Skip to main content
SpringerLink
Log in

Adipose mesenchymal stem cells-derived exosomes alleviate osteoarthritis by transporting microRNA -376c-3p and targeting the WNT-beta-catenin signaling axis

  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Osteoarthritis (OA), one of the major diseases afflicting the elderly, is a type of degenerative joint disease related to cartilage and synovium. This study aimed to clarify the role and mechanism of adipose mesenchymal stem cell (ADSC)-derived exosomes (Exos) in OA-induced chondrocyte degradation and synovial hyperplasia, thus improving the quality of life of patients. The rat OA model, chondrocytes, synovial fibroblast models and immunofluorescence were applied to observe the in vivo and in vitro functions of human ADSC (hADSC)-derived Exos in OA and its possible regulatory signaling pathways. Bioinformatics software and luciferase reporter assay were carried out to verify the mechanism of microRNA-376c-3p (miR-376c-3p) in hADSC-derived Exos in OA in vitro. Moreover, Safranine O-Fast Green Cartilage staining, Masson staining, immunohistochemistry and immunofluorescence were conducted to verify the role of miR-376c-3p in hADSC-derived Exos in OA in vivo. hADSC-derived Exos mitigated OA-induced chondrocyte degradation and synovial fibrosis both in vivo and in vitro models by repressing the WNT-beta-catenin signaling pathway. For the mechanism exploration in vitro, miR-376c-3p was raised in hADSC-derived Exos and mediated the fibrosis of synovial fibroblasts in OA, and miR-376c-3p targeted the 3’-untranslated region of WNT3 or WNT9a. Meanwhile, the in vivo experiments also corroborated that the miR-376c-3p in hADSC-derived Exos mitigated OA-induced chondrocyte degradation and synovial fibrosis. MiR-376c-3p in hADSC-derived Exos repressed the WNT-beta-catenin pathway by targeting WNT3 or WNT9a, and then mitigating OA-induced chondrocyte degradation and synovial fibrosis, thereby providing theoretical basis for clinical implementation of treatment.

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
Fig. 7

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

OA:

Osteoarthritis

ADSC:

Adipose mesenchymal stem cell

Exos:

Exosomes

hADSC:

Human ADSC

ECM:

Extracellular matrix

MSCs:

Mesenchymal stem cells

MIA:

Monosodium iodoacetate

PBS:

Phosphate buffer saline

LPS:

Lipopolysaccharide

IHC:

Immunohistochemistry

EdU:

5-Ethynyl-2-deoxyuridine

HPF:

High-power field

CT:

Cycle threshold

SDS-PAGE:

Sulfate-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride

3′UTR:

3′-Untranslated region

miRNAs:

MicroRNAs

SO:

Safranine O-fast green cartilage

NC:

Negative control

References

  1. Chen D, Shen J, Zhao W, Wang T, Han L, Hamilton JL, Im HJ (2017) Osteoarthritis: toward a comprehensive understanding of pathological mechanism. Bone Res 5:16044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Loeser RF, Goldring SR, Scanzello CR, Goldring MB (2012) Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 64(6):1697–1707

    Article  PubMed  PubMed Central  Google Scholar 

  3. Reesink HL, Sutton RM, Shurer CR, Peterson RP, Tan JS, Su J, Paszek MJ, Nixon AJ (2017) Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility. Stem Cell Res Ther 8(1):243

    Article  PubMed  PubMed Central  Google Scholar 

  4. Steenvoorden MM, Bank RA, Ronday HK, Toes RE, Huizinga TW, DeGroot J (2007) Fibroblast-like synoviocyte-chondrocyte interaction in cartilage degradation. Clin Exp Rheumatol 25(2):239–245

    CAS  PubMed  Google Scholar 

  5. Fosang AJ, Rogerson FM, East CJ, Stanton H (2008) ADAMTS-5: the story so far. Eur Cell Mater 15:11–26

    Article  CAS  PubMed  Google Scholar 

  6. Lee AS, Ellman MB, Yan D, Kroin JS, Cole BJ, van Wijnen AJ, Im HJ (2013) A current review of molecular mechanisms regarding osteoarthritis and pain. Gene 527(2):440–447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Remst DF, Blaney Davidson EN, van der Kraan PM (2015) Unravelling osteoarthritis-related synovial fibrosis: a step closer to solving joint stiffness. Rheumatol (Oxford) 54(11):1954–1963

    Article  CAS  Google Scholar 

  8. Chang X, Shen J, Yang H, Xu Y, Gao W, Wang J, Zhang H, He S (2016) Upregulated expression of CCR3 in osteoarthritis and CCR3 mediated activation of fibroblast-like synoviocytes. Cytokine 77:211–219

    Article  PubMed  Google Scholar 

  9. Gupta A, Niger C, Buo AM, Eidelman ER, Chen RJ, Stains JP (2014) Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture. BMC Musculoskelet Disord 15:425

    Article  PubMed  PubMed Central  Google Scholar 

  10. Johnson K, Zhu S, Tremblay MS, Payette JN, Wang J, Bouchez LC, Meeusen S, Althage A, Cho CY, Wu X, Schultz PG (2012) A stem cell-based approach to cartilage repair. Science 336(6082):717–721

    Article  CAS  PubMed  Google Scholar 

  11. Vinatier C, Guicheux J (2016) Cartilage tissue engineering: from biomaterials and stem cells to osteoarthritis treatments. Ann Phys Rehabil Med 59(3):139–144

    Article  CAS  PubMed  Google Scholar 

  12. Mamidi MK, Das AK, Zakaria Z, Bhonde R (2016) Mesenchymal stromal cells for cartilage repair in osteoarthritis. Osteoarthr Cartil 24(8):1307–1316

    Article  CAS  Google Scholar 

  13. ter Huurne M, Schelbergen R, Blattes R, Blom A, de Munter W, Grevers LC, Jeanson J, Noël D, Casteilla L, Jorgensen C, van den Berg W, van Lent PL (2012) Antiinflammatory and chondroprotective effects of intraarticular injection of adipose-derived stem cells in experimental osteoarthritis. Arthritis Rheum 64(11):3604–3613

    Article  PubMed  Google Scholar 

  14. Zhang R, Ma J, Han J, Zhang W, Ma J (2019) Mesenchymal stem cell related therapies for cartilage lesions and osteoarthritis. Am J Trans Res 11(10):6275–6289

    CAS  Google Scholar 

  15. Herberts CA, Kwa MS, Hermsen HP (2011) Risk factors in the development of stem cell therapy. J Transl Med 9:29

    Article  PubMed  PubMed Central  Google Scholar 

  16. Kourembanas S (2015) Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol 77:13–27

    Article  CAS  PubMed  Google Scholar 

  17. Théry C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9(8):581–593

    Article  PubMed  Google Scholar 

  18. Chang YH, Wu KC, Harn HJ, Lin SZ, Ding DC (2018) Exosomes and stem cells in degenerative disease diagnosis and therapy. Cell Transplant 27(3):349–363

    Article  PubMed  PubMed Central  Google Scholar 

  19. Xin H, Li Y, Chopp M (2014) Exosomes/miRNAs as mediating cell-based therapy of stroke. Front Cell Neurosci 8:377

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhu Y, Wang Y, Zhao B, Niu X, Hu B, Li Q, Zhang J, Ding J, Chen Y, Wang Y (2017) Comparison of exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells and synovial membrane-derived mesenchymal stem cells for the treatment of osteoarthritis. Stem Cell Res Ther 8(1):64

    Article  PubMed  PubMed Central  Google Scholar 

  21. Hong P, Yang H, Wu Y, Li K, Tang Z (2019) The functions and clinical application potential of exosomes derived from adipose mesenchymal stem cells: a comprehensive review. Stem Cell Res Ther 10(1):242

    Article  PubMed  PubMed Central  Google Scholar 

  22. Guo J, Hu H, Gorecka J, Bai H, He H, Assi R, Isaji T, Wang T, Setia O, Lopes L, Gu Y, Dardik A (2018) Adipose-derived mesenchymal stem cells accelerate diabetic wound healing in a similar fashion as bone marrow-derived cells. Am J Physiol Cell Physiol 315(6):C885-c896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu X, Zhu W, Wang L, Wu J, Ding F, Song Y (2019) miR-145–5p suppresses osteogenic differentiation of adipose-derived stem cells by targeting semaphorin 3A. In vitro cellular and developmental biology. Animal 55(3):189–202

    CAS  Google Scholar 

  24. Deng S, Zhou X, Ge Z, Song Y, Wang H, Liu X, Zhang D (2019) Exosomes from adipose-derived mesenchymal stem cells ameliorate cardiac damage after myocardial infarction by activating S1P/SK1/S1PR1 signaling and promoting macrophage M2 polarization. Int J Biochem Cell Biol 114:105564

    Article  CAS  PubMed  Google Scholar 

  25. Philpott HT, McDougall JJ (2020) Combatting joint pain and inflammation by dual inhibition of monoacylglycerol lipase and cyclooxygenase-2 in a rat model of osteoarthritis. Arthritis Res Ther 22(1):9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. He L, He T, Xing J, Zhou Q, Fan L, Liu C, Chen Y, Wu D, Tian Z, Liu B, Rong L (2020) Bone marrow mesenchymal stem cell-derived exosomes protect cartilage damage and relieve knee osteoarthritis pain in a rat model of osteoarthritis. Stem Cell Res Ther 11(1):276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ko JY, Lee MS, Lian WS, Weng WT, Sun YC, Chen YS, Wang FS (2017) MicroRNA-29a counteracts synovitis in knee osteoarthritis pathogenesis by targeting VEGF. Sci Rep 7(1):3584

    Article  PubMed  PubMed Central  Google Scholar 

  28. Fang S, Xu C, Zhang Y, Xue C, Yang C, Bi H, Qian X, Wu M, Ji K, Zhao Y, Wang Y, Liu H, Xing X (2016) Umbilical cord-derived mesenchymal stem cell-derived exosomal MicroRNAs suppress myofibroblast differentiation by inhibiting the transforming growth factor-β/SMAD2 pathway during wound healing. Stem Cells Transl Med 5(10):1425–1439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chapel DB, Schulte JJ, Husain AN, Krausz T (2020) Application of immunohistochemistry in diagnosis and management of malignant mesothelioma. Trans Lung Cancer Res 9(Suppl 1):S3-s27

    Article  CAS  Google Scholar 

  30. Li L, Li M, Pang Y, Wang J, Wan Y, Zhu C, Yin Z (2019) Abnormal thyroid hormone receptor signaling in osteoarthritic osteoblasts regulates microangiogenesis in subchondral bone. Life Sci 239:116975

    Article  CAS  PubMed  Google Scholar 

  31. Flomerfelt FA, Gress RE (2016) Analysis of cell proliferation and homeostasis using EdU labeling. Methods Mol Biol 1323:211–220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wang L, Yang J, Wang H, Wang W, Liang X (2020) Highly expressed ribosomal protein L34 predicts poor prognosis in acute myeloid leukemia and could be a potential therapy target. Aging Pathobiol Ther 2(1):32–37

    Article  CAS  Google Scholar 

  33. Mao G, Zhang Z, Hu S, Zhang Z, Chang Z, Huang Z, Liao W, Kang Y (2018) Exosomes derived from miR-92a-3p-overexpressing human mesenchymal stem cells enhance chondrogenesis and suppress cartilage degradation via targeting WNT5A. Stem Cell Res Ther 9(1):247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Niu Y, Zhou B, Wan C et al (2020) Down-regulation of miR-181a promotes microglial M1 polarization through increasing expression of NDRG. Aging Pathobiol Ther 2(1):52–57

    Article  CAS  Google Scholar 

  35. Kimura K, Hohjoh H, Fukuoka M, Sato W, Oki S, Tomi C, Yamaguchi H, Kondo T, Takahashi R, Yamamura T (2018) Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis. Nat Commun 9(1):17

    Article  PubMed  PubMed Central  Google Scholar 

  36. Lietman C, Wu B, Lechner S, Shinar A, Sehgal M, Rossomacha E, Datta P, Sharma A, Gandhi R, Kapoor M, Young PP (2018) Inhibition of Wnt/β-catenin signaling ameliorates osteoarthritis in a murine model of experimental osteoarthritis. JCI insight. https://doi.org/10.1172/jci.insight.96308

    Article  PubMed  PubMed Central  Google Scholar 

  37. Johnsen KB, Gudbergsson JM, Skov MN, Pilgaard L, Moos T, Duroux M (2014) A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy. Biochem Biophys Acta 1846(1):75–87

    CAS  PubMed  Google Scholar 

  38. Rani S, Ryan AE, Griffin MD, Ritter T (2015) Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther 23(5):812–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yeo RW, Lai RC, Zhang B, Tan SS, Yin Y, Teh BJ, Lim SK (2013) Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv Drug Deliv Rev 65(3):336–341

    Article  CAS  PubMed  Google Scholar 

  40. Zhou Y, Wang T, Hamilton JL, Chen D (2017) Wnt/β-catenin signaling in osteoarthritis and in other forms of arthritis. Curr Rheumatol Rep 19(9):53

    Article  PubMed  PubMed Central  Google Scholar 

  41. Deshmukh V, O’Green AL, Bossard C, Seo T, Lamangan L, Ibanez M, Ghias A, Lai C, Do L, Cho S, Cahiwat J, Chiu K, Pedraza M, Anderson S, Harris R, Dellamary L, Kc S, Barroga C, Melchior B, Tam B, Kennedy S, Tambiah J, Hood J, Yazici Y (2019) Modulation of the Wnt pathway through inhibition of CLK2 and DYRK1A by lorecivivint as a novel, potentially disease-modifying approach for knee osteoarthritis treatment. Osteoarthr Cartil 27(9):1347–1360

    Article  CAS  Google Scholar 

  42. Yazici Y, McAlindon TE, Fleischmann R, Gibofsky A, Lane NE, Kivitz AJ, Skrepnik N, Armas E, Swearingen CJ, DiFrancesco A, Tambiah JRS, Hood J, Hochberg MC (2017) A novel Wnt pathway inhibitor, SM04690, for the treatment of moderate to severe osteoarthritis of the knee: results of a 24-week, randomized, controlled, phase 1 study. Osteoarthr Cartil 25(10):1598–1606

    Article  CAS  Google Scholar 

  43. Chen L, Heikkinen L, Wang C, Yang Y, Sun H, Wong G (2019) Trends in the development of miRNA bioinformatics tools. Brief Bioinform 20(5):1836–1852

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Correia de Sousa M, Gjorgjieva M, Dolicka D, Sobolewski C, Foti M (2019) Deciphering miRNAs’ action through miRNA editing. Int J Mol Sci 20(24):6249

    Article  PubMed  PubMed Central  Google Scholar 

  45. Mayourian J, Ceholski DK, Gorski PA, Mathiyalagan P, Murphy JF, Salazar SI, Stillitano F, Hare JM, Sahoo S, Hajjar RJ, Costa KD (2018) Exosomal microRNA-21-5p mediates mesenchymal stem cell paracrine effects on human cardiac tissue contractility. Circ Res 122(7):933–944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang R, Xu B, Xu H (2018) TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b. Cell Cycle 17(24):2756–2765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Tao SC, Yuan T, Zhang YL, Yin WJ, Guo SC, Zhang CQ (2017) Exosomes derived from miR-140-5p-overexpressing human synovial mesenchymal stem cells enhance cartilage tissue regeneration and prevent osteoarthritis of the knee in a rat model. Theranostics 7(1):180–195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang K, Jin J, Ma T, Zhai H (2017) MiR-376c-3p regulates the proliferation, invasion, migration, cell cycle and apoptosis of human oral squamous cancer cells by suppressing HOXB7. Biomed Pharmacother 91:517–525

    Article  CAS  PubMed  Google Scholar 

  49. Zhang H, Zhou J, Zhang M, Yi Y, He B (2019) Upregulation of miR-376c-3p alleviates oxygen-glucose deprivation-induced cell injury by targeting ING5. Cell Mol Biol Lett 24:67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This study is supported by grants from the National Natural Science Foundation of China (No. 81802164).

Author information

Authors and Affiliations

  1. Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University, No. 1, Jianshe East Road, Zhengzhou, 450052, Henan, People’s Republic of China

    Feng Li, Zhiming Xu, Zheng Xie, Xing Sun, Chengxiang Li, Yangyang Chen, Jianzhong Xu & Guofu Pi

Authors
  1. Feng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhiming Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zheng Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xing Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chengxiang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yangyang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jianzhong Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guofu Pi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

FL, JX and GP contributed to the conception of the study and wrote the paper; ZX, ZX and XS contributed significantly to analysis and manuscript preparation; CL and YC participated in the design of the study. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Feng Li, Jianzhong Xu or Guofu Pi.

Ethics declarations

Competing interest

All authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Institute Research Medical Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

Consent for publication

Not applicable.

Research involving in human and animal participants

This article contained studies with human adipose tissues for hADSCs isolation, in accordance with all principles of the Declaration of Helsinki, and the written informed consent was obtained from each participant. Rats were used for in vivo OA model establishment, and all animal experiments were conducted according to the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) and the IACUC (Institutional Animal Care and Use Committee) guidelines.

Additional information

Publisher's Note

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

Supplementary Information

Below is the link to the electronic supplementary material.

10495_2022_1787_MOESM1_ESM.jpg

Supplementary file1 (JPG 998 kb)—Identification of hADSC and hADSC-derived Exos. A Identification of hADSCs by flow cytometry. B Detection of the osteogenic and adipogenic differentiation capabilities to identify hADSCs (Scale Bar = 100 μm). C Transmission electron microscope identification of hADSC-derived Exos (Scale Bar = 200 nm) and analysis of the CD63 and TSG101 protein levels. D Flow cytometry to detect endocytosis of hADSC-derived Exos.

10495_2022_1787_MOESM2_ESM.tif

Supplementary file2 (TIF 25532 kb)—The synovial fibroblasts of OA rats were treated with TGF-β1 and/or hADSC-derived Exos. Western blot was carried out to quantify the protein levels of α-SMA, Col III, WNT3, WNT9a and β-catenin.

10495_2022_1787_MOESM3_ESM.jpg

Supplementary file3 (JPG 728 kb)—Analysis of hADSC-derived Exos on the WNT-beta-catenin signaling pathway. A Detection of WNT1, WNT2, WNT8a, WNT8b, WNT10a and WNT10b mRNA levels in IL-1β-induced chondrocytes or synovial fibroblasts. Unpaired two-tailed Student’s t-test, N = 3. B The chondrocytes induced by IL-1β were treated with RNase, respectively. Detection of WNT1 and WNT2 protein levels. *P<0.05, **P<0.01 versus IL-1β.

10495_2022_1787_MOESM4_ESM.jpg

Supplementary file4 (JPG 2044 kb)—A Software available online (TargetScan) predicted miRNAs that targeted WNT3 and WNT9a both in rats and humans. B Detection of miR-19b-3p, miR-532-3p, miR-376c-3p and miR-342-3p in hADSC-derived Exos by qRT-PCR (miR-19a-3p expression was applied as a control group). C IL-1β and hADSC-derived Exos co-processed chondrocytes. LPS and hADSC-derived Exos or TGF-β1 and hADSC-derived Exos co-processed synovial fibroblasts. Fibroblast or chondrocytes were treated with Exos, followed by ActD treatment. DMSO vehicle was used as the control. Detection of miR-376c-3p expression. Unpaired two-tailed Student’s t-test, N=3. D The binding sites between miR-376c-3p and WNT3 or WNT9a's 3′UTR region in rats or humans and the detection of the luciferase activity of WNT3 or WNT9a by luciferase reporter assay. Unpaired two-tailed Student’s t-test, N=3. E Detection of WNT3 and WNT9a mRNA levels in IL-1β-induced chondrocytes combined with 10 ug/ml hADSC-derived Exos and/or transfected with miR-19a-3p inhibitor by qRT-PCR. One-way ANOVA analysis combined with Tukey's post-hoc, N=3. F Western blot detection of MMP13, Collagen II, WNT3, WNT9a and β-catenin in chondrocytes, and α-SMA and Collagen III, WNT3, WNT9a and β-catenin in fibroblasts. *P<0.05, **P<0.01 versus miR-19a-3p, chondrocyte, fibroblast, control, IL-1β. ##P<0.01 versus IL-1β + Exo.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, F., Xu, Z., Xie, Z. et al. Adipose mesenchymal stem cells-derived exosomes alleviate osteoarthritis by transporting microRNA -376c-3p and targeting the WNT-beta-catenin signaling axis. Apoptosis 28, 362–378 (2023). https://doi.org/10.1007/s10495-022-01787-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-022-01787-0

Keywords

Search

Navigation