Skip to main content

Advertisement

Log in

Low intensity ultrasound stimulates osteoblast migration at different frequencies

  • Short communication
  • Published:
Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45 kHz ultrasound (25 mW/cm2) or pulsed 1 MHz ultrasound (250 mW/cm2) for 30 min followed by 2 days’ culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2 days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing.

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.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  1. 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 48:33033–33038

    Article  Google Scholar 

  2. Claes L, Willie B (2007) The enhancement of bone regeneration by ultrasound. Progr Biophys Mol Biol 93:384–398

    Article  Google Scholar 

  3. Dyson M, Brookes M (1983) Stimulation of bone repair by ultrasound. Ultrasound Med Biol Suppl 2:61–66

    Google Scholar 

  4. Ter Haar G (2007) Therapeutic applications of ultrasound. Progr Biophys Mol Biol 93:111–129

    Article  Google Scholar 

  5. Khan Y, Laurencin CT (2008) Fracture repair with ultrasound: clinical and cell-based evaluation. J Bone Joint Surg Am 90A:138–144

    Article  Google Scholar 

  6. Yang RS, Lin WL, Chen YZ, Tang CH, Huang TH, Lu BY, Fu WM (2005) Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts. Bone 36:276–283

    Article  PubMed  CAS  Google Scholar 

  7. Dalecki D (2004) Mechanical bioeffects of ultrasound. Ann Rev Biomed Eng 6:229–248

    Article  CAS  Google Scholar 

  8. Reher P, Doan N, Bradnock B, Meghji S, Harris M (1999) Effect of ultrasound on the production of IL-8, basic FGF and VEGF. Cytokine 11:416–423

    Article  PubMed  CAS  Google Scholar 

  9. Scheven BA, Man J, Millard JL, Cooper PR, Lea SC, Walmsley AD, Smith AJ (2009) VEGF and odontoblast-like cells: stimulation by low frequency ultrasound. Arch Oral Biol 54:185–191

    Article  PubMed  CAS  Google Scholar 

  10. Harle J, Mayia F, Olsen I, Salih V (2005) Effects of ultrasound on transforming growth factor-beta genes in bone cells. Eur Cell Mater 10:70–76

    PubMed  CAS  Google Scholar 

  11. Park K, Hoffmeister B, Han DK, Hasty K (2007) Therapeutic ultrasound effects on interleukin-1 beta stimulated cartilage construct in vitro. Ultrasound Med Biol 33:286–295

    Article  PubMed  Google Scholar 

  12. 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 16:671–680

    Article  PubMed  CAS  Google Scholar 

  13. Griffin XL, Costello I, Costa ML (2008) The role of low intensity pulsed ultrasound therapy in the management of acute fractures: a systematic review. J Trauma 65:1446–1452

    Article  PubMed  Google Scholar 

  14. Katano M, Naruse K, Uchida K, Mikuni-Takagaki Y, Takaso M, Itoman M, Urabe K (2011) Low intensity pulsed ultrasound accelerates delayed healing process by reducing the time required for the completion of endochondral ossification in the aged mouse femur fracture model. Exp Anim 60:385–395

    Article  PubMed  CAS  Google Scholar 

  15. Lai CH, Chuang CC, Li JKJ, Chen SC, Chang WHS (2011) Effects of ultrasound on osteotomy healing in a rabbit fracture model. Ultrasound Med Biol 37:1635–1643

    Article  PubMed  Google Scholar 

  16. Rutten S, Nolte PA, Korstjens CM, van Duin MA, Klein-Nulend J (2008) Low-intensity pulsed ultrasound increases bone volume, osteoid thickness and mineral apposition rate in the area of fracture healing in patients with a delayed union of the osteotomized fibula. Bone 43:348–354

    Article  PubMed  Google Scholar 

  17. Choi BH, Woo JI, Min BH, Park SR (2006) Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture. J Biomed Mater Res Part A 79A:858–864

    Article  CAS  Google Scholar 

  18. Harle J, Salih V, Mayia F, Knowles JC, Olsen I (2001) Effects of ultrasound on the growth and function of bone and periodontal ligament cells in vitro. Ultrasound Med Biol 27:579–586

    Article  PubMed  CAS  Google Scholar 

  19. 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 25:392–399

    Article  PubMed  Google Scholar 

  20. Korstjens CM, Nolte PA, Burger EH, Albers GH, Semeins CM, Aartman IH, Goei SW, Klein-Nulend J (2004) Stimulation of bone cell differentiation by low-intensity ultrasound—a histomorphometric in vitro study. J Orthop Res 22:495–500

    Article  PubMed  CAS  Google Scholar 

  21. Naruse K, Miyauchi A, Itoman M, Mikuni-Takagaki Y (2003) Distinct anabolic response of osteoblast to low-intensity pulsed ultrasound. J Bone Miner Res 18:360–369

    Article  PubMed  CAS  Google Scholar 

  22. Reher P, Doan N, Bradnock B, Meghji S, Harris M (1998) Therapeutic ultrasound for osteoradionecrosis: an in vitro comparison between 1 MHz and 45 kHz machines. Eur J Cancer 34:1962–1968

    Article  PubMed  CAS  Google Scholar 

  23. Takayama T, Suzuki N, Ikeda K, Shimada T, Suzuki A, Maeno M, Otsuka K, Ito K (2007) Low-intensity pulsed ultrasound stimulates osteogenic differentiation in ROS 17/2.8 cells. Life Sci 80:965–971

    Article  PubMed  CAS  Google Scholar 

  24. Unsworth J, Kaneez S, Harris S, Ridgway J, Fenwick S, Chenery D, Harrison A (2007) Pulsed low intensity ultrasound enhances mineralisation in preosteoblast cells. Ultrasound Med Biol 33:1468–1474

    Article  PubMed  Google Scholar 

  25. Tsai W-C, Chen JY-S, Pang J-HS, Hsu C-C, Lin M-S, Chieh L-W (2008) Therapeutic ultrasound stimulation of tendon cell migration. Conn Tissue Res 49:367–373

    Article  CAS  Google Scholar 

  26. Liang C–C, Park AY, Guan J-L (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protocols 2:329–333

    Article  CAS  Google Scholar 

  27. Hill GE, Fenwick S, Matthews BJ, Chivers RA, Southgate J (2005) The effect of low-intensity pulsed ultrasound on repair of epithelial cell monolayers in vitro. Ultrasound Med Biol 31:1701–1706

    Article  PubMed  Google Scholar 

  28. McCarroll JA, Phillips PA, Kumar RK, Park S, Pirola RC, Wilson JS, Apte MV (2004) Pancreatic stellate cell migration: role of the phosphatidylinositol 3-kinase (PI3-kinase) pathway. Biochem Pharmacol 67:1215–1225

    Article  PubMed  CAS  Google Scholar 

  29. Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198

    Article  PubMed  CAS  Google Scholar 

  30. Patel US, Walmsley AD, Scheven BA (2012) An in vitro set-up to study low-frequency ultrasound effects on dental cells. AIP Conf Proc (in press)

  31. Man J, RM Shelton, PR Cooper, Scheven BA (2012) Low-intensity low-frequency ultrasound promotes proliferation and differentiation of odontoblast-like cells. J Endod 38:608–613

    Google Scholar 

  32. Shelton RM, Landini G (1997) Image analysis of primary bone-derived cells on different polystyrene surfaces. Cell Mater 7:147–159

    Google Scholar 

  33. Lee JG, Kay EDP (2006) FGF-2-induced wound healing in corneal endothelial cells requires Cdc42 activation and Rho inactivation through the phosphatidylinositol 3-kinase pathway. Invest Ophthalmol Vis Sci 47:1376–1386

    Article  PubMed  Google Scholar 

  34. Hauser J, Hauser M, Muhr G, Esenwein S (2009) Ultrasound-induced modifications of cytoskeletal components in osteoblast-like SAOS-2 cells. J Orthop Res 27:286–294

    Article  PubMed  Google Scholar 

  35. Ng GYF, Fung DTC (2007) The effect of therapeutic ultrasound intensity on the ultrastructural morphology of tendon repair. Ultrasound Med Biol 33:1750–1754

    Article  PubMed  Google Scholar 

  36. Angle SR, Sena K, Sumner DR, Virdi AS (2011) Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound. Ultrasonics 51:281–288

    Article  PubMed  CAS  Google Scholar 

  37. Dalla-Bona DA, Tanaka E, Inubushi T, Oka H, Ohta A, Okada H, Miyauchi M, Takata T, Tanne K (2008) Cementoblast response to low- and high-intensity ultrasound. Arch Oral Biol 53:318–323

    Article  PubMed  CAS  Google Scholar 

  38. Li JK, Lin JC, Liu HC, Sun JS, Ruaan RC, Shih C, Chang WH (2006) Comparison of ultrasound and electromagnetic field effects on osteoblast growth. Ultrasound Med Biol 32:769–775

    Article  PubMed  Google Scholar 

  39. Alter A, Rozenszajn LA, Miller HI, Rosenschein U (1998) Ultrasound inhibits the adhesion and migration of smooth muscle cells in vitro. Ultrasound Med Biol 24:711–721

    Article  PubMed  CAS  Google Scholar 

  40. Hsu S-K, Huang W-T, Liu B-S, Li S-M, Chen H-T, Chang C-J (2011) Effects of near-field ultrasound stimulation on new bone formation and osseointegration of dental titanium implants in vitro and in vivo. Ultrasound Med Biol 37:403–416

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by a University of Birmingham School of Dentistry PhD grant (Ms J. Man).

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ben A. Scheven.

About this article

Cite this article

Man, J., Shelton, R.M., Cooper, P.R. et al. Low intensity ultrasound stimulates osteoblast migration at different frequencies. J Bone Miner Metab 30, 602–607 (2012). https://doi.org/10.1007/s00774-012-0368-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00774-012-0368-y

Keywords

Navigation