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Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Nature Protocols (2024)Cite this article

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Abstract

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100–400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young’s modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young’s modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1–2 d.

Key points

  • The function of pulmonary alveoli is dependent on their mechanical robustness and response to external forces. The Young’s modulus E (stiffness) of their basement membranes is higher than that of the surrounding cell layers.

  • This protocol describes how to prepare lung sections from humans or mice and perform atomic force microscopy experiments. Challenges in data analysis—including filtering to focus specifically on basement membrane values—are addressed using the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox.

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Fig. 1: Overview of the protocol.
Fig. 2: Identifying a suitable alveolar wall for the determination of the BM stiffness.
Fig. 3: Identifying the BM in the slope channel image of the overview force map.
Fig. 4: Checking that the program has correctly determined the contact point.
Fig. 5: Spatial filtering of the Young’s modulus values.
Fig. 6: BM’s Young’s modulus results of human lung samples.
Fig. 7: BM’s Young’s modulus results of murine lung samples.

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Data availability

All raw data and derived data used to generate graphs presented in this manuscript are available from the corresponding authors upon reasonable request. The force maps are available at https://figshare.com/articles/journal_contribution/Force_Maps/24591198.

Code availability

All codes used in this manuscript are available in an open-source repository on GitHub: https://github.com/CANTERhm/CANTER_Processing_Tool.

References

  1. Chaudhuri, O., Cooper-White, J., Janmey, P. A., Mooney, D. J. & Shenoy, V. B. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature 584, 535–546 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Eyckmans, J., Boudou, T., Yu, X. & Chen, C. S. A hitchhiker’s guide to mechanobiology. Dev. Cell 21, 35–47 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Holle, A. W. et al. Cell–extracellular matrix mechanobiology: forceful tools and emerging needs for basic and translational research. Nano Lett. 18, 1–8 (2018).

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Iskratsch, T., Wolfenson, H. & Sheetz, M. P. Appreciating force and shape-the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol. 15, 825–833 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ladoux, B. & Mege, R. M. Mechanobiology of collective cell behaviours. Nat. Rev. Mol. Cell Biol. 18, 743–757 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Wang, J. H. & Thampatty, B. P. An introductory review of cell mechanobiology. Biomech. Model. Mechanobiol. 5, 1–16 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Barriga, E. H., Franze, K., Charras, G. & Mayor, R. Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo. Nature 554, 523–527 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Murphy, W. L., McDevitt, T. C. & Engler, A. J. Materials as stem cell regulators. Nat. Mater. 13, 547–557 (2014).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vining, K. H. & Mooney, D. J. Mechanical forces direct stem cell behaviour in development and regeneration. Nat. Rev. Mol. Cell Biol. 18, 728–742 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Swift, J. et al. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science 341, 1240104 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Koser, D. E. et al. Mechanosensing is critical for axon growth in the developing brain. Nat. Neurosci. 19, 1592–1598 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Prein, C. et al. Structural and mechanical properties of the proliferative zone of the developing murine growth plate cartilage assessed by atomic force microscopy. Matrix Biol. 50, 1–15 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Alvey, C. et al. Mechanosensing of solid tumors by cancer-attacking macrophages. Biophys. J. 114, 654a–654a (2018).

    Article  ADS  Google Scholar 

  14. Bras, M. M., Radmacher, M., Sousa, S. R. & Granja, P. L. Melanoma in the eyes of mechanobiology. Front. Cell Dev. Biol. 8, 54 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Bras, M. M., Sousa, S. R., Carneiro, F., Radmacher, M. & Granja, P. L. Mechanobiology of colorectal cancer. Cancers https://doi.org/10.3390/cancers14081945 (2022).

  16. Fleischhauer, L. et al. Nano-scale mechanical properties of the articular cartilage zones in a mouse model of post-traumatic osteoarthritis. Appl. Sci. 12, 2596 (2022).

    Article  MathSciNet  CAS  Google Scholar 

  17. Rianna, C., Radmacher, M. & Kumar, S. Direct evidence that tumor cells soften when navigating confined spaces. Mol. Biol. Cell 31, 1726–1734 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Streitberger, K. J. et al. How tissue fluidity influences brain tumor progression. Proc. Natl Acad. Sci. USA 117, 128–134 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Stylianou, A., Lekka, M. & Stylianopoulos, T. AFM assessing of nanomechanical fingerprints for cancer early diagnosis and classification: from single cell to tissue level. Nanoscale 10, 20930–20945 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Suresh, S. Biomechanics and biophysics of cancer cells. Acta Biomater. 3, 413–438 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rudiger, D. et al. Cell-based strain remodeling of a nonfibrous matrix as an organizing principle for vasculogenesis. Cell Rep. 32, 108015 (2020).

    Article  PubMed  Google Scholar 

  22. Bertalan, G. et al. Mechanical behavior of the hippocampus and corpus callosum: An attempt to reconcile ex vivo with in vivo and micro with macro properties. J. Mech. Behav. Biomed. Mater. 138, 105613 (2023).

    Article  PubMed  Google Scholar 

  23. Franze, K. Atomic force microscopy and its contribution to understanding the development of the nervous system. Curr. Opin. Genet. Dev. 21, 530–537 (2011).

    Article  MathSciNet  CAS  PubMed  Google Scholar 

  24. Koser, D. E., Moeendarbary, E., Hanne, J., Kuerten, S. & Franze, K. CNS cell distribution and axon orientation determine local spinal cord mechanical properties. Biophys. J. 108, 2137–2147 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schaeffer, J., Weber, I. P., Thompson, A. J., Keynes, R. J. & Franze, K. Axons in the Chick embryo follow soft pathways through developing somite segments. Front. Cell Dev. Biol. 10, 917589 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Fritsch, A. et al. Are biomechanical changes necessary for tumour progression? Nat. Phys. 6, 730–732 (2010).

    Article  CAS  Google Scholar 

  27. Guck, J. et al. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88, 3689–3698 (2005).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ilina, O. et al. Cell-cell adhesion and 3D matrix confinement determine jamming transitions in breast cancer invasion. Nat. Cell Biol. 22, 1103–1115 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Irianto, J., Pfeifer, C. R., Ivanovska, I. L., Swift, J. & Discher, D. E. Nuclear lamins in cancer. Cell. Mol. Bioeng. 9, 258–267 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Reuten, R. et al. Basement membrane stiffness determines metastases formation. Nat. Mater. 20, 892–903 (2021).

    Article  ADS  CAS  PubMed  Google Scholar 

  31. Seltmann, K., Fritsch, A. W., Kas, J. A. & Magin, T. M. Keratins significantly contribute to cell stiffness and impact invasive behavior. Proc. Natl Acad. Sci. USA 110, 18507–18512 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Alsteens, D. et al. Atomic force microscopy-based characterization and design of biointerfaces. Nat. Rev. Mater. https://doi.org/10.1038/natrevmats.2017.8 (2017).

  33. Domke, J. & Radmacher, M. Measuring the elastic properties of thin polymer films with the atomic force microscope. Langmuir 14, 3320–3325 (1998).

    Article  CAS  Google Scholar 

  34. Huth, S., Sindt, S. & Selhuber-Unkel, C. Automated analysis of soft hydrogel microindentation: Impact of various indentation parameters on the measurement of Young’s modulus. PLoS ONE 14, e0220281 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Krieg, M. et al. Atomic force microscopy-based mechanobiology. Nat. Revi. Phys. 1, 41–57 (2019).

    Article  ADS  Google Scholar 

  36. Radmacher, M., Fritz, M., Kacher, C. M., Cleveland, J. P. & Hansma, P. K. Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys. J. 70, 556–567 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Radmacher, M., Tillamnn, R. W., Fritz, M. & Gaub, H. E. From molecules to cells: imaging soft samples with the atomic force microscope. Science 257, 1900–1905 (1992).

    Article  ADS  CAS  PubMed  Google Scholar 

  38. Stolz, M. et al. Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy. Nat. Nanotechnol. 4, 186–192 (2009).

    Article  ADS  CAS  PubMed  Google Scholar 

  39. van de Vijver, M. J. et al. A gene-expression signature as a predictor of survival in breast cancer. N. Engl. J. Med. 347, 1999–2009 (2002).

    Article  PubMed  Google Scholar 

  40. Higgins, J. P. et al. Gene expression in the normal adult human kidney assessed by complementary DNA microarray. Mol. Biol. Cell 15, 649–656 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhao, H. et al. Gene expression profiling predicts survival in conventional renal cell carcinoma. PLoS Med. 3, e13 (2006).

    Article  PubMed  Google Scholar 

  42. Nicolau, M., Tibshirani, R., Borresen-Dale, A. L. & Jeffrey, S. S. Disease-specific genomic analysis: identifying the signature of pathologic biology. Bioinformatics 23, 957–965 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. CANTER Processing Toolbox v. 5.6.0 GitHub (2022).

  44. Binnig, G. Atomic force microscope and method for imaging surfaces with atomic resolution. USA patent US4724318A (1986).

  45. Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986).

    Article  ADS  CAS  PubMed  Google Scholar 

  46. Florin, E. L., Moy, V. T. & Gaub, H. E. Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  47. Hansma, P. K. et al. Tapping mode atomic force microscopy in liquids. Appl. Phys. Lett. 64, 1738–1740 (1994).

    Article  ADS  CAS  Google Scholar 

  48. Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  49. Radmacher, M., Fritz, M., Hansma, H. G. & Hansma, P. K. Direct observation of enzyme activity with the atomic force microscope. Science 265, 1577–1579 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  50. Radmacher, M., Fritz, M. & Hansma, P. K. Imaging soft samples with the atomic force microscope: gelatin in water and propanol. Biophys. J. 69, 264–270 (1995).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rief, M., Clausen-Schaumann, H. & Gaub, H. E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).

    Article  CAS  PubMed  Google Scholar 

  52. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    Article  CAS  PubMed  Google Scholar 

  53. Drake, B. et al. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science 243, 1586–1589 (1989).

    Article  ADS  CAS  PubMed  Google Scholar 

  54. Loparic, M. et al. Micro- and nanomechanical analysis of articular cartilage by indentation-type atomic force microscopy: validation with a gel-microfiber composite. Biophys. J. 98, 2731–2740 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Rotsch, C. & Radmacher, M. Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78, 520–535 (2000).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. Clausen-Schaumann, H., Rief, M., Tolksdorf, C. & Gaub, H. E. Mechanical stability of single DNA molecules. Biophys. J. 78, 1997–2007 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lekka, M. et al. Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy. Eur. Biophys. J. 28, 312–316 (1999).

    Article  CAS  PubMed  Google Scholar 

  58. Weisenhorn, A. L., Khorsandi, M., Kasas, S., Gotzos, V. & Butt, H. J. Deformation and height anomaly of soft surfaces studied with an AFM. Nanotechnology 4, 106 (1993).

    Article  ADS  CAS  Google Scholar 

  59. Rotsch, C., Jacobson, K. & Radmacher, M. Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy. Proc. Natl Acad. Sci. USA 96, 921–926 (1999).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. Goldmann, W. H. & Ezzell, R. M. Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology. Exp. Cell Res. 226, 234–237 (1996).

    Article  CAS  PubMed  Google Scholar 

  61. Radmacher, M. Measuring the elastic properties of biological samples with the AFM. IEEE Eng. Med. Biol. Mag. 16, 47–57 (1997).

    Article  CAS  PubMed  Google Scholar 

  62. Kinney, J. H., Balooch, M., Marshall, S. J., Marshall, G. W. Jr & Weihs, T. P. Atomic force microscope measurements of the hardness and elasticity of peritubular and intertubular human dentin. J. Biomech. Eng. 118, 133–135 (1996).

    Article  CAS  PubMed  Google Scholar 

  63. Lundkvist, A. et al. Viscoelastic properties of healthy human artery measured in saline solution by AFM-based indentation technique. MRS Online Proc. Library 436, 353–358 (1996).

    Article  Google Scholar 

  64. Tao, N. J., Lindsay, S. M. & Lees, S. Measuring the microelastic properties of biological material. Biophys. J. 63, 1165–1169 (1992).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. Aro, E. et al. Severe extracellular matrix abnormalities and chondrodysplasia in mice lacking collagen prolyl 4-hydroxylase isoenzyme II in combination with a reduced amount of isoenzyme I. J. Biol. Chem. 290, 16964–16978 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Plodinec, M. et al. The nanomechanical signature of breast cancer. Nat. Nanotechnol. 7, 757–765 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  67. Stolz, M. et al. Dynamic elastic modulus of porcine articular cartilage determined at two different levels of tissue organization by indentation-type atomic force microscopy. Biophys. J. 86, 3269–3283 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  68. Akhtar, R., Sherratt, M. J., Cruickshank, J. K. & Derby, B. Characterizing the elastic properties of tissues. Mater. Today 14, 96–105 (2011).

    Article  CAS  Google Scholar 

  69. Junior, C. et al. Baseline stiffness modulates the non-linear response to stretch of the extracellular matrix in pulmonary fibrosis. Int. J. Mol. Sci. https://doi.org/10.3390/ijms222312928 (2021).

  70. Junior, C. et al. Multi-step extracellular matrix remodelling and stiffening in the development of idiopathic pulmonary fibrosis. Int. J. Mol. Sci. https://doi.org/10.3390/ijms24021708 (2023).

  71. Sicard, D., Fredenburgh, L. E. & Tschumperlin, D. J. Measured pulmonary arterial tissue stiffness is highly sensitive to AFM indenter dimensions. J. Mech. Behav. Biomed. Mater. 74, 118–127 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  72. Zemla, J. et al. AFM-based nanomechanical characterization of bronchoscopic samples in asthma patients. J. Mol. Recognit. 31, e2752 (2018).

    Article  PubMed  Google Scholar 

  73. Becke, T. D. et al. Single molecule force spectroscopy reveals two-domain binding mode of pilus-1 tip protein RrgA of Streptococcus pneumoniae to fibronectin. ACS Nano 12, 549–558 (2018).

    Article  CAS  PubMed  Google Scholar 

  74. Becke, T. D. et al. Pilus-1 backbone protein RrgB of Streptococcus pneumoniae binds collagen I in a force-dependent way. ACS Nano 13, 7155–7165 (2019).

    Article  CAS  PubMed  Google Scholar 

  75. Pill, M. F., East, A. L. L., Marx, D., Beyer, M. K. & Clausen-Schaumann, H. Mechanical activation drastically accelerates amide bond hydrolysis, matching enzyme activity. Angew. Chem. Int. Ed. Engl. 58, 9787–9790 (2019).

    Article  CAS  PubMed  Google Scholar 

  76. Schmidt, S. W., Filippov, P., Kersch, A., Beyer, M. K. & Clausen-Schaumann, H. Single-molecule force-clamp experiments reveal kinetics of mechanically activated silyl ester hydrolysis. ACS Nano 6, 1314–1321 (2012).

    Article  CAS  PubMed  Google Scholar 

  77. Docheva, D. et al. Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy. J. Cell Mol. Med. 12, 537–552 (2008).

    Article  PubMed  Google Scholar 

  78. Docheva, D., Padula, D., Schieker, M. & Clausen-Schaumann, H. Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells. Biochem. Biophys. Res. Commun. 402, 361–366 (2010).

    Article  CAS  PubMed  Google Scholar 

  79. Kiderlen, S. et al. Age related changes in cell stiffness of tendon stem/progenitor cells and a rejuvenating effect of ROCK-inhibition. Biochem. Biophys. Res. Commun. 509, 839–844 (2019).

    Article  CAS  PubMed  Google Scholar 

  80. Reuten, R. et al. Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes. Nat. Commun. 7, 13515 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  81. Yin, H. et al. Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells. Biomaterials 236, 119802 (2020).

    Article  ADS  CAS  PubMed  Google Scholar 

  82. Ferreira, S. A. et al. Bi-directional cell-pericellular matrix interactions direct stem cell fate. Nat. Commun. 9, 4049 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  83. Norman, M. D. A., Ferreira, S. A., Jowett, G. M., Bozec, L. & Gentleman, E. Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy. Nat. Protoc. 16, 2418–2449 (2021).

    Article  CAS  PubMed  Google Scholar 

  84. Alberton, P. et al. Aggrecan hypomorphism compromises articular cartilage biomechanical properties and is associated with increased incidence of spontaneous osteoarthritis. Int. J. Mol. Sci. https://doi.org/10.3390/ijms20051008 (2019).

  85. Alberton, P. et al. Aggrecan is critical in maintaining the cartilage matrix biomechanics which in turn influences the correct development of the growth plate. Osteoarthr. Cartil. 27, S178–S178 (2019).

    Article  Google Scholar 

  86. Gronau, T. et al. Forced exercise-induced osteoarthritis is attenuated in mice lacking the small leucine-rich proteoglycan decorin. Ann. Rheum. Dis. 76, 442–449 (2017).

    Article  CAS  PubMed  Google Scholar 

  87. Hartmann, B. et al. Early detection of cartilage degeneration: a comparison of histology, fiber bragg grating-based micro-indentation, and atomic force microscopy-based nano-indentation. Int. J. Mol. Sci. https://doi.org/10.3390/ijms21197384 (2020).

  88. Rellmann, Y. et al. ER Stress in ERp57 knockout knee joint chondrocytes induces osteoarthritic cartilage degradation and osteophyte formation. Int. J. Mol. Sci.https://doi.org/10.3390/ijms23010182 (2021).

  89. Kamper, M. et al. Early changes in morphology, bone mineral density and matrix composition of vertebrae lead to disc degeneration in aged collagen IX −/−mice. Matrix Biol. 49, 132–143 (2016).

    Article  CAS  PubMed  Google Scholar 

  90. Lin, D. et al. Loss of tenomodulin expression is a risk factor for age-related intervertebral disc degeneration. Aging Cell 19, e13091 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Dex, S. et al. Tenomodulin is required for tendon endurance running and Collagen I Fibril Adaptation to Mechanical Load. EBioMedicine 20, 240–254 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  92. Li, P. et al. Mice lacking the matrilin family of extracellular matrix proteins develop mild skeletal abnormalities and are susceptible to age-associated osteoarthritis. Int. J. Mol. Sci. https://doi.org/10.3390/ijms21020666 (2020).

  93. Muschter, D. et al. Sensory neuropeptides are required for bone and cartilage homeostasis in a murine destabilization-induced osteoarthritis model. Bone 133, 115181 (2020).

    Article  CAS  PubMed  Google Scholar 

  94. Seifer, P. et al. The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model. Osteoarthr. Cartil. 29, 78–88 (2021).

    Article  CAS  Google Scholar 

  95. Westermann, L. M. et al. Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma. Dis. Model Mech.https://doi.org/10.1242/dmm.046425 (2020).

  96. Franke, O. et al. Mechanical properties of hyaline and repair cartilage studied by nanoindentation. Acta Biomater. 3, 873–881 (2007).

    Article  CAS  PubMed  Google Scholar 

  97. Braet, F., Rotsch, C., Wisse, E. & Radmacher, M. Comparison of fixed and living liver endothelial cells by atomic force microscopy. Appl. Phys. A 66, S575–S578 (1998).

    Article  ADS  CAS  Google Scholar 

  98. Fiore, V. F. et al. Mechanics of a multilayer epithelium instruct tumour architecture and function. Nature 585, 433–439 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  99. Glentis, A. et al. Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane. Nat. Commun. 8, 924 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  100. Koester, J. et al. Niche stiffening compromises hair follicle stem cell potential during ageing by reducing bivalent promoter accessibility. Nat. Cell Biol. 23, 771–781 (2021).

    Article  CAS  PubMed  Google Scholar 

  101. Last, J. A., Liliensiek, S. J., Nealey, P. F. & Murphy, C. J. Determining the mechanical properties of human corneal basement membranes with atomic force microscopy. J. Struct. Biol. 167, 19–24 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Wareham, L. K. et al. Lysyl oxidase-like 1 deficiency alters ultrastructural and biomechanical properties of the peripapillary sclera in mice. Matrix Biol. 16, 100120 (2022).

    Article  CAS  Google Scholar 

  103. Liyanage, S. et al. Optimization and validation of cryostat temperature conditions for trans-reflectance mode FTIR microspectroscopic imaging of biological tissues. MethodsX 4, 118–127 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E. & Chu, S. Observation of a single-beam gradient force optical trap for dielectric particles. Opt. Lett. 11, 288 (1986).

    Article  ADS  CAS  PubMed  Google Scholar 

  105. Marago, O. M., Jones, P. H., Gucciardi, P. G., Volpe, G. & Ferrari, A. C. Optical trapping and manipulation of nanostructures. Nat. Nanotechnol. 8, 807–819 (2013).

    Article  ADS  CAS  PubMed  Google Scholar 

  106. Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat. Methods 5, 491–505 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Marchi, G. et al. Microindentation sensor system based on an optical fiber Bragg grating for the mechanical characterization of articular cartilage by stress-relaxation. Sens. Actuators B Chem. 252, 440–449 (2017).

    Article  CAS  Google Scholar 

  108. Wakitani, S. et al. Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel. Tissue Eng. 4, 429–444 (1998).

    Article  CAS  PubMed  Google Scholar 

  109. Moutos, F. T., Freed, L. E. & Guilak, F. A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage. Nat. Mater. 6, 162–167 (2007).

    Article  ADS  CAS  PubMed  Google Scholar 

  110. Schwarz, S. et al. Contactless vibrational analysis of transparent hydrogel structures using laser-doppler vibrometry. Exp. Mech. 60, 1067–1078 (2020).

    Article  CAS  Google Scholar 

  111. Bhave, G., Colon, S. & Ferrell, N. The sulfilimine cross-link of collagen IV contributes to kidney tubular basement membrane stiffness. Am. J. Physiol. Ren. Physiol. 313, F596–F602 (2017).

    Article  CAS  Google Scholar 

  112. Fisher, R. F. & Wakely, J. The elastic constants and ultrastructural organization of a basement membrane (lens capsule). Proc. R. Soc. Lond. B 193, 335–358 (1976).

    Article  ADS  CAS  PubMed  Google Scholar 

  113. Wisdom, K. M. et al. Covalent cross-linking of basement membrane-like matrices physically restricts invasive protrusions in breast cancer cells. Matrix Biol. 85-86, 94–111 (2020).

    Article  CAS  PubMed  Google Scholar 

  114. Del Campo, L. et al. Vascular smooth muscle cell-specific progerin expression in a mouse model of Hutchinson-Gilford progeria syndrome promotes arterial stiffness: therapeutic effect of dietary nitrite. Aging Cell 18, e12936 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Di Russo, J. et al. Endothelial basement membrane laminin 511 is essential for shear stress response. EMBO J. 36, 183–201 (2017).

    Article  PubMed  Google Scholar 

  116. Steppan, J. et al. Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening. Am. J. Physiol. Heart Circ. Physiol. 317, H49–H59 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Wenceslau, C. F. et al. Guidelines for the measurement of vascular function and structure in isolated arteries and veins. Am. J. Physiol. Heart Circ. Physiol. 321, H77–H111 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Florin, E. L., Pralle, A., Horber, J. K. & Stelzer, E. H. Photonic force microscope based on optical tweezers and two-photon excitation for biological applications. J. Struct. Biol. 119, 202–211 (1997).

    Article  CAS  PubMed  Google Scholar 

  119. Catala-Castro, F., Schaffer, E. & Krieg, M. Exploring cell and tissue mechanics with optical tweezers. J. Cell Sci. https://doi.org/10.1242/jcs.259355 (2022).

  120. Cuthbertson, R. A. & Mandel, T. E. Anatomy of the mouse retina. Capillary basement membrane thickness. Invest. Ophthalmol. Vis. Sci. 27, 1653–1658 (1986).

    CAS  PubMed  Google Scholar 

  121. Vracko, R., Thorning, D. & Huang, T. W. Basal lamina of alveolar epithelium and capillaries: quantitative changes with aging and in diabetes mellitus. Am. Rev. Respir. Dis. 120, 973–983 (1979).

    CAS  PubMed  Google Scholar 

  122. Rico, F. et al. Probing mechanical properties of living cells by atomic force microscopy with blunted pyramidal cantilever tips. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 72, 021914 (2005).

    Article  PubMed  Google Scholar 

  123. Chizhik, S. A., Wierzcholski, K., Trushko, A. V., Zhytkova, M. A. & Miszczak, A. Properties of cartilage on micro- and nanolevel. Adv. Tribol. 2010, 1–8 (2010).

    Article  Google Scholar 

  124. Mak, A. F., Lai, W. M. & Mow, V. C. Biphasic indentation of articular cartilage–I. Theoretical analysis. J. Biomech. 20, 703–714 (1987).

    Article  CAS  PubMed  Google Scholar 

  125. Matzelle, T. R. et al. Micromechanical properties of “smart” gels: studies by scanning force and scanning electron microscopy of PNIPAAm. J. Phys. Chem. B 106, 2861–2866 (2002).

    Article  CAS  Google Scholar 

  126. Bilodeau, G. G. Regular pyramid punch problem. J. Appl. Mech. 59, 519–523 (1992).

    Article  ADS  Google Scholar 

  127. Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B. & Chadwick, R. S. Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophys. J. 82, 2798–2810 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  ADS  CAS  PubMed  Google Scholar 

  129. Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  ADS  CAS  PubMed  Google Scholar 

  130. Hell, S. W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

    Article  CAS  PubMed  Google Scholar 

  131. Friedl, P., Wolf, K., von Andrian, U. H. & Harms, G. Biological second and third harmonic generation microscopy. Curr. Protoc. Cell Biol. 4, 4.15 (2007).

    Google Scholar 

  132. Evans, C. L. & Xie, X. S. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. 1, 883–909 (2008).

    Article  CAS  Google Scholar 

  133. Lu, F., Jin, M. & Belkin, M. A. Tip-enhanced infrared nanospectroscopy via molecular expansion force detection. Nat. Photonics 8, 307–312 (2014).

    Article  ADS  CAS  Google Scholar 

  134. Ruggeri, F. S. et al. Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Abeta42 oligomers. Nat. Commun. 12, 688 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  135. Ruggeri, F. S., Mannini, B., Schmid, R., Vendruscolo, M. & Knowles, T. P. J. Single molecule secondary structure determination of proteins through infrared absorption nanospectroscopy. Nat. Commun. 11, 2945 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  136. Schillers, H. et al. Standardized nanomechanical atomic force microscopy procedure (SNAP) for measuring soft and biological samples. Sci. Rep. 7, 5117 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  137. Hutter, J. L. & Bechhoefer, J. Calibration of atomic‐force microscope tips. Rev. Sci. Instrum. 64, 1868–1873 (1993).

    Article  ADS  CAS  Google Scholar 

  138. Bedzhov, I. & Zernicka-Goetz, M. Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–1044 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Kyprianou, C. et al. Basement membrane remodelling regulates mouse embryogenesis. Nature 582, 253–258 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  140. Saraswathibhatla, A., Indana, D. & Chaudhuri, O. Cell–extracellular matrix mechanotransduction in 3D. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-023-00583-1 (2023).

  141. Sherwood, D. R. Basement membrane remodeling guides cell migration and cell morphogenesis during development. Curr. Opin. Cell Biol. 72, 19–27 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Candiello, J. et al. Biomechanical properties of native basement membranes. FEBS J. 274, 2897–2908 (2007).

    Article  CAS  PubMed  Google Scholar 

  143. Halfter, W. et al. The bi-functional organization of human basement membranes. PLoS ONE 8, e67660 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  144. Halfter, W. et al. New concepts in basement membrane biology. FEBS J. 282, 4466–4479 (2015).

    Article  CAS  PubMed  Google Scholar 

  145. Henrich, P. B. et al. Nanoscale topographic and biomechanical studies of the human internal limiting membrane. Invest. Ophthalmol. Vis. Sci. 53, 2561–2570 (2012).

    Article  PubMed  Google Scholar 

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Acknowledgements

B.H., L.F. and H.C.-S. acknowledge funding from the Bavarian State Ministry of Science and the Arts through the Bavarian Research Focus ‘Herstellung und biophysikalische Charakterisierung von dreidimensionalen Geweben (CANTER)’ and the Bavarian Academic Forum (BayWISS)—Doctoral Consortium ‘Health Research’. The development of the data analysis software CANTER processing toolbox was funded by the German Research Foundation as part of subproject 1 (CL 409/4-1/2) of the research consortium ‘Exploring articular cartilage and subchondral bone degeneration and regeneration in osteoarthritis – ExCarBon’ (FOR2407-1/2). H.C.-S. acknowledges funding from the German Research Foundation through the major instrumentation campaign GGA-HAW (INST 99/38-1). This work was further supported by the Danish Cancer Society (R204-A12454 (R.R.)).

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Authors and Affiliations

  1. Munich University of Applied Sciences, Center for Applied Tissue Engineering and Regenerative Medicine – CANTER, Munich, Germany

    Bastian Hartmann, Lutz Fleischhauer & Hauke Clausen-Schaumann

  2. Center for Nanoscience, Munich, Germany

    Bastian Hartmann, Lutz Fleischhauer & Hauke Clausen-Schaumann

  3. Experimental and Clinical Pharmacology and Toxicology, Medical Faculty, University of Freiburg, Freiburg, Germany

    Monica Nicolau & Raphael Reuten

  4. Department of Obstetrics and Gynecology, Medical Center, University of Freiburg, Freiburg, Germany

    Monica Nicolau, Florin-Andrei Taran & Raphael Reuten

  5. Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Thomas Hartvig Lindkær Jensen

  6. Department of Pathology, Rigshospitalet, Copenhagen, Denmark

    Thomas Hartvig Lindkær Jensen

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Contributions

All authors developed experimental protocols and designed experiments. B.H., L.F. and T.H.L.J. conducted the experiments. B.H., L.F. and M.N. developed the data analysis tools. B.H., M.N. and F.-A.T. analyzed the data. H.C.-S. and R.R. conceived the ideas and contributed to experimental interpretation. B.H., H.C.-S. and R.R. wrote the manuscript. All authors revised the manuscript.

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Correspondence to Hauke Clausen-Schaumann or Raphael Reuten.

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Key reference using this protocol

Reuten, R. et al. Nat. Mater. 20, 892–903 (2021): https://doi.org/10.1038/s41563-020-00894-0

Key data used in this protocol

Reuten, R. et al. Nat. Mater. 20, 892–903 (2021): https://doi.org/10.1038/s41563-020-00894-0

Extended data

Supplementary information

Supplementary Information

Suplementary discussion and Figs. 1–20.

Source data

Source Data Fig. 5

Young’s modulus values histograms.

Source Data Fig. 6

Log-transformed Young’s modulus values used to generate the histograms and the QQ-plot.

Source Data Fig. 7

Log-transformed Young’s modulus values of the histograms. Young’s modulus values (individual values and summary values) of the box plots in Fig. 7d. Standard deviation values of the box plots in Fig. 7e.

Source Data Extended Data Fig. 1

Young’s modulus values from Net4 WT and KO mice splitted into female and male (individual values and summary values) of the box plots in Extended Data Fig. 1.

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Hartmann, B., Fleischhauer, L., Nicolau, M. et al. Profiling native pulmonary basement membrane stiffness using atomic force microscopy. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00955-7

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