Früher|Nächster

Die Verbindung von immunmodulatorischen, angiogenen und osteogenen Fähigkeiten in einem piezoelektrischen Hydrogel-Gewebeingenieurgerüst für die Militärmedizin

  • role： Contribute equally, First author同等贡献者, 第一作者
  • Affiliation：

    Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China

    Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325000, Zhejiang, China

Ping Wu12,  
  • role： Contribute equally同等贡献者
  • Affiliation：

    Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China

Lin Shen1,  
  • role： Contribute equally同等贡献者
  • Affiliation：

    Department of Spine Surgery and Musculoskeletal Tumor, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

Hui-Fan Liu3,  
  • role： Contribute equally同等贡献者
  • Affiliation：

    National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

Xiang-Hui Zou4,  
  • Affiliation：

    Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China

Juan Zhao1,  
  • Affiliation：

    Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China

Yu Huang1,  
  • Affiliation：

    Department of Spine Surgery and Musculoskeletal Tumor, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

Yu-Fan Zhu3,  
  • Affiliation：

    Department of Overseas Education College, Jimei University, Xiamen 361021, Fujian, China

Zhao-Yu Li5,  
  • Affiliation：

    National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

Chao Xu4,  
  • Affiliation：

    School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China

Li-Hua Luo6,  
  • Affiliation：

    National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China

Zhi-Qiang Luo4,  
  • role： Corresponding author通信作者
  • Affiliation：

    Department of Spine Surgery and Musculoskeletal Tumor, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

  • Email：Wuwuminhao1991@whu.edu.cn
  • Introduction：*Wuwuminhao1991@whu.edu.cn;
Min-Hao Wu3*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Department of Spine Surgery and Musculoskeletal Tumor, Zhongnan Hospital of Wuhan University, Wuhan 430071, China

  • Email：orthopedics@whu.edu.cn
  • Introduction：orthopedics@whu.edu.cn;
Lin Cai3*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325000, Zhejiang, China

  • Email：xiaokunli@wmu.edu.cn
  • Introduction：xiaokunli@wmu.edu.cn;
Xiao-Kun Li2*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Key Laboratory of Imaging Diagnosis and Minimally Invasive Intervention Research, the Fifth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China

    Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325000, Zhejiang, China

  • Email：wzhouguang@gmail.com
  • Introduction：wzhouguang@gmail.com
Zhou-Guang Wang12*

Resümee

Background:Most bone-related injuries to grassroots troops are caused by training or accidental injuries. To establish preventive measures to reduce all kinds of trauma and improve the combat effectiveness of grassroots troops, it is imperative to develop new strategies and scafolds to promote bone regeneration.Methods:In this study, a porous piezoelectric hydrogel bone scafold was fabricated by incorporating polydopamine (PDA)-modified ceramic hydroxyapatite (PDA-hydroxyapatite, PHA) and PDA-modified barium titanate (PDA-BaTiO3, PBT) nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. The physical and chemical properties of the Cs/Gel/PHA scafold with 0–10 wt% PBT were analyzed. Cell and animal experiments were performed to characterize the immunomodulatory, angiogenic, and osteogenic capabilities of the piezoelectric hydrogel scafold in vitro and in vivo.Results:The incorporation of BaTiO3 into the scafold improved its mechanical properties and increased self-generated electricity. Due to their endogenous piezoelectric stimulation and bioactive constituents, the prepared Cs/Gel/PHA/PBT hydrogels exhibited cytocompatibility as well as immunomodulatory, angiogenic, and osteogenic capabilities; they not only effectively induced macrophage polarization to M2 phenotype but also promoted the migration, tube formation, and angiogenic differentiation of human umbilical vein endothelial cells (HUVECs) and facilitated the migration, osteo-differentiation, and extracellular matrix (ECM) mineralization of MC3T3-E1 cells. The in vivo evaluations showed that these piezoelectric hydrogels with versatile capabilities significantly facilitated new bone formation in a rat large-sized cranial injury model. The underlying molecular mechanism can be partly attributed to the immunomodulation of the Cs/Gel/PHA/PBT hydrogels as shown via transcriptome sequencing analysis, and the PI3K/Akt signaling axis plays an important role in regulating macrophage M2 polarization.Conclusions:The piezoelectric Cs/Gel/PHA/PBT hydrogels developed here with favorable immunomodulation, angiogenesis, and osteogenesis functions may be used as a substitute in periosteum injuries, thereby offering the novel strategy of applying piezoelectric stimulation in bone tissue engineering for the enhancement of combat efectiveness in grassroots troops.

Schlüsselwort

Piezoelectric hydrogel;Tissue engineering scafold;Immunomodulation;angiogenesis;osteogenic differentiation

References

  1. 1..
    Xu PC, Xuan M, Cheng B. Effects and mechanism of platelet-rich plasma on military drill injury: a review. Mil Med Res. 2020;7(1):56.
  2. 2..
    Mathieu L, Mourtialon R, Durand M, de Rousiers A, de L'Escalopier N, Collombet JM. Masquelet technique in military practice: specificities and future directions for combat-related bone defect reconstruction. Mil Med Res. 2022;9(1):48.
  3. 3..
    Arif ZU, Khalid MY, Noroozi R, Sadeghianmaryan A, Jalalvand M, Hossain M. Recent advances in 3D-printed polylactide and polycaprolactonebased biomaterials for tissue engineering applications. Int J Biol Macromol. 2022;218:930-68.
  4. 4..
    Arif ZU, Khalid MY, Ahmed W, Arshad H. A review on four-dimensional (4D) bioprinting in pursuit of advanced tissue engineering applications. Bioprinting. 2022;27:e00203.
  5. 5..
    Zhang J, Tong D, Song H, Ruan R, Sun Y, Lin Y, et al. Osteoimmunity regulating biomimetically hierarchical scaffold for augmented bone regeneration. Adv Mater. 2022;34(36):e2202044.
  6. 6..
    Liu S, Wang YN, Yu L, Li J, Ge S. Development of a thermosensitive hydrogel loaded with DTT and SDF-1 facilitating in situ periodontal bone regeneration. Chem Eng J. 2022;432:134308.
  7. 7..
    Arif ZU, Khalid MY, Sheikh MF, Zolfagharian A, Bodaghi M. Biopolymeric sustainable materials and their emerging applications. J Environ Chem Eng. 2022;10(4):108159.
  8. 8..
    Das R, Curry EJ, Le TT, Awale G, Liu Y, Li S, et al. Biodegradable nanofiber bone-tissue scaffold as remotely-controlled and self-powering electrical stimulator. Nano Energy. 2020;76:105028.
  9. 9..
    Wu H, Dong H, Tang Z, Chen Y, Liu Y, Wang M, et al. Electrical stimulation of piezoelectric BaTiO3 coated Ti6Al4V scaffolds promotes anti-inflammatory polarization of macrophages and bone repair via MAPK/JNK inhibition and OXPHOS activation. Biomaterials. 2023;293:121990.
  10. 10..
    Xiong Y, Mi BB, Lin Z, Hu YQ, Yu L, Zha KK, et al. The role of the immune microenvironment in bone, cartilage, and soft tissue regeneration: from mechanism to therapeutic opportunity. Mil Med Res. 2022;9(1):65.
  11. 11..
    Kong Y, Liu F, Ma B, Duan J, Yuan W, Sang Y, et al. Wireless localized electrical stimulation generated by an ultrasound-driven piezoelectric discharge regulates proinflammatory macrophage polarization. Adv Sci (Weinh). 2021;8(13):2100962.
  12. 12..
    Du Y, Guo JL, Wang J, Mikos AG, Zhang S. Hierarchically designed bone scaffolds: from internal cues to external stimuli. Biomaterials. 2019;218:119334.
  13. 13..
    Tandon B, Blaker JJ, Cartmell SH. Piezoelectric materials as stimulatory biomedical materials and scaffolds for bone repair. Acta Biomater. 2018;73:1-20.
  14. 14..
    Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat Rev Rheumatol. 2015;11(1):45–54.
  15. 15..
    Cafarelli A, Marino A, Vannozzi L, Puigmartí-Luis J, Pané S, Ciofani G, et al. Piezoelectric nanomaterials activated by ultrasound: the pathway from discovery to future clinical adoption. ACS Nano. 2021;15(7):11066–86.
  16. 16..
    Goldstein C, Sprague S, Petrisor BA. Electrical stimulation for fracture healing: current evidence. J Orthop Trauma. 2010;24(Suppl 1):S62–5.
  17. 17..
    Mobini S, Leppik L, Thottakkattumana Parameswaran V, Barker JH. In vitro effect of direct current electrical stimulation on rat mesenchymal stem cells. PeerJ. 2017;5:e2821.
  18. 18..
    Dai X, Heng BC, Bai Y, You F, Sun X, Li Y, et al. Restoration of electrical microenvironment enhances bone regeneration under diabetic conditions by modulating macrophage polarization. Bioact Mater. 2021;6(7):2029–38.
  19. 19..
    Chen P, Xu C, Wu P, Liu K, Chen F, Chen Y, et al. Wirelessly powered electrical-stimulation based on biodegradable 3D piezoelectric scaffolds promotes the spinal cord injury repair. ACS Nano. 2022;16:16513-28.
  20. 20..
    Singh A, Raghav A, Shiekh PA, Kumar A. Transplantation of engineered exosomes derived from bone marrow mesenchymal stromal cells ameliorate diabetic peripheral neuropathy under electrical stimulation. Bioact Mater. 2021;6(8):2231–49.
  21. 21..
    Zhao Y, Liang Y, Ding S, Zhang K, Mao HQ, Yang Y. Application of conductive PPy/SF composite scaffold and electrical stimulation for neural tissue engineering. Biomaterials. 2020;255:120164.
  22. 22..
    Wu P, Tong Z, Luo L, Zhao Y, Chen F, Li Y, et al. Comprehensive strategy of conduit guidance combined with VEGF producing Schwann cells accelerates peripheral nerve repair. Bioact Mater. 2021;6(10):3515–27.
  23. 23..
    Srirussamee K, Mobini S, Cassidy NJ, Cartmell SH. Direct electrical stimulation enhances osteogenesis by inducing Bmp2 and Spp1 expressions from macrophages and preosteoblasts. Biotechnol Bioeng. 2019;116(12):3421–32.
  24. 24..
    Kim IS, Song JK, Zhang YL, Lee TH, Cho TH, Song YM, et al. Biphasic electric current stimulates proliferation and induces VEGF production in osteoblasts. Biochim Biophys Acta. 2006;1763(9):907–16.
  25. 25..
    Chen P, Wu P, Wan X, Wang Q, Xu C, Yang M, et al. Ultrasound-driven electrical stimulation of peripheral nerves based on implantable piezoelectric thin film nanogenerators. Nano Energy. 2021;86:106123.
  26. 26..
    Wu P, Chen P, Xu C, Wang Q, Zhang F, Yang K, et al. Ultrasound-driven in vivo electrical stimulation based on biodegradable piezoelectric nanogenerators for enhancing and monitoring the nerve tissue repair. Nano Energy. 2022;102:107707.
  27. 27..
    Dobashi Y, Yao D, Petel Y, Nguyen TN, Sarwar MS, Thabet Y, et al. Piezoionic mechanoreceptors: force-induced current generation in hydrogels. Science. 2022;376(6592):502–7.
  28. 28..
    Zhu Y, Liang H, Liu X, Wu J, Yang C, Wong TM, et al. Regulation of macrophage polarization through surface topography design to facilitate implant-to-bone osteointegration. Sci Adv. 2021;7(14):eabf6654.
  29. 29..
    Wu M, Zhang Y, Wu P, Chen F, Yang Z, Zhang S, et al. Mussel-inspired multifunctional surface through promoting osteogenesis and inhibiting osteoclastogenesis to facilitate bone regeneration. NPJ Regen Med. 2022;7(1):29.
  30. 30..
    Dong X, Wu P, Yan L, Liu K, Wei W, Cheng Q, et al. Oriented nanofibrous P(MMD-co-LA)/Deferoxamine nerve scaffold facilitates peripheral nerve regeneration by regulating macrophage phenotype and revascularization. Biomaterials. 2022;280:121288.
  31. 31..
    Zhong Z, Wu X, Wang Y, Li M, Li Y, Liu X, et al. Zn/Sr dual ions-collagen co-assembly hydroxyapatite enhances bone regeneration through procedural osteo-immunomodulation and osteogenesis. Bioact Mater. 2022;10:195-206.
  32. 32..
    Wang Z, Liu Z, Zhao G, Zhang Z, Zhao X, Wan X, et al. Stretchable unsymmetrical piezoelectric BaTiO3 composite hydrogel for triboelectric nanogenerators and multimodal sensors. ACS Nano. 2022;16(1):1661–70.
  33. 33..
    Liu H, Shi Y, Zhu Y, Wu P, Deng Z, Dong Q, et al. Bioinspired piezoelectric periosteum to augment bone regeneration via synergistic immunomodulation and osteogenesis. ACS Appl Mater Interfaces. 2023;15(9):12273–93.
  34. 34..
    Ji X, Yuan X, Ma L, Bi B, Zhu H, Lei Z, et al. Mesenchymal stem cell-loaded thermosensitive hydroxypropyl chitin hydrogel combined with a three dimensional-printed poly(ε-caprolactone) /nano-hydroxyapatite scaffold to repair bone defects via osteogenesis, angiogenesis and immunomodulation. Theranostics. 2020;10(2):725–40.
  35. 35..
    Qiu P, Li M, Chen K, Fang B, Chen P, Tang Z, et al. Periosteal matrix-derived hydrogel promotes bone repair through an early immune regulation coupled with enhanced angio- and osteogenesis. Biomaterials. 2020;227:119552.
  36. 36..
    Zheng ZW, Chen YH, Wu DY, Wang JB, Lv MM, Wang XS, et al. Development of an accurate and proactive immunomodulatory strategy to improve bone substitute material-mediated osteogenesis and angiogenesis. Theranostics. 2018;8(19):5482–500.
  37. 37..
    Wu M, Wang Y, Liu H, Chen F, Zhang Y, Wu P, et al. Engineering musselinspired multifunctional nanocomposite hydrogels to orchestrate osteoimmune microenvironment and promote bone healing. Mater Design. 2023;227:111705.
  38. 38..
    Liu H, Chen F, Zhang Y, Wu P, Yang Z, Zhang S, et al. Facile fabrication of biomimetic silicified gelatin scaffolds for angiogenesis and bone regeneration by a bioinspired polymer-induced liquid precursor. Mater Design. 2022;222:111070.
  39. 39..
    Wu M, Chen F, Liu H, Wu P, Yang Z, Zhang Z, et al. Bioinspired sandwich-like hybrid surface functionalized scaffold capable of regulating osteogenesis, angiogenesis, and osteoclastogenesis for robust bone regeneration. Mater Today Bio. 2022;17:100458.
  40. 40..
    Lu G, Xu Y, Liu Q, Chen M, Sun H, Wang P, et al. An instantly fixable and self-adaptive scaffold for skull regeneration by autologous stem cell recruitment and angiogenesis. Nat Commun. 2022;13(1):2499.
  41. 41..
    Duan Y, Zheng H, Li Z, Yao Y, Ding J, Wang X, et al. Unsaturated polyurethane films grafted with enantiomeric polylysine promotes macrophage polarization to a M2 phenotype through PI3K/Akt1/mTOR axis. Biomaterials. 2020;246:120012.
  42. 42..
    Xiang L, Cui W. Biomedical application of photo-crosslinked gelatin hydrogels. J Leather Sci Eng. 2021;3(1):3.
  43. 43..
    Duan WL, Zhang LN, Bohara R, Martin-Saldaña S, Yang F, Zhao YY, et al. Adhesive hydrogels in osteoarthritis: from design to application. Mil Med Res. 2023;10(1):4.
  44. 44..
    Qi L, Mu L, Guo X, Liu A, Chen C, Ye Q, et al. Fast expandable chitosanfibers cryogel from ambient drying for noncompressible bleeding control and in situ tissue regeneration. Adv Funct Mater. 2023;33(16):2212231.
  45. 45..
    Nieto-Suárez M, López-Quintela MA, Lazzari M. Preparation and characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-assembly. Carbohydr Polym. 2016;141:175-83.
  46. 46..
    Wei S, Ma JX, Xu L, Gu XS, Ma XL. Biodegradable materials for bone defect repair. Mil Med Res. 2020;7(1):54.
  47. 47..
    Prokhorov E, Bárcenas GL, Sánchez BLE, Franco B, Padilla-Vaca F, Landaverde MH, et al. Chitosan-BaTiO3 nanostructured piezopolymer for tissue engineering. Colloids Surf B Biointerfaces. 2020;196:111296.
  48. 48..
    Zhang Q, Qiao Y, Li C, Lin J, Han H, Li X, et al. Chitosan/gelatin-tannic acid decorated porous tape suture with multifunctionality for tendon healing. Carbohydr Polym. 2021;268:118246.
  49. 49..
    Park B, Shin JH, Ok J, Park S, Jung W, Jeong C, Choy S, et al. Cuticular pad-inspired selective frequency damper for nearly dynamic noise-free bioelectronics. Science. 2022;376(6593):624–9.
  50. 50..
    Tu C, Lu H, Zhou T, Zhang W, Deng L, Cao W, et al. Promoting the healing of infected diabetic wound by an anti-bacterial and nanoenzyme-containing hydrogel with inflammation-suppressing, ROSscavenging, oxygen and nitric oxide-generating properties. Biomaterials. 2022;286:121597.
  51. 51..
    Su N, Villicana C, Yang F. Immunomodulatory strategies for bone regeneration: a review from the perspective of disease types. Biomaterials. 2022;286:121604.
  52. 52..
    Li D, Chen K, Tang H, Hu S, Xin L, Jing X, et al. A logic-based diagnostic and therapeutic hydrogel with multistimuli responsiveness to orchestrate diabetic bone regeneration. Adv Mater. 2022;34(11):e2108430.
  53. 53..
    Xu L, Gao S, Zhou R, Zhou F, Qiao Y, Qiu D. Bioactive pore-forming bone adhesives facilitating cell ingrowth for fracture healing. Adv Mater. 2020;32(10):e1907491.
  54. 54..
    Christman KL. Biomaterials for tissue repair. Science. 2019; 363(6425):340–1.
  55. 55..
    Granito RN, Muniz Renno AC, Yamamura H, de Almeida MC, Menin Ruiz PL, Ribeiro DA. Hydroxyapatite from fish for bone tissue engineering: a promising approach. Int J Mol Cell Med. 2018;7(2):80–90.
  56. 56..
    Genchi GG, Ceseracciu L, Marino A, Labardi M, Marras S, Pignatelli F, et al. P(VDF-TrFE)/BaTiO3 nanoparticle composite films mediate piezoelectric stimulation and promote differentiation of SH-SY5Y neuroblastoma cells. Adv Healthc Mater. 2016;5(14):1808–20.
  57. 57..
    Shi K, Huang X, Sun B, Wu Z, He J, Jiang P. Cellulose/BaTiO3 aerogel paper based flexible piezoelectric nanogenerators and the electric coupling with triboelectricity. Nano Energy. 2019;57:450-8.
  58. 58..
    Kim T, Kim HJ, Choi W, Lee YM, Pyo JH, Lee J, et al. Deep brain stimulation by blood-brain-barrier-crossing piezoelectric nanoparticles generating current and nitric oxide under focused ultrasound. Nat Biomed Eng. 2023;7(2):149–63.
  59. 59..
    Liu D, Li L, Shi BL, Shi B, Li MD, Qiu Y, et al. Ultrasound-triggered piezocatalytic composite hydrogels for promoting bacterial-infected wound healing. Bioact Mater. 2023;24:96-111.
  60. 60..
    Sun J, Guo H, Ribera J, Wu C, Tu K, Binelli M, et al. Sustainable and biodegradable wood sponge piezoelectric nanogenerator for sensing and energy harvesting applications. ACS Nano. 2020;14(11):14665–74.
  61. 61..
    Wang R, Gao S, Yang Z, Li Y, Chen W, Wu B, et al. Engineered and laserprocessed chitosan biopolymers for sustainable and biodegradable triboelectric power generation. Adv Mater. 2018;30(11):1706267.
  62. 62..
    Fan FR, Tang W, Wang ZL. Flexible nanogenerators for energy harvesting and self-powered electronics. Adv Mater. 2016;28(22):4283–305.
  63. 63..
    Li H, Zhao C, Wang X, Meng J, Zou Y, Noreen S, et al. Fully bioabsorbable capacitor as an energy storage unit for implantable medical electronics. Adv Sci (Weinh). 2019;6(6):1801625.
  64. 64..
    Huang X, Xing J, Wang Z, Han J, Wang R, Li C, et al. 0D/1D heterojunction implant with electro-mechanobiological coupling cues promotes osteogenesis. Adv Funct Mater. 2021;31(50):2106249.
  65. 65..
    He Q, Yuan S, Tang H, Wang S, Mu Z, Li D, et al. Safeguarding osteointegration in diabetic patients: a potent “chain armor” coating for scavenging ROS and macrophage reprogramming in a microenvironment-responsive manner. Adv Funct Mater. 2021;31(31):2101611.
  66. 66..
    Xu C, Chang Y, Xu Y, Wu P, Mu C, Nie A, et al. Silicon-phosphorus-nanosheets-integrated 3D-printable hydrogel as a bioactive and biodegradable scaffold for vascularized bone regeneration. Adv Healthc Mater. 2022;11(6):e2101911.
  67. 67..
    Liu Z, Wan X, Wang ZL, Li L. Electroactive biomaterials and systems for cell fate determination and tissue regeneration: design and applications. Adv Mater. 2021;33(32):e2007429.
  68. 68..
    Liu X, Chen M, Luo J, Zhao H, Zhou X, Gu Q, et al. Immunopolarization-regulated 3D printed-electrospun fibrous scaffolds for bone regeneration. Biomaterials. 2021;276:121037.
  69. 69..
    Ma B, Yang Y, Li Z, Zhao D, Zhang W, Jiang Y, et al. Modular bioinformatics analysis demonstrates that a Toll-like receptor signaling pathway is involved in the regulation of macrophage polarization. Mol Med Rep. 2018;18(5):4313–20.
  70. 70..
    Chen W, Liu Y, Chen J, Ma Y, Song Y, Cen Y, et al. The Notch signaling pathway regulates macrophage polarization in liver diseases. Int Immunopharmacol. 2021;99:107938

Lesen Sie die ganze Passage

The above content is generated by Large Model Translation. The translated content is for reference only. We do not assume any commercial or legal responsibilty for any consequences arising from the use of our website