Skip to main content
Log in

Does cellular elastic modulus change due to subculture and fixation: an atomic force microscopy study of nucleus pulposus cells in vitro

  • Regular Article
  • Published:
The European Physical Journal Plus Aims and scope Submit manuscript

Abstract

Cervical spinal degenerative disease (CSDD) is the progressive deterioration of cervical intervertebral disk (IVD) and is strongly related to the degeneration, necrosis, and apoptosis of nucleus pulposus cells (NPCs). The cellular mechanics of cervical NPCs is essential for the biomechanics of degenerative IVD and the pathogenesis of CSDD. Atomic force microscopy (AFM) can be used to quantify the cellular morphology and mechanics. However, the cellular subculture and experimental conditions may influence the cellular elastic modulus of the NPCs. In this study, cervical nucleus pulposus cells were cultured in vitro and their cellular elastic modulus were measured using AFM to explore the influence of cell subculture and cell fixation on the elastic modulus. To this end, the cellular morphology and mechanical properties of the HNPCs were successfully quantified by AFM. The elastic modulus of the primary NPCs was maintained with P2 generation (2 passages in vitro) cells during the in vitro culture and the elastic modulus increased after fixation. The results of this study lay the foundation for further research. P2 generation, living NPCs are recommended as the experimental subject for exploring the cellular mechanics mechanisms of cervical degenerative disease.

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

Access this article

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

Instant access to the full article PDF.

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

Similar content being viewed by others

Data availability

No data associated in the manuscript.

References

  1. N. Theodore, Degenerative cervical spondylosis. N. Engl. J. Med. 383(2), 159–168 (2020)

    Article  Google Scholar 

  2. W. Cui, B. Wu, B. Liu, D. Li, L. Wang, S. Ma, Adjacent segment motion following multi-level ACDF: a kinematic and clinical study in patients with zero-profile anchored spacer or plate. Eur. Spine J. 28(10), 2408–2416 (2019)

    Article  Google Scholar 

  3. C. Feng, H. Liu, M. Yang, Y. Zhang, B. Huang, Y. Zhou, Disc cell senescence in intervertebral disc degeneration: causes and molecular pathways. Cell Cycle 15(13), 1674–1684 (2016)

    Article  Google Scholar 

  4. N. Theodore, A.K. Ahmed, T. Fulton et al., Genetic predisposition to symptomatic lumbar disk herniation in pediatric and young adult patients. Spine 44(11), E640–E649 (2019)

    Article  Google Scholar 

  5. W. Brinjikji, P.H. Luetmer, B. Comstock et al., Systematic literature review of imaging features of spinal degeneration in asymptomatic populations. AJNR Am. J. Neuroradiol. 36(4), 811–816 (2015)

    Article  Google Scholar 

  6. R.P. Schlenk, T. Stewart, E.C. Benzel, The biomechanics of iatrogenic spinal destabilization and implant failure. Neurosurg. Focus 15(3), E2 (2003)

    Article  Google Scholar 

  7. N.G. Rodriguez-Martinez, L. Perez-Orribo, S. Kalb et al., The role of obesity in the biomechanics and radiological changes of the spine: an in vitro study. J. Neurosurg. Spine 24(4), 615–623 (2016)

    Article  Google Scholar 

  8. X. Chen, H. Chen, B.L. Li et al., Dynamic elastic modulus assessment of the early degeneration model of an intervertebral disc in cynomolgus monkeys with one strike loading. Comput. Methods Progr. Biomed. 224, 106982 (2022)

    Article  Google Scholar 

  9. M. Bergert, S. Lembo, S. Sharma et al., Cell surface mechanics gate embryonic stem cell differentiation. Cell Stem Cell 28(2), 209–216 (2021)

    Article  Google Scholar 

  10. M. Tartibi, Y.X. Liu, G.Y. Liu, K. Komvopoulos, Single-cell mechanics–an experimental-computational method for quantifying the membrane-cytoskeleton elasticity of cells. Acta Biomater. 27, 224–235 (2015)

    Article  Google Scholar 

  11. S. Roberts, H. Evans, J. Trivedi, J. Menage, Histology and pathology of the human intervertebral disc. J. Bone Joint Surg. Am. 88(Suppl 2), 10–14 (2006)

    Google Scholar 

  12. D. Chen, D. Xia, Z. Pan et al., Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo. Cell Death Dis. 7(10), e2441 (2016)

    Article  Google Scholar 

  13. P.P. Vergroesen, I. Kingma, K.S. Emanuel et al., Mechanics and biology in intervertebral disc degeneration: a vicious circle. Osteoarthr. Cartil.. Cartil. 23(7), 1057–1070 (2015)

    Article  Google Scholar 

  14. D.J. Muller, A.C. Dumitru, C. Lo Giudice et al., Atomic force microscopy-based force spectroscopy and multiparametric imaging of biomolecular and cellular systems. Chem. Rev. 121(19), 11701–11725 (2021)

    Article  Google Scholar 

  15. E. Hecht, P. Knittel, E. Felder, P. Dietl, B. Mizaikoff, C. Kranz, Combining atomic force-fluorescence microscopy with a stretching device for analyzing mechanotransduction processes in living cells. Analyst 137(22), 5208–5214 (2012)

    Article  ADS  Google Scholar 

  16. S. Matsunaga, T. Onishi, T. Sakou, Significance of occipitoaxial angle in subaxial lesion after occipitocervical fusion. Spine 26(2), 161–165 (2001)

    Article  Google Scholar 

  17. M. Li, N. Xi, L. Liu, Peak force tapping atomic force microscopy for advancing cell and molecular biology. Nanoscale 13(18), 8358–8375 (2021)

    Article  Google Scholar 

  18. H. Wang, Q. Xie, X.G. Xu, Super-resolution mid-infrared spectro-microscopy of biological applications through tapping mode and peak force tapping mode atomic force microscope. Adv. Drug Deliv. Rev. 180, 114080 (2022)

    Article  Google Scholar 

  19. X. Zhao, Y. Zhong, T. Ye, D. Wang, B. Mao, Discrimination between cervical cancer cells and normal cervical cells based on longitudinal elasticity using atomic force microscopy. Nanoscale Res. Lett. 10(1), 482 (2015)

    Article  ADS  Google Scholar 

  20. R. Vargas-Pinto, H. Gong, A. Vahabikashi, M. Johnson, The effect of the endothelial cell cortex on atomic force microscopy measurements. Biophys. J. 105(2), 300–309 (2013)

    Article  ADS  Google Scholar 

  21. F.M. Hecht, J. Rheinlaender, N. Schierbaum, W.H. Goldmann, B. Fabry, T.E. Schäffer, Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale. Soft Matter 11(23), 4584–4591 (2015)

    Article  ADS  Google Scholar 

  22. M. Li, L. Liu, N. Xi, Y. Wang, Atomic force microscopy studies on cellular elastic and viscoelastic properties. Sci. China Life Sci. 61(1), 57–67 (2018)

    Article  ADS  Google Scholar 

  23. D. Alsteens, D.J. Müller, Y.F. Dufrêne, Multiparametric atomic force microscopy imaging of biomolecular and cellular systems. Acc. Chem. Res. 50(4), 924–931 (2017)

    Article  Google Scholar 

  24. D. Sang, C.F. Du, B. Wu et al., The effect of cervical intervertebral disc degeneration on the motion path of instantaneous center of rotation at degenerated and adjacent segments: a finite element analysis. Comput. Biol. Med. 134, 104426 (2021)

    Article  Google Scholar 

  25. B. Liu, Z. Liu, T. VanHoof, J. Kalala, Z. Zeng, X. Lin, Kinematic study of the relation between the instantaneous center of rotation and degenerative changes in the cervical intervertebral disc. Eur. Spine J. 23(11), 2307–2313 (2014)

    Article  Google Scholar 

  26. W. Wang, Z. Guo, S. Yang, H. Wang, W. Ding, Upregulation of miR-199 attenuates TNF-alpha-induced human nucleus pulposus cell apoptosis by downregulating MAP3K5. Biochem. Biophys. Res. Commun. 505(3), 917–924 (2018)

    Article  Google Scholar 

  27. S. Chen, X. Lv, B. Hu et al., RIPK1/RIPK3/MLKL-mediated necroptosis contributes to compression-induced rat nucleus pulposus cells death. Apoptosis 22(5), 626–638 (2017)

    Article  Google Scholar 

  28. X. Xu, D. Wang, C. Zheng et al., Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane. Theranostics 9(8), 2252–2267 (2019)

    Article  Google Scholar 

  29. Q. Xiang, L. Kang, J. Wang et al., CircRNA-CIDN mitigated compression loading-induced damage in human nucleus pulposus cells via miR-34a-5p/SIRT1 axis. EBioMedicine 53, 102679 (2020)

    Article  Google Scholar 

  30. P. Cazzanelli, K. Wuertz-Kozak, MicroRNAs in intervertebral disc degeneration, apoptosis, inflammation, and mechanobiology. Int. J. Mol. Sci. 21(10), 3601 (2020)

    Article  Google Scholar 

  31. Y. Zhang, B. Yang, J. Wang et al., Cell senescence: a nonnegligible cell state under survival stress in pathology of intervertebral disc degeneration. Oxid. Med. Cell. Longev.. Med. Cell. Longev. 2020, 9503562 (2020)

    Google Scholar 

  32. Z.H. Yu, Y.C. Ji, K. Li et al., Stiffness of the extracellular matrix affects apoptosis of nucleus pulposus cells by regulating the cytoskeleton and activating the TRPV2 channel protein. Cell. Signal. 84, 110005 (2021)

    Article  Google Scholar 

  33. D. Bosnakovski, M. Mizuno, G. Kim, S. Takagi, M. Okumura, T. Fujinaga, Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen type II extracellular matrix on MSC chondrogenesis. Biotechnol. Bioeng. 93(6), 1152–1163 (2006)

    Article  Google Scholar 

  34. T.G. Kuznetsova, M.N. Starodubtseva, N.I. Yegorenkov, S.A. Chizhik, R.I. Zhdanov, Atomic force microscopy probing of cell elasticity. Micron 38(8), 824–833 (2007)

    Article  Google Scholar 

  35. C. Rotsch, M. Radmacher, Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78(1), 520–535 (2000)

    Article  ADS  Google Scholar 

  36. F. Eghiaian, A. Rigato, S. Scheuring, Structural, mechanical, and dynamical variability of the actin cortex in living cells. Biophys. J. 108(6), 1330–1340 (2015)

    Article  ADS  Google Scholar 

  37. B. Codan, V. Martinelli, L. Mestroni, O. Sbaizero, Atomic force microscopy of 3T3 and SW-13 cell lines: an investigation of cell elasticity changes due to fixation. Mater. Sci. Eng. C Mater. Biol. Appl. 33(6), 3303–3308 (2013)

    Article  Google Scholar 

  38. K. Hayashi, M. Iwata, Stiffness of cancer cells measured with an AFM indentation method. J. Mech. Behav. Biomed. Mater. 49, 105–111 (2015)

    Article  Google Scholar 

  39. A.S. Kamruzzahan, F. Kienberger, C.M. Stroh et al., Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM. Biol. Chem. 385(10), 955–960 (2004)

    Article  Google Scholar 

  40. E.H. Zhou, F. Xu, S.T. Quek et al., A power-law rheology-based finite element model for single cell deformation. Biomech. Model. Mechanobiol.. Model. Mechanobiol. 11(7), 1075–1084 (2012)

    Article  Google Scholar 

Download references

Acknowledgements

The Authors thank all the patients for their contributions.

Funding

This work was supported by the Beijing Municipal Natural Science Foundation (Grant NO.7222051); the National Natural Science Foundation of China (Grant NO. 82272524; NO. 81972084) and the High Level Public Health Technology Talent Construction Project (Grant NO. Leading Talent-02–05).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Baoge Liu.

Ethics declarations

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

The study was approved by the Institutional Review (Ethic number, IRB: #KY 2022–248-02).

Consent to participate

Informed consent was obtained from all participants.

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

Xiao, B., Zhou, T., Huang, J. et al. Does cellular elastic modulus change due to subculture and fixation: an atomic force microscopy study of nucleus pulposus cells in vitro. Eur. Phys. J. Plus 138, 960 (2023). https://doi.org/10.1140/epjp/s13360-023-04609-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1140/epjp/s13360-023-04609-7

Navigation