Skip to main content

Advertisement

SpringerLink
Log in

Zoledronic Acid Deteriorates Soft and Hard Tissue Healing of Murine Tooth Extraction Sockets in a Dose-Dependent Manner

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

The pathophysiology, histopathology, and immunopathology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) Stage 0 remain unclear. The aim of this study was to investigate the effects of high-dose bisphosphonates on tooth extraction socket healing by creating a murine model of BRONJ Stage 0-like lesions using 8-week-old female C57BL/6J mice. Zoledronic acid (Zol) was administered subcutaneously twice a week for 7 weeks at doses of 0.1 mg/kg/week (moderate dose; Zol-M), 0.5 mg/kg/week (high dose; Zol-H1), and 1.0 mg/kg/week (higher dose; Zol-H2). Saline was used as a control (VC). Both maxillary first molars were extracted 3 weeks after drug treatment. Maxillae, long bones, and sera were collected 4 weeks post-extraction (n = 7 mice/group). Microcomputed tomography, histological, immunohistochemical, and ELISA analyses were performed. A ceiling effect for Zol was noted at the Zol-H1 dose. Osseous healing of extraction sites was significantly impaired with increased necrotic bone and the number of empty lacunae in a Zol dose-dependent manner. Zol significantly decreased epithelial thickness, due to a decrease in thickness of the stratum spinosum, in both Zol-H1 and Zol-H2. Both Zol-H1 and Zol-H2 significantly suppressed the distribution of F4/80+ macrophages in the connective tissue of tooth extraction sockets, although gross healing appeared to be normal. Intriguingly, both Zol-H1 and Zol-H2 significantly increased the numbers of TRAP+ mononuclear cells and detached osteoclasts in the connective tissue and bone marrow of extraction sites compared to VC and Zol-M, correlated with serum TRAcP5b levels. The created murine model of BRONJ Stage 0-like lesions becoming more severe in a dose-dependent manner may help to understand the pathophysiology and histopathology of BRONJ Stage 0 in humans.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 68:394–424

    Article  Google Scholar 

  2. Cooper C, Campion G, Melton LJ (1992) Hip fractures in the elderly: a world-wide projection. Osteoporos Int 2:285–289

    CAS  PubMed  Google Scholar 

  3. Reginster JY, Burlet N (2006) Osteoporosis: a still increasing prevalence. Bone 38:S4-9

    PubMed  Google Scholar 

  4. Baron R, Ferrari S, Russell RG (2011) Denosumab and bisphosphonates: different mechanisms of action and effects. Bone 48:677–692

    CAS  PubMed  Google Scholar 

  5. Marx RE (2003) Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic. J Oral Maxillofac Surg 61:1115–1117

    PubMed  Google Scholar 

  6. Aghaloo TL, Felsenfeld AL, Tetradis S (2010) Osteonecrosis of the jaw in a patient on Denosumab. J Oral Maxillofac Surg 68:959–963

    PubMed  PubMed Central  Google Scholar 

  7. Taylor KH, Middlefell LS, Mizen KD (2010) Osteonecrosis of the jaws induced by anti-RANK ligand therapy. Br J Oral Maxillofac Surg 48:221–223

    CAS  PubMed  Google Scholar 

  8. Khan AA, Morrison A, Hanley DA, Felsenberg D, McCauley LK, O’Ryan F, Reid IR, Ruggiero SL, Taguchi A, Tetradis S, Watts NB, Brandi ML, Peters E, Guise T, Eastell R, Cheung AM, Morin SN, Masri B, Cooper C, Morgan SL, Obermayer-Pietsch B, Langdahl BL, Al Dabagh R, Davison KS, Kendler DL, Sándor GK, Josse RG, Bhandari M, El Rabbany M, Pierroz DD, Sulimani R, Saunders DP, Brown JP, Compston J, International Task Force on Osteonecrosis of the Jaw (2015) Diagnosis and management of osteonecrosis of the jaw: a systematic review and international consensus. J Bone Miner Res 30:3–23

    PubMed  Google Scholar 

  9. Ruggiero SL, Dodson TB, Fantasia J, Goodday R, Aghaloo T, Mehrotra B, O’Ryan F, American Association of Oral and Maxillofacial Surgeons (2014) American Association of Oral and Maxillofacial Surgeons position paper on medication-related osteonecrosis of the jaw–2014 update. J Oral Maxillofac Surg 72:1938–1956

    PubMed  Google Scholar 

  10. Chang J, Hakam AE, McCauley LK (2018) Current understanding of the pathophysiology of osteonecrosis of the jaw. Curr Osteoporos Rep 16:584–595

    CAS  PubMed  Google Scholar 

  11. Advisory Task Force on Bisphosphonate-Related Ostenonecrosis of the Jaws, American Association of Oral and Maxillofacial Surgeons (2007) American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws. J Oral Maxillofac Surg 65:369–376

    Google Scholar 

  12. Svejda B, Muschitz C, Gruber R, Brandtner C, Svejda C, Gasser RW, Santler G, Dimai HP (2016) Position paper on medication-related osteonecrosis of the jaw (MRONJ). Wien Med Wochenschr 166:68–74

    CAS  PubMed  Google Scholar 

  13. Kim KM, Rhee Y, Kwon YD, Kwon TG, Lee JK, Kim DY (2015) Medication related osteonecrosis of the jaw: 2015 position statement of the Korean society for bone and mineral research and the Korean Association of Oral and Maxillofacial Surgeons. J Bone Metab 22:151–165

    PubMed  PubMed Central  Google Scholar 

  14. Yoneda T, Hagino H, Sugimoto T, Ohta H, Takahashi S, Soen S, Taguchi A, Nagata T, Urade M, Shibahara T, Toyosawa S, Japanese Allied Committee on Osteonecrosis of the Jaw (2017) Antiresorptive agent-related osteonecrosis of the jaw: position paper 2017 of the Japanese Allied Committee on Osteonecrosis of the Jaw. J Bone Miner Metab 35:6–19

    CAS  PubMed  Google Scholar 

  15. Fedele S, Porter SR, D’Aiuto F, Aljohani S, Vescovi P, Manfredi M, Arduino PG, Broccoletti R, Musciotto A, Di Fede O, Lazarovici TS, Campisi G, Yarom N (2010) Nonexposed variant of bisphosphonate-associated osteonecrosis of the jaw: a case series. Am J Med 123:1060–1064

    CAS  PubMed  Google Scholar 

  16. Kuroshima S, Sasaki M, Nakajima K, Tamaki S, Hayano H, Sawase T (2018) Prevalence of bisphosphonate-related osteonecrosis of the jaw-like lesions is increased in a chemotherapeutic dose-dependent manner in mice. Bone 112:177–186

    CAS  PubMed  Google Scholar 

  17. Akita Y, Kuroshima S, Nakajima K, Hayano H, Kanai R, Sasaki M, Sawase T (2018) Effect of anti-angiogenesis induced by chemotherapeutic monotherapy, chemotherapeutic/bisphosphonate combination therapy and anti-VEGFA mAb therapy on tooth extraction socket healing in mice. J Bone Miner Metab 36:547–559

    CAS  PubMed  Google Scholar 

  18. Hayano H, Kuroshima S, Sasaki M, Tamaki S, Inoue M, Ishisaki A, Sawase T (2020) Distinct immunopathology in the early stages between different antiresorptives-related osteonecrosis of the jaw-like lesions in mice. Bone 135:115308

    CAS  PubMed  Google Scholar 

  19. Stavropoulos A, Bertl K, Pietschmann P, Pandis N, Schiødt M, Klinge B (2018) The effect of antiresorptive drugs on implant therapy: systematic review and meta-analysis. Clin Oral Implants Res 29(Suppl 18):54–92

    PubMed  Google Scholar 

  20. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Müller R (2010) Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25:1468–1486

    PubMed  Google Scholar 

  21. Tamaki S, Kuroshima S, Hayano H, Nakajima K, Kakehashi H, Ishisaki A, Sawase T (2020) Dynamic polarization shifting from M1 to M2 macrophages in reduced osteonecrosis of the jaw-like lesions by cessation of anti-RANKL antibody in mice. Bone 141:115560

    CAS  PubMed  Google Scholar 

  22. Kuroshima S, Sasaki M, Nakajima K, Tamaki S, Hayano H, Sawase T (2018) Transplantation of noncultured stromal vascular fraction cells of adipose tissue ameliorates osteonecrosis of the jaw-like lesions in mice. J Bone Miner Res 33:154–166

    CAS  PubMed  Google Scholar 

  23. Yamashita J, Koi K, Yang DY, McCauley LK (2011) Effect of zoledronate on oral wound healing in rats. Clin Cancer Res 17:1405–1414

    CAS  PubMed  Google Scholar 

  24. Kuroshima S, Go VA, Yamashita J (2012) Increased numbers of nonattached osteoclasts after long-term zoledronic acid therapy in mice. Endocrinology 153:17–28

    CAS  PubMed  Google Scholar 

  25. Ibrahim A, Scher N, Williams G, Sridhara R, Li N, Chen G, Leighton J, Booth B, Gobburu JV, Rahman A, Hsieh Y, Wood R, Vause D, Pazdur R (2003) Approval summary for zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin Cancer Res 9:2394–2399

    CAS  PubMed  Google Scholar 

  26. Daubiné F, Le Gall C, Gasser J, Green J, Clézardin P (2007) Antitumor effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis. J Natl Cancer Inst 99:322–330

    PubMed  Google Scholar 

  27. Katsumi H, Liu S, Tanaka Y, Hitomi K, Hayashi R, Hirai Y, Kusamori K, Quan YS, Kamiyama F, Sakane T, Yamamoto A (2012) Development of a novel self-dissolving microneedle array of alendronate, a nitrogen-containing bisphosphonate: evaluation of transdermal absorption, safety, and pharmacological effects after application in rats. J Pharm Sci 101:3230–3238

    CAS  PubMed  Google Scholar 

  28. Hung TT, Chan J, Russell PJ, Power CA (2011) Zoledronic acid preserves bone structure and increases survival but does not limit tumour incidence in a prostate cancer bone metastasis model. PLoS One 6:e19389

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kuroshima S, Yamashita J (2013) Chemotherapeutic and antiresorptive combination therapy suppressed lymphangiogenesis and induced osteonecrosis of the jaw-like lesions in mice. Bone 56:101–109

    CAS  PubMed  Google Scholar 

  30. Kobayashi Y, Hiraga T, Ueda A, Wang L, Matsumoto-Nakano M, Hata K, Yatani H, Yoneda T (2010) Zoledronic acid delays wound healing of the tooth extraction socket, inhibits oral epithelial cell migration, and promotes proliferation and adhesion to hydroxyapatite of oral bacteria, without causing osteonecrosis of the jaw, in mice. J Bone Miner Metab 28:165–175

    CAS  PubMed  Google Scholar 

  31. Kikuiri T, Kim I, Yamaza T, Akiyama K, Zhang Q, Li Y, Chen C, Chen W, Wang S, Le AD, Shi S (2010) Cell-based immunotherapy with mesenchymal stem cells cures bisphosphonate-related osteonecrosis of the jaw-like disease in mice. J Bone Miner Res 25:1668–1679

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kang B, Cheong S, Chaichanasakul T, Bezouglaia O, Atti E, Dry SM, Pirih FQ, Aghaloo TL, Tetradis S (2013) Periapical disease and bisphosphonates induce osteonecrosis of the jaws in mice. J Bone Miner Res 28:1631–1640

    CAS  PubMed  Google Scholar 

  33. Zhao Y, Wang L, Liu Y, Akiyama K, Chen C, Atsuta I, Zhou T, Duan X, Jin Y, Shi S (2012) Technetium-99 conjugated with methylene diphosphonate ameliorates ovariectomy-induced osteoporotic phenotype without causing osteonecrosis in the jaw. Calcif Tissue Int 91:400–408

    CAS  PubMed  Google Scholar 

  34. Yarom N, Shapiro CL, Peterson DE, Van Poznak CH, Bohlke K, Ruggiero SL, Migliorati CA, Khan A, Morrison A, Anderson H, Murphy BA, Alston-Johnson D, Mendes RA, Beadle BM, Jensen SB, Saunders DP (2019) Medication-related osteonecrosis of the Jaw: MASCC/ISOO/ASCO clinical practice guideline. J Clin Oncol 37:2270–2290

    PubMed  Google Scholar 

  35. Ravosa MJ, Ning J, Liu Y, Stack MS (2011) Bisphosphonate effects on the behaviour of oral epithelial cells and oral fibroblasts. Arch Oral Biol 56:491–498

    CAS  PubMed  Google Scholar 

  36. Bullock G, Miller C, McKechnie A, Hearnden V (2020) Synthetic hydroxyapatite inhibits bisphosphonate toxicity to the oral mucosa in vitro. Mater (Basel) 13:2086

    CAS  Google Scholar 

  37. Tseng HC, Kanayama K, Kaur K, Park SH, Park S, Kozlowska A, Sun S, McKenna CE, Nishimura I, Jewett A (2015) Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation. Oncotarget 6:20002–20025

    PubMed  PubMed Central  Google Scholar 

  38. Park S, Kanayama K, Kaur K, Tseng HC, Banankhah S, Quje DT, Sayre JW, Jewett A, Nishimura I (2015) Osteonecrosis of the jaw developed in mice: disease variants regulated By γδ T cells in oral mucosal barrier immunitY. J Biol Chem 290:17349–17366

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gkouveris I, Hadaya D, Soundia A, Bezouglaia O, Chau Y, Dry SM, Pirih FQ, Aghaloo TL, Tetradis S (2019) Vasculature submucosal changes at early stages of osteonecrosis of the jaw (ONJ). Bone 123:234–245

    PubMed  PubMed Central  Google Scholar 

  40. Pozzi S, Vallet S, Mukherjee S, Cirstea D, Vaghela N, Santo L, Rosen E, Ikeda H, Okawa Y, Kiziltepe T, Schoonmaker J, Xie W, Hideshima T, Weller E, Bouxsein ML, Munshi NC, Anderson KC, Raje N (2009) High-dose zoledronic acid impacts bone remodeling with effects on osteoblastic lineage and bone mechanical properties. Clin Cancer Res 15:5829–5839

    CAS  PubMed  Google Scholar 

  41. Vieira AE, Repeke CE, Ferreira Junior SEB, Colavite PM, Biguetti CC, Oliveira RC, Assis GF, Taga R, Trombone AP, Garlet GP (2015) Intramembranous bone healing process subsequent to tooth extraction in mice: micro-computed tomography, histomorphometric and molecular characterization. PLoS One 10:e0128021

    PubMed  PubMed Central  Google Scholar 

  42. Soundia A, Hadaya D, Mallya SM, Aghaloo TL, Tetradis S (2018) Radiographic predictors of bone exposure in patients with stage 0 medication-related osteonecrosis of the jaws. Oral Surg Oral Med Oral Pathol Oral Radiol 126:537–544

    PubMed  PubMed Central  Google Scholar 

  43. Gross C, Weber M, Creutzburg K, Möbius P, Preidl R, Amann K, Wehrhan F (2017) Osteoclast profile of medication-related osteonecrosis of the jaw secondary to bisphosphonate therapy: a comparison with osteoradionecrosis and osteomyelitis. J Transl Med 15:128

    PubMed  PubMed Central  Google Scholar 

  44. Weinstein RS, Roberson PK, Manolagas SC (2009) Giant osteoclast formation and long-term oral bisphosphonate therapy. N Engl J Med 360:53–62

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Jain N, Weinstein RS (2009) Giant osteoclasts after long-term bisphosphonate therapy: diagnostic challenges. Nat Rev Rheumatol 5:341–346

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Mac-Way F, Trombetti A, Noel C, Lafage-Proust MH (2014) Giant osteoclasts in patients under bisphosphonates. BMC Clin Pathol 14:31

    PubMed  PubMed Central  Google Scholar 

  47. Biguetti CC, De Oliva AH, Healy K, Mahmoud RH, Custódio IDC, Constantino DH, Ervolino E, Duarte MAH, Fakhouri WD, Matsumoto MA (2019) Medication-related osteonecrosis of the jaws after tooth extraction in senescent female mice treated with zoledronic acid: microtomographic, histological and immunohistochemical characterization. PLoS One 14:e0214173

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Matsumoto MA, de Abreu Furquim EM, Gonçalves A, Santiago-Júnior JF, Saraiva PP, Cardoso CL, Munerato MS, Okamoto R (2017) Aged rats under zoledronic acid therapy and oral surgery. J Craniomaxillofac Surg 45:781–787

    PubMed  Google Scholar 

  49. Alakangas A, Selander K, Mulari M, Halleen J, Lehenkari P, Mönkkönen J, Salo J, Väänänen K (2002) Alendronate disturbs vesicular trafficking in osteoclasts. Calcif Tissue Int 70:40–47

    CAS  PubMed  Google Scholar 

  50. Mori Y, Kasai H, Ose A, Serada M, Ishiguro M, Shiraki M, Tanigawara Y (2018) Modeling and simulation of bone mineral density in Japanese osteoporosis patients treated with zoledronic acid using tartrate-resistant acid phosphatase 5b, a bone resorption marker. Osteoporos Int 29:1155–1163

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kim JW, Cha IH, Kim SJ, Kim MR (2016) Biomarkers for bisphosphonate-related osteonecrosis of the jaw. Clin Implant Dent Relat Res 18:281–291

    PubMed  Google Scholar 

  52. Bagan L, Jiménez Y, Leopoldo M, Rubert A, Bagan J (2017) Serum levels of RANKL and OPG, and the RANKL/OPG ratio in bisphosphonate-related osteonecrosis of the jaw: are they useful biomarkers for the advanced stages of osteonecrosis? Med Oral Patol Oral Cir Bucal 22:e542–e547

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Tsuboi H, Matsui Y, Hayashida K, Yamane S, Maeda-Tanimura M, Nampei A, Hashimoto J, Suzuki R, Yoshikawa H, Ochi T (2003) Tartrate resistant acid phosphatase (TRAP) positive cells in rheumatoid synovium may induce the destruction of articular cartilage. Ann Rheum Dis 62:196–203

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Thumbigere-Math V, Michalowicz BS, de Jong EP, Griffin TJ, Basi DL, Hughes PJ, Tsai ML, Swenson KK, Rockwell L, Gopalakrishnan R (2015) Salivary proteomics in bisphosphonate-related osteonecrosis of the jaw. Oral Dis 21:46–56

    CAS  PubMed  Google Scholar 

  55. Basi DL, Hughes PJ, Thumbigere-Math V, Sabino M, Mariash A, Lunos SA, Jensen E, Gopalakrishnan R (2011) Matrix metalloproteinase-9 expression in alveolar extraction sockets of Zoledronic acid-treated rats. J Oral Maxillofac Surg 69:2698–2707

    PubMed  Google Scholar 

  56. Manzano-Moreno FJ, Ramos-Torrecillas J, De Luna-Bertos E, Ruiz C, García-Martínez O (2015) High doses of bisphosphonates reduce osteoblast-like cell proliferation by arresting the cell cycle and inducing apoptosis. J Craniomaxillofac Surg 43:396–401

    PubMed  Google Scholar 

  57. Manzano-Moreno FJ, Ramos-Torrecillas J, De Luna-Bertos E, Reyes-Botella C, Ruiz C, García-Martínez O (2015) Nitrogen-containing bisphosphonates modulate the antigenic profile and inhibit the maturation and biomineralization potential of osteoblast-like cells. Clin Oral Investig 19:895–902

    PubMed  Google Scholar 

  58. Koch FP, Merkel C, Al-Nawas B, Smeets R, Ziebart T, Walter C, Wagner W (2011) Zoledronate, ibandronate and clodronate enhance osteoblast differentiation in a dose dependent manner–a quantitative in vitro gene expression analysis of Dlx5, Runx2, OCN, MSX1 and MSX2. J Craniomaxillofac Surg 39:562–569

    PubMed  Google Scholar 

Download references

Acknowledgements

Authors like to thank Associate Prof. Dr. Y Shibata (Nagasaki University) for supporting this study.

Funding

This work was supported by grant support from JSPS KAKENHI (Grant Nos. 18H02994 and 16H05534). All authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Author information

Author notes

  1. Ryohei Kozutsumi and Shinichiro Kuroshima equally contributed to this work.

Authors and Affiliations

  1. Department of Applied Prosthodontics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 852-8588, Japan

    Ryohei Kozutsumi, Shinichiro Kuroshima, Haruka Kaneko, Muneteru Sasaki & Takashi Sawase

  2. Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Shiwa-gun, Iwate, 028-3694, Japan

    Akira Ishisaki

Authors
  1. Ryohei Kozutsumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shinichiro Kuroshima
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haruka Kaneko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Muneteru Sasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Akira Ishisaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Takashi Sawase
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SK designed the research; RK, SK, HK, MS, AI and TS performed the research; RK and SK analyzed the data; RK and SK wrote the paper; RK, SK, IA and TS contributed to the interpretation of the results.

Corresponding author

Correspondence to Shinichiro Kuroshima.

Ethics declarations

Conflict of interest

R.K., S.K., H.K., M.S., A.I. and T.S declare that they have no conflicts of interest.

Ethical Approval

All animal studies were performed in accordance with protocols approved by the Institutional Committee on Animal Resources.

Human and Animal Rights and Informed Consent

Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University, with approval from the Ethics Committee for Animal Research.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kozutsumi, R., Kuroshima, S., Kaneko, H. et al. Zoledronic Acid Deteriorates Soft and Hard Tissue Healing of Murine Tooth Extraction Sockets in a Dose-Dependent Manner. Calcif Tissue Int 110, 104–116 (2022). https://doi.org/10.1007/s00223-021-00890-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-021-00890-9

Keywords

Search

Navigation