Medical & Biological Engineering & Computing

, Volume 57, Issue 4, pp 819–835 | Cite as

Frequency-induced morphology alterations in microconfined biological cells

  • Hritwick BanerjeeEmail author
  • Bibhas Roy
  • Kaustav Chaudhury
  • Babji Srinivasan
  • Suman Chakraborty
  • Hongliang RenEmail author
Original Article


Low-intensity therapeutic ultrasound has demonstrated an impetus in bone signaling and tissue healing for decades now. Though this technology is clinically well proven, still there are breaches in studies to understand the fundamental principle of how osteoblast tissue regenerates physiologically at the cellular level with ultrasound interaction as a form of acoustic wave stimuli. Through this article, we illustrate an analysis for cytomechanical changes of cell membrane periphery as a basic first physical principle for facilitating late downstream biochemical pathways. With the help of in situ single-cell direct analysis in a microfluidic confinement, we demonstrate that alteration of low-intensity pulse ultrasound (LIPUS) frequency would physically perturb cell membrane and establish inherent cell oscillation. We experimentally demonstrate here that, at LIPUS resonance near 1.7 MHz (during 1–3 MHz alteration), cell membrane area would expand to 6.85 ± 0.7% during ultrasound exposure while it contracts 44.68 ± 0.8% in post actuation. Conversely, cell cross-sectional area change (%) from its previous morphology during and after switching off LIPUS was reversibly different before and after resonance. For instance, at 1.5 MHz, LIPUS exposure produced 1.44 ± 0.5% expansion while in contrast 2 MHz instigates 1.6 ± 0.3% contraction. We conclude that alteration of LIPUS frequency from 1–3 MHz keeping other ultrasound parameters like exposure time, pulse repetition frequency (PRF), etc., constant, if applied to a microconfined biological single living cell, would perturb physical structure reversibly based on the system resonance during and post exposure ultrasound pulsing. We envision, in the near future, our results would constitute the foundation of mechanistic effects of low-intensity therapeutic ultrasound and its allied potential in medical applications.

Graphical Abstract

Frequency Dependent Characterization of Area Strain in Cell Membrane by Microfluidic Based Single Cell Analysis


Cellular morphology Microfluidic confinement Ultrasound therapy LIPUS 



All experiments were performed in Department of Biotechnology, Indian Institute of Technology Kharagpur, India, under supervision of Professor Tapas Kumar Maiti. We would also like to thank Dr. Dario Carugo, University of Southampton, for helping and guiding to optimize the ultrasound setup for our ultrasound-cell interaction study. For assisting the biological experiments, we would like to extend our sincere acknowledgement to Mr. Joyjyoti Das, Dr. Birendra Behera, and Dr. Nilanjana Bose Chakraborty.

Compliance with Ethical Standards

Conflict of interests

The authors declare that they have no conflicts of interest and there was no funding associated with this research.

Supplementary material

(MP4 11.3 MB)

(MP4 20.7 MB)

(MP4 8.09 MB)

11517_2018_1908_MOESM4_ESM.pdf (1.1 mb)
(PDF 1.06 MB)


  1. 1.
    Lowry WE, Quan WL (2010) Roadblocks en route to the clinical application of induced pluripotent stem cells. J Cell Sci 0(5):643–651Google Scholar
  2. 2.
    Haar GT (2007) Therapeutic applications of ultrasound. Prog Biophys Mol Biol 93(1):111–129Google Scholar
  3. 3.
    Wood RW, Loomis AL (1927) Xxxviii. The physical and biological effects of high-frequency sound-waves of great intensity. The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science 4(22):417–436Google Scholar
  4. 4.
    Baker KG, Robertson VJ, Duck FA (2001) A review of therapeutic ultrasound: biophysical effects. Phys Ther 81(7):1351–1358Google Scholar
  5. 5.
    Speed CA (2001) Therapeutic ultrasound in soft tissue lesions. Rheumatology 40(12):1331–1336Google Scholar
  6. 6.
    Artho PA, Thyne JG, Warring BP, Willis CD, Brismee J-M, Latman NS (2002) A calibration study of therapeutic ultrasound units. Phys Ther 82(3):257–263Google Scholar
  7. 7.
    Alhadlaq A, Mao JJ (2004) Mesenchymal stem cells: isolation and therapeutics. Stem Cells Dev 13 (4):436–448Google Scholar
  8. 8.
    Saini V, Yadav S, McCormick S (2011) Low-intensity pulsed ultrasound modulates shear stress induced pghs-2 expression and pge2 synthesis in mlo-y4 osteocyte-like cells. Ann Biomed Eng 39(1):378–393Google Scholar
  9. 9.
    Bose N, Zhang X, Maiti TK, Chakraborty S (2015) The role of acoustofluidics in targeted drug delivery. Biomicrofluidics 9(5):052609Google Scholar
  10. 10.
    Daftary GS, Taylor HS (2006) Endocrine regulation of hox genes. Endocr Rev 27(4):331–355Google Scholar
  11. 11.
    Tabuchi Y, Ando H, Takasaki I, Feril LB, Zhao Q-L, Ogawa R, Kudo N, Tachibana K, Kondo T (2007) Identification of genes responsive to low intensity pulsed ultrasound in a human leukemia cell line molt-4. Cancer Lett 246(1):149–156Google Scholar
  12. 12.
    de Albornoz PM, Khanna A, Longo UG, Forriol F, Maffulli N (2011) The evidence of low-intensity pulsed ultrasound for in vitro, animal and human fracture healing. Br Med Bull 100(1):39–57Google Scholar
  13. 13.
    Cermik D, Karaca M, Taylor HS (2001) Hoxa10 expression is repressed by progesterone in the myometrium: differential tissue-specific regulation of hox gene expression in the reproductive tract. J Clin Endocrinol Metab 86 (7):3387–3392Google Scholar
  14. 14.
    Zacherl M, Gruber G, Radl R, Rehak PH, Windhager R (2009) No midterm benefit from low intensity pulsed ultrasound after chevron osteotomy for hallux valgus. Ultrasound Med Biol 35(8):1290–1297Google Scholar
  15. 15.
    Tan MK, Friend JR, Yeo LY (2009) Interfacial jetting phenomena induced by focused surface vibrations. Phys Rev Lett 103(2):024501Google Scholar
  16. 16.
    Shaw A, Hodnett M (2008) Calibration and measurement issues for therapeutic ultrasound. Ultrasonics 48(4):234–252Google Scholar
  17. 17.
    Hauser J, Hauser M, Muhr G, Esenwein S (2009) Ultrasound-induced modifications of cytoskeletal components in osteoblast-like saos-2 cells. J Orthop Res 27(3):286–294Google Scholar
  18. 18.
    Mizrahi N, Zhou EH, Lenormand G, Krishnan R, Weihs D, Butler JP, Weitz DA, Fredberg JJ, Kimmel E (2012) Low intensity ultrasound perturbs cytoskeleton dynamics. Soft Matter 8(8):2438–2443Google Scholar
  19. 19.
    Noriega S, Hasanova G, Subramanian A (2013) The effect of ultrasound stimulation on the cytoskeletal organization of chondrocytes seeded in three-dimensional matrices. Cells Tissues Organs 197(1):14–26Google Scholar
  20. 20.
    Zhang S, Cheng J, Qin Y-X (2012) Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force. PLoS One 7(6):e38343Google Scholar
  21. 21.
    Mahoney CM, Morgan MR, Harrison A, Humphries MJ, Bass MD (2009) Therapeutic ultrasound bypasses canonical syndecan-4 signaling to activate rac1. J Biol Chem 284(13):8898–8909Google Scholar
  22. 22.
    Roper J, Harrison A, Bass MD (2012) Induction of adhesion-dependent signals using low-intensity ultrasound. J Vis Exp: JoVE (63):4024.
  23. 23.
    Zhou S, Schmelz A, Seufferlein T, Li Y, Zhao J, Bachem MG (2004) Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts. J Biol Chem 279(52):54463–54469Google Scholar
  24. 24.
    Hu Y, Wan JMF, Alfred CH (2014) Cytomechanical perturbations during low-intensity ultrasound pulsing. Ultrasound Med Biol 40(7):1587–1598Google Scholar
  25. 25.
    Banerjee H (2014) Frequency driven alteration in cellular morphology during ultrasound pulsing in a microfluidic confinement. PhD thesis, Indian Institute of Technology, GandhinagarGoogle Scholar
  26. 26.
    Das T, Chakraborty S (2013) Perspective: flicking with flow: can microfluidics revolutionize the cancer research? Biomicrofluidics 7(1):011811Google Scholar
  27. 27.
    Banerjee H, Suhail M, Ren H (2018) Hydrogel actuators and sensors for biomedical soft robots: brief overview with impending challenges. Biomimetics 3(3):15Google Scholar
  28. 28.
    Claes L, Willie B (2007) The enhancement of bone regeneration by ultrasound. Prog Biophys Mol Biol 93 (1):384–398Google Scholar
  29. 29.
    Duck FA (2007) Medical and non-medical protection standards for ultrasound and infrasound. Prog Biophys Mol Biol 93(1):176–191Google Scholar
  30. 30.
    Das T, Maiti TK, Chakraborty S (2011) Augmented stress-responsive characteristics of cell lines in narrow confinements. Integr Biol 3(6):684–695Google Scholar
  31. 31.
    Santini MT, Rainaldi G, Romano R, Ferrante A, Clemente S, Motta A, Indovina PL (2004) Mg-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a 1h nmr study. FEBS Lett 557(1–3):148–154Google Scholar
  32. 32.
    El-Ali J, Sorger PK, Jensen KF (2006) Cells on chips. Nature 442(7101):403–411Google Scholar
  33. 33.
    Feril LB, Kondo T, Cui Z-G, Tabuchi Y, Zhao Q-L, Ando H, Misaki T, Yoshikawa H, Umemura S-I (2005) Apoptosis induced by the sonomechanical effects of low intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 221(2):145–152Google Scholar
  34. 34.
    Ward TH, Cummings J, Dean E, Greystoke A, Hou J-M, Backen A, Ranson M, Dive C (2008) Biomarkers of apoptosis. Br J Cancer 99(6):841Google Scholar
  35. 35.
    Li X, Kierfeld J, Lipowsky R (2009) Actin polymerization and depolymerization coupled to cooperative hydrolysis. Phys Rev Lett 103(4):048102Google Scholar
  36. 36.
    Wang J, Boja ES, Tan W, Tekle E, Fales HM, English S, Mieyal JJ, Chock PB (2001) Reversible glutathionylation regulates actin polymerization in a431 cells. J Biol Chem 276(51):47763–47766Google Scholar
  37. 37.
    Yonezawa Naoto, Nishida E, Sakai H (1985) Ph control of actin polymerization by cofilin. J Biol Chem 260(27):14410–14412Google Scholar
  38. 38.
    Cárdenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiol 146(4):1611–1621Google Scholar
  39. 39.
    Krasovitski B, Frenkel V, Shoham S, Kimmel E (2011) Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects. Proc Natl Acad Sci 108(8):3258–3263Google Scholar
  40. 40.
    Van der Meer SM, Versluis M, Lohse D, Chin CT, Bouakaz A, De Jong N (2004) The resonance frequency of sonovue/spl trade/as observed by high-speed optical imaging. In: Ultrasonics symposium IEEE, vol 1, p 2004Google Scholar
  41. 41.
    Zinin PV, Allen JS III (2009) Deformation of biological cells in the acoustic field of an oscillating bubble. Phys Rev E 79(2): 021910Google Scholar
  42. 42.
    Bausch AR, Ziemann F, Boulbitch AA, Jacobson K, Sackmann E (1998) Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys J 75(4):2038–2049Google Scholar
  43. 43.
    Hopkins PM, Bodenham AR, Reeves ST (2008) Practical ultrasound in anesthesia for critical care and pain management. Taylor & Francis USGoogle Scholar
  44. 44.
    Iwashina T, Mochida J, Miyazaki T, Watanabe T, Iwabuchi S, Ando K, Hotta T, Sakai D (2006) Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate. Biomaterials 27(3):354–361Google Scholar
  45. 45.
    Khan Y, Laurencin CT (2008) Fracture repair with ultrasound: clinical and cell-based evaluation. JBJS 90((Supplement_1)):138–144Google Scholar
  46. 46.
    Norvell SM, Alvarez M, Bidwell JP, Pavalko FM (2004) Fluid shear stress induces β-catenin signaling in osteoblasts. Calcif Tissue Int 75(5):396–404Google Scholar
  47. 47.
    Mishra D (2013) Osteoblast microtissues as profunctional modules for bone tissue engineering applications. PhD thesis, IIT KharagpurGoogle Scholar
  48. 48.
    Azuma Y, Ito M, Harada Y, Takagi H, Ohta T, Jingushi S (2001) Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus. J Bone Miner Res 16(4):671–680Google Scholar
  49. 49.
    Iwai T, Harada Y, Imura K, Iwabuchi S, Murai J, Hiramatsu K, Myoui A, Yoshikawa H, Tsumaki N (2007) Low-intensity pulsed ultrasound increases bone ingrowth into porous hydroxyapatite ceramic. J Bone Miner Metab 25(6):392–399Google Scholar
  50. 50.
    Hongmei Yu, Meyvantsson I, Shkel IA, Beebe DJ (2005) Diffusion dependent cell behavior in microenvironments. Lab Chip 5(10):1089–1095Google Scholar
  51. 51.
    Yang L, Effler JC, Kutscher BL, Sullivan SE, Robinson DN, Iglesias PA (2008) Modeling cellular deformations using the level set formalism. BMC Syst Biol 2(1):68Google Scholar
  52. 52.
    Zohar O, Ikeda M, Shinagawa H, Inoue H, Nakamura H, Elbaum D, Alkon DL, Yoshioka T (1998) Thermal imaging of receptor-activated heat production in single cells. Biophys J 74(1):82– 89Google Scholar
  53. 53.
    Bruus H (2012) Acoustofluidics 7: the acoustic radiation force on small particles. Lab Chip 12(6):1014–1021Google Scholar
  54. 54.
    Wells PNT (1975) Absorption and dispersion of ultrasound in biological tissue. Ultrasound Med Biol 1 (4):369–376Google Scholar
  55. 55.
    van Wamel A, Bouakaz A, Versluis M, de Jong N (2004) Micromanipulation of endothelial cells: ultrasound-microbubble-cell interaction. Ultrasound in Med Biol 3(9):1255–1258Google Scholar
  56. 56.
    Mundi R, Petis S, Kaloty R, Shetty V, Bhandari M (2009) Low-intensity pulsed ultrasound: fracture healing. Indian Journal of Orthopaedics 43(2):132Google Scholar
  57. 57.
    Mettin R, Akhatov I, Parlitz U, Ohl CD, Lauterborn W (1997) Bjerknes forces between small cavitation bubbles in a strong acoustic field. Physical Rev E 56(3):2924Google Scholar
  58. 58.
    Pounder NM, Harrison AJ (2008) Low intensity pulsed ultrasound for fracture healing: a review of the clinical evidence and the associated biological mechanism of action. Ultrasonics 48(4):330–338Google Scholar
  59. 59.
    Xing J (2016) Design of low-intensity pulsed ultrasound device intensity sensor and its application to enhance vaccine production. PhD thesis, University of AlbertaGoogle Scholar
  60. 60.
    Fávaro-Pípi E, Feitosa SM, Ribeiro DA, Bossini P, Oliveira P, Parizotto NA, Renno ACM (2010) Comparative study of the effects of low-intensity pulsed ultrasound and low-level laser therapy on bone defects in tibias of rats. Lasers Med Sci 25(5):727–732Google Scholar
  61. 61.
    Gebauer D, Mayr E, Orthner E, Ryaby JP (2005) Low-intensity pulsed ultrasound: effects on nonunions. Ultrasound in Med Biol 31(10):1391–1402Google Scholar
  62. 62.
    Schuster A, Schwab T, Bischof M, Klotz M, Lemor R, Degel C, Schäfer K-H (2013) Cell specific ultrasound effects are dose and frequency dependent. Annals of Anatomy-Anatomischer Anzeiger 195(1):57–67Google Scholar
  63. 63.
    Miller DL, Smith NB, Bailey MR, Czarnota GJ, Hynynen K, Makin IRS (2012) Overview of therapeutic ultrasound applications and safety considerations. J Ultrasound Med 31(4):623– 634Google Scholar
  64. 64.
    Kopechek JA, Kim H, McPherson DD, Holland CK (2010) Calibration of the 1-mhz sonitron ultrasound system. Ultrasound in Med Biol 36(10):1762–1766Google Scholar
  65. 65.
    Leskinen JJ, Hynynen K (2012) Study of factors affecting the magnitude and nature of ultrasound exposure with in vitro set-ups. Ultrasound in Med Biol 38(5):777–794Google Scholar
  66. 66.
    Pietak A, Levin M (2017) Bioelectric gene and reaction networks: computational modelling of genetic, biochemical and bioelectrical dynamics in pattern regulation. Journal of The Royal Society Interface 14(134):20170425Google Scholar
  67. 67.
    Maddala J, Srinivasan B, Bithi SS, Vanapalli SA, Rengaswamy R (2012) Design of a model-based feedback controller for active sorting and synchronization of droplets in a microfluidic loop. AICHE J 58(7):2120–2130Google Scholar
  68. 68.
    Banerjee H, Srinivasan B (2013) Modelling, optimization and control of droplet based microfluidic technology for single-cell high-throughput screeningGoogle Scholar

Copyright information

© International Federation for Medical and Biological Engineering 2018

Authors and Affiliations

  1. 1.Department of Electrical EngineeringIndian Institute of Technology GandhinagarGandhinagarIndia
  2. 2.Department of Mechanical EngineeringIndian Institute of Technology KharagpurKharagpurIndia
  3. 3.Department of Biomedical Engineering, Faculty of EngineeringNational University of SingaporeSingaporeSingapore
  4. 4.Singapore Institute for Neurotechnology (SINAPSE), Centre for Life SciencesNational University of SingaporeSingaporeSingapore
  5. 5.Department of BiotechnologyIndian Institute of Technology KharagpurKharagpurIndia
  6. 6.Mechanobiology InstituteNational University of Singapore, T-LabSingaporeSingapore
  7. 7.National Institute of Technology RourkelaOdishaIndia
  8. 8.Department of Chemical EngineeringIndian Institute of Technology GandhinagarGandhinagarIndia
  9. 9.School of Medical Science and TechnologyIndian Institute of Technology KharagpurKharagpurIndia
  10. 10.National University of Singapore (Suzhou) Research Institute (NUSRI)SuzhouChina

Personalised recommendations