ndash;3 days, and cell viability was assessed via the CCK-8 assay. ZA demonstrated significant toxicity at 50 \u0026micro;M within 24 hours, with further reductions observed after 48 and 72 hours. Even at 1 \u0026micro;M, ZA exhibited measurable cytotoxicity by day 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which was consistent with its strong inhibitory effects on fibroblast proliferation and extracellular matrix (ECM) production in vitro [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIB showed minimal toxicity on day 1, but significant cytotoxicity was evident at 50 \u0026micro;M by days 2 and 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, ALN induced marked cytotoxicity at 50 \u0026micro;M after 48\u0026ndash;72 hours of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), which aligns with previous studies showing its dose-dependent inhibitory effects on fibroblast viability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo ensure clinically relevant conditions, the following effective concentrations were selected for subsequent experiments: ZA (10 \u0026micro;M), IB (50 \u0026micro;M), and ALN (50 \u0026micro;M) under a 3-day continuous treatment protocol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHypoxia exacerbates bisphosphonate-induced cytotoxicity in gingival fibroblasts\u003c/h2\u003e\u003cp\u003eAfter 3 days of BP treatment, the HGnFs were exposed to normoxia, hypoxia (1% O₂), or hyperbaric oxygen (HBO; 2.4 ATA O₂ for 90 min). Hypoxia significantly reduced fibroblast proliferation, particularly in ALN-treated cells, compared with that in the ZA- and IB-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These findings indicate that ALN-treated fibroblasts are more sensitive to oxygen deprivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHBO treatment did not fully restore proliferation in BP-treated fibroblasts, suggesting that oxygen supplementation alone is insufficient to counteract BP-induced cytotoxicity. IB-treated cells exhibited comparable viability across all oxygen conditions, indicating that \u003cem\u003eIB-mediated cytotoxicity is independent of oxygen levels\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHypoxia impairs fibroblast migration and wound healing in BP-treated cells\u003c/h2\u003e\u003cp\u003eTo assess wound healing, HGnFs were subjected to a scratch wound assay, with closure quantified via \u003cem\u003eImageJ\u003c/em\u003e-based area measurement, ensuring objective analysis.\u003c/p\u003e\u003cp\u003eUnder \u003cem\u003enormoxia and HBO\u003c/em\u003e conditions, \u003cem\u003efibroblasts treated with low BP\u003c/em\u003e concentrations closed the wound within \u003cem\u003e48 hours\u003c/em\u003e. However, higher BP concentrations significantly impaired migration, leading to persistent wound gaps even after 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUnder \u003cem\u003ehypoxic\u003c/em\u003e conditions, fibroblasts fail to achieve full wound closure regardless of the BP concentration, confirming that oxygen deprivation severely limits fibroblast migration. Notably, alendronate at 50 \u0026micro;M almost completely halted migration by 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and continued to markedly hinder fibroblast migration at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), representing the most potent inhibitory effect among all BPs tested under hypoxia. HBO partially improved wound healing, particularly in ZA- and high ALN-treated cells, but remained insufficient to fully reverse BP-induced migration deficits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eImportantly, \u003cem\u003eIB-treated fibroblasts exhibited impaired migration only under hypoxia\u003c/em\u003e, suggesting that \u003cem\u003eoxygen fluctuations affect IB effects differently than ALN or ZA do.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eOxygen tension modulates fibronectin, collagen type I, and HIF-1α expression in BP-treated fibroblasts\u003c/h2\u003e\u003cp\u003eWestern blot analysis was conducted to evaluate changes in ECM protein expression in response to oxygen fluctuations.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHypoxia significantly increased intracellular fibronectin\u003c/em\u003e expression in BP-treated fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Compared with normoxia, \u003cem\u003eHBO also increased fibronectin\u003c/em\u003e levels, but this increase was \u003cem\u003enot statistically significant\u003c/em\u003e. Both \u003cem\u003ehypoxia and HBO inhibited type I collagen expression\u003c/em\u003e, suggesting that \u003cem\u003eoxygen tension influences ECM integrity\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). \u003cem\u003eHIF-1α\u003c/em\u003e expression was \u003cem\u003estrongly upregulated under hypoxia in all BP-treated\u003c/em\u003e groups, whereas \u003cem\u003eHBO did not alter HIF-1α expression\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, D). These findings indicate that \u003cem\u003ehypoxia enhances cellular stress\u003c/em\u003e, leading to \u003cem\u003edisruptions in ECM protein regulation.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHypoxia and HBO influence extracellular protein secretion in BP-treated fibroblasts\u003c/h2\u003e\u003cp\u003eTo evaluate extracellular protein secretion, conditioned media were collected and analyzed. \u003cem\u003eHypoxia significantly reduced fibronectin secretion\u003c/em\u003e, whereas \u003cem\u003eHBO increased fibronectin levels\u003c/em\u003e in the extracellular matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Both \u003cem\u003ehypoxia and HBO suppressed type I collagen secretion\u003c/em\u003e, resulting in BP-induced \u003cem\u003eECM degradation\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). Notably, \u003cem\u003ehypoxia and HBO significantly reduced extracellular HIF-1α levels\u003c/em\u003e, suggesting that \u003cem\u003eoxygen-related stress responses are altered posttranslationally\u003c/em\u003e rather than at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings \u003cem\u003eunderscore the interplay between oxygen tension, bisphosphonate exposure, and ECM remodeling in fibroblasts\u003c/em\u003e, highlighting \u003cem\u003epotential limitations in HBO\u003c/em\u003e therapy for BP-induced cytotoxicity.\u003c/p\u003e\u003cp\u003eDetailed physicochemical properties of the bisphosphonates are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that oxygen tension critically modulates bisphosphonate (BP)-induced toxicity in gingival fibroblasts (HGnFs). Hypoxia exacerbates BP-induced cytotoxicity and impaired wound healing, particularly in alendronate (ALN)-treated cells, whereas hyperbaric oxygen (HBO) therapy has partial but limited reparative effects. The observed reductions in cell viability, migration, and ECM protein secretion highlight the impact of oxygen availability on soft tissue repair.\u003c/p\u003e\u003cp\u003eHGnFs play a key role in MRONJ progression, as loss of gingival integrity precedes bone exposure and necrosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Prior studies have shown that BPs disrupt fibroblast proliferation, adhesion, and ECM remodeling, contributing to MRONJ [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among the BPs tested, ALN exhibited the greatest cytotoxicity under hypoxia, which is consistent with its prolonged retention in bone and soft tissues [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While HIF-1α upregulation may mediate an adaptive response, intracellular fibronectin accumulation fails to restore ECM function due to impaired secretion, likely contributing to wound healing deficits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHypoxia markedly impaired fibroblast migration across all BP-treated groups, with alendronate at 50 \u0026micro;M exerting the most potent effect\u0026mdash;almost completely halting migration by 24 h and continuing to markedly hinder closure at 48 h. This severe inhibition, objectively quantified by ImageJ-based scratch wound analysis, underscores that oxygen deprivation is a critical factor limiting fibroblast-mediated tissue repair and that ALN\u0026rsquo;s impact is disproportionately strong compared with that of other BPs. HBO partially restored migration and fibronectin secretion, particularly in ZA- and high ALN-treated cells, but its effects were insufficient to fully reverse BP-induced dysfunction[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These findings reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHypoxia markedly impaired fibroblast migration, particularly in ALN- and zoledronate-treated cells, as shown by objective scratch wound analysis via ImageJ-based measurements. These findings confirm that oxygen deprivation is a key factor limiting fibroblast-mediated tissue repair. HBO partially restored migration and fibronectin secretion, but its effects were insufficient to fully reverse BP-induced dysfunction. These results reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling.\u003c/p\u003e\u003cp\u003eWestern blot and conditioned media analyses confirmed that hypoxia reduced fibronectin secretion, whereas HBO increased extracellular fibronectin levels. Type I collagen secretion was suppressed under both hypoxia and HBO, resulting in BP-induced ECM degradation. While fibronectin and collagen I are crucial ECM components, cytokines, integrins, and matrix metalloproteinases (MMPs) also regulate fibroblast function. Upregulated MMP activity under BPs contributes to ECM remodeling but is insufficient to fully restore wound healing [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similarly, BP exposure inhibits collagen synthesis and increases inflammatory cytokines, further impairing tissue regeneration [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe suppression of type I collagen and the intracellular accumulation but extracellular deficiency of fibronectin under hypoxia highlights oxygen tension as a key regulator of ECM assembly. HBO partially enhances fibronectin secretion, supporting cell adhesion and migration, but fails to restore collagen synthesis, limiting its reparative potential [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings suggest that additional molecular mediators beyond fibronectin and collagen I contribute to BP-induced dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results have important clinical implications for MRONJ management, particularly in hypoxic environments such as tunnels, submarines, or high-altitude workplaces, where oxygen deprivation may worsen BP-related soft tissue damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. While HBO enhances fibronectin secretion and migration, its limited impact on collagen synthesis suggests that combination therapies targeting oxidative stress, collagen restoration, or BP toxicity may be necessary [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile this study provides mechanistic insights, the in vitro model does not fully replicate the complexity of in vivo wound healing. Future research should explore coculture models, animal studies, and advanced transcriptomic profiling to further investigate BP‒hypoxia interactions. Additionally, investigating upstream signaling pathways (e.g., the PI3K/AKT and NF-κB pathways) may provide a more comprehensive understanding of ECM remodeling in BP-treated fibroblasts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHypoxia markedly impaired fibroblast migration, particularly in ALN- and ZA-treated cells, as shown in objective scratch wound analysis using ImageJ-based measurements. This confirms that oxygen deprivation is a key factor limiting fibroblast-mediated tissue repair. Notably, despite its oral administration and perceived lower potency, alendronate exerted the most pronounced inhibitory effects on fibroblast migration and ECM integrity under hypoxic conditions, almost completely halting migration at 24 h and severely hindering closure at 48 h. HBO partially restored migration and fibronectin secretion, but its effects remained insufficient to fully reverse BP-induced dysfunction. These results reinforce that proliferation inhibition alone does not fully explain delayed wound healing, as BP exposure disrupts multiple cellular pathways regulating ECM remodeling. Collectively, our findings suggest that in hypoxic environments\u0026mdash;such as those occurring in elderly or systemically compromised patients\u0026mdash;routine alendronate use may pose a higher-than-anticipated risk to oral soft tissue healing, and that HBO may offer partial but clinically meaningful mitigation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that hypoxia markedly intensifies bisphosphonate-induced cytotoxicity and extracellular matrix disruption in human gingival fibroblasts, with Alendronate producing the most severe inhibitory effects on fibroblast migration and ECM integrity. Notably, under hypoxic conditions, high-dose Alendronate nearly abolished cell migration within 24 hours and continued to impair it at 48 hours, surpassing the effects of Zoledronate and Ibandronate. Although hyperbaric oxygen therapy partially restored fibronectin secretion and modestly improved migration, it was insufficient to fully reverse bisphosphonate-induced damage. These findings underscore that even routinely prescribed oral bisphosphonates, such as Alendronate, may carry significant soft tissue healing risks in hypoxic conditions\u0026mdash;particularly in elderly or systemically compromised patients\u0026mdash;and suggest that HBO therapy may serve as a supportive but incomplete countermeasure.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eALN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAlendronate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZoledronate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIbandronate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBPs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBisphosphonates\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHGnFs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman gingival fibroblasts\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eExtracellular matrix\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHBO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHyperbaric oxygen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHIF-1α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHypoxia-inducible factor 1-alpha\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCK-8\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCell Counting Kit-8\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHBO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHyperbaric oxygen at 2.4 atmospheres absolute (ATA)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMRONJ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMedication-related osteonecrosis of the jaw\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files. The original uncropped Western blot images are not available due to the multi-marker blotting approach and the time elapsed since data collection. All Western blot experiments were independently repeated three to four times, yielding consistent and reproducible results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the personal self-funding of the corresponding author, Edward Chengchuan Ko. No external funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.C.K. conceived and designed the study, performed experiments, analysed data, and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eJ. J. revised the manuscript.\u003c/p\u003e\n\u003cp\u003eS.H. provided the hyperbaric oxygen chamber.\u003c/p\u003e\n\u003cp\u003eY.K., H.C., Y.K., C.T. and C.H. provided clinical correlation and interpretation.\u003c/p\u003e\n\u003cp\u003eJ.K. contributed clinical insights regarding bisphosphonate therapy in cancer patients.\u003c/p\u003e\n\u003cp\u003eC.W. and F.W. performed the laboratory work and analysed the results.\u003c/p\u003e\n\u003cp\u003eW.L., T.T. and K.H. supervised the study and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Miss Maruko Wanchih Huang for her efforts in building our laboratory. We also extend our gratitude to The Liberty Lab, a non-profit research institution dedicated to promoting interinstitutional and international collaborations. We welcome and sincerely appreciate any additional support or grants that may help further advance our research efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEdward Chengchuan Ko, Ph.D., DDS, MS \u0026ndash; Corresponding author. Email: [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRuggiero SL, Dodson TB, Aghaloo T, Carlson ER, Ward BB, Kademani D. American Association of Oral and Maxillofacial Surgeons' Position Paper on Medication-Related Osteonecrosis of the Jaws-2022 Update. J Oral Maxillofac Surg. 2022;80(5):920\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCampisi G, Mauceri R, Bertoldo F, Bettini G, Biasotto M, Colella G, Consolo U, Di Fede O, Favia G, Fusco V et al. Medication-Related Osteonecrosis of Jaws (MRONJ) Prevention and Diagnosis: Italian Consensus Update 2020. Int J Environ Res Public Health 2020, 17(16).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRocho FR, Bonatto V, Lameiro RF, Lameira J, Leitao A, Montanari CA. A patent review on cathepsin K inhibitors to treat osteoporosis (2011\u0026ndash;2021). Expert Opin Ther Pat. 2022;32(5):561\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuirguis RH, Tan LP, Hicks RM, Hasan A, Duong TD, Hu X, Hng JYS, Hadi MH, Owuama HC, Matthyssen T et al. In Vitro Cytotoxicity of Antiresorptive and Antiangiogenic Compounds on Oral Tissues Contributing to MRONJ: Systematic Review. Biomolecules 2023, 13(6).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang SH, Hu MH, Lo WY, Sun YH, Wu CC, Yang KC. The influence of oxygen concentration on the extracellular matrix production of human nucleus pulposus cells during isolation-expansion process. J Biomed Mater Res A. 2017;105(6):1575\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTretter V, Zach ML, Bohme S, Ullrich R, Markstaller K, Klein KU. Investigating Disturbances of Oxygen Homeostasis: From Cellular Mechanisms to the Clinical Practice. Front Physiol. 2020;11:947.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBayraktar S, Ustun C, Kehr NS. Oxygen Delivery Biomaterials in Wound Healing Applications. Macromol Biosci. 2024;24(3):e2300363.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGolz L, Memmert S, Rath-Deschner B, Jager A, Appel T, Baumgarten G, Gotz W, Frede S. Hypoxia and \u003cem\u003eP. gingivalis\u003c/em\u003e synergistically induce HIF-1 and NF-kappaB activation in PDL cells and periodontal diseases. \u003cem\u003eMediators Inflamm\u003c/em\u003e 2015, 2015:438085.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalter C, Klein MO, Pabst A, Al-Nawas B, Duschner H, Ziebart T. Influence of bisphosphonates on endothelial cells, fibroblasts, and osteogenic cells. Clin Oral Investig. 2010;14(1):35\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSoydan SS, Araz K, Senel FV, Yurtcu E, Helvacioglu F, Dagdeviren A, Tekindal MA, Sahin F. Effects of alendronate and pamidronate on apoptosis and cell proliferation in cultured primary human gingival fibroblasts. Hum Exp Toxicol. 2015;34(11):1073\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLandesberg R, Cozin M, Cremers S, Woo V, Kousteni S, Sinha S, Garrett-Sinha L, Raghavan S. Inhibition of oral mucosal cell wound healing by bisphosphonates. J Oral Maxillofac Surg. 2008;66(5):839\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAcil Y, Arndt ML, Gulses A, Wieker H, Naujokat H, Ayna M, Wiltfang J. Cytotoxic and inflammatory effects of alendronate and zolendronate on human osteoblasts, gingival fibroblasts and osteosarcoma cells. J Craniomaxillofac Surg. 2018;46(4):538\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRyu MH, Park HM, Chung J, Lee CH, Park HR. Hypoxia-inducible factor-1alpha mediates oral squamous cell carcinoma invasion via upregulation of alpha5 integrin and fibronectin. Biochem Biophys Res Commun. 2010;393(1):11\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSimon MJ, Niehoff P, Kimmig B, Wiltfang J, Acil Y. Expression profile and synthesis of different collagen types I, II, III, and V of human gingival fibroblasts, osteoblasts, and SaOS-2 cells after bisphosphonate treatment. Clin Oral Investig. 2010;14(1):51\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAcil Y, Moller B, Niehoff P, Rachko K, Gassling V, Wiltfang J, Simon MJ. The cytotoxic effects of three different bisphosphonates in vitro on human gingival fibroblasts, osteoblasts and osteogenic sarcoma cells. J Craniomaxillofac Surg. 2012;40(8):e229\u0026ndash;235.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJeon C, Oh KC, Park KH, Moon HS. Effects of ultraviolet treatment and alendronate immersion on osteoblast-like cells and human gingival fibroblasts cultured on titanium surfaces. Sci Rep. 2019;9(1):2581.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBasso FG, Cardoso LM, Ribeiro IM, Rizzi E, Pansani TN, Hebling J, de Souza Costa CA. Influence of bisphosphonates on oral implantology: Sodium alendronate and zoledronic acid enhance the synthesis and activity of matrix metalloproteinases by gingival fibroblasts seeded on titanium. Arch Oral Biol. 2021;127:105134.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKruger TB, Herlofson BB, Lian AM, Syversen U, Reseland JE. Alendronate and omeprazole in combination reduce angiogenic and growth signals from osteoblasts. Bone Rep. 2021;14:100750.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTipton DA, Seshul BA, Dabbous M. Effect of bisphosphonates on human gingival fibroblast production of mediators of osteoclastogenesis: RANKL, osteoprotegerin and interleukin-6. J Periodontal Res. 2011;46(1):39\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBasso FG, Pansani TN, Soares DG, Cardoso LM, Hebling J, de Souza Costa CA. Influence of bisphosphonates on the adherence and metabolism of epithelial cells and gingival fibroblasts to titanium surfaces. Clin Oral Investig. 2018;22(2):893\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang HL, Weber D, McCauley LK. Effect of long-term oral bisphosphonates on implant wound healing: literature review and a case report. J Periodontol. 2007;78(3):584\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hypoxia, Fibronectin, Cytotoxicity, Bisphosphonates, Alendronate, Wound healing","lastPublishedDoi":"10.21203/rs.3.rs-7338302/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7338302/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThis study aimed to evaluate how oxygen tension influences the effects of three bisphosphonates\u0026mdash;Alendronate (ALN), Zoledronate (ZA), and Ibandronate (IB)\u0026mdash;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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eHGnFs 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHypoxia significantly reduced viability and migration in all bisphosphonate-treated groups, with ALN showing the most pronounced cytotoxicity. Under hypoxia, ALN at 50 \u0026micro;M 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eDespite 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.\u003c/p\u003e","manuscriptTitle":"Hypoxia Aggravates Alendronate-Induced Cytotoxicity and Extracellular Matrix Disruption in Gingival Fibroblasts: A Comparative In Vitro Study of Three Bisphosphonates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 09:56:35","doi":"10.21203/rs.3.rs-7338302/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30eccd40-b906-4a46-9ca6-25451d1cec73","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-08T14:37:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 09:56:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7338302","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7338302","identity":"rs-7338302","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}