In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering

Ultrasound stimulation produces significant multifunctional effects that are directly relevant to alveolar bone formation, which is necessary for periodontal healing and regeneration. We focused to find out effects of specific duty cycles and the percentage of time that ultrasound is being generated over one on/off pulse period, under ultrasound stimulation. Low-intensity pulsed ultrasound ((LIPUS) 1 MHz) with duty cycles of 20% and 50% was used in this study, and human alveolar bone-derived mesenchymal stem cells (hABMSCs) were treated with an intensity of 50 mW/cm2 and exposure time of 10 min/day. hABMSCs exposed at duty cycles of 20% and 50% had similar cell viability (O.D.), which was higher (*P < 0.05) than that of control cells. The alkaline phosphatase (ALP) was significantly enhanced at 1 week with LIPUS treatment in osteogenic cultures as compared to control. Gene expressions showed significantly higher expression levels of CD29, CD44, COL1, and OCN in the hABMSCs under LIPUS treatment when compared to control after two weeks of treatment. The effects were partially controlled by LIPUS treatment, indicating that modulation of osteogenesis in hABMSCs was related to the specific stimulation. Furthermore, mineralized nodule formation was markedly increased after LIPUS treatment than that seen in untreated cells. Through simple staining methods such as Alizarin red and von Kossa staining, calcium deposits generated their highest levels at about 3 weeks. These results suggest that LIPUS could enhance the cell viability and osteogenic differentiation of hABMSCs, and could be part of effective treatment methods for clinical applications.

[1]  I Binderman,et al.  Bone remodelling induced by physical stress is prostaglandin E2 mediated. , 1980, Biochimica et biophysica acta.

[2]  R F Kilcoyne,et al.  Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound. , 1994, The Journal of bone and joint surgery. American volume.

[3]  Wenchuan Chen,et al.  Ultrasound: a potential technique to improve osseointegration of dental implants. , 2008, Medical hypotheses.

[4]  M. Schaffler,et al.  Prevention of fracture healing in rats by an inhibitor of angiogenesis. , 2001, Bone.

[5]  J B Pond,et al.  The role of ultrasound-induced cavitation in the 'in vitro' stimulation of collagen synthesis in human fibroblasts. , 1980, Ultrasonics.

[6]  Nicola Maffulli,et al.  The effects of LIPUS on soft-tissue healing: a review of literature. , 2008, British medical bulletin.

[7]  W. Harvey,et al.  The role of cavitation in the in vitro stimulation of protein synthesis in human fibroblasts by ultrasound. , 1978, Ultrasound in medicine & biology.

[8]  N S El-Said,et al.  Selective release of the flexor origin with transfer of flexor carpi ulnaris in cerebral palsy. , 2001, The Journal of bone and joint surgery. British volume.

[9]  S Meghji,et al.  Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts. , 2002, Bone.

[10]  J F Greenleaf,et al.  Low‐intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression , 1999, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[11]  Y. Azuma,et al.  Low-intensity pulsed ultrasound accelerates bone maturation in distraction osteogenesis in rabbits , 2000 .

[12]  Hani El-Mowafi,et al.  The effect of low-intensity pulsed ultrasound on callus maturation in tibial distraction osteogenesis , 2005, International Orthopaedics.

[13]  W. Harvey,et al.  The stimulation of protein synthesis in human fibroblasts by therapeutic ultrasound. , 1975, Rheumatology and rehabilitation.

[14]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[15]  D E Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton. , 1993, Science.

[16]  Gerry M Raghoebar,et al.  Ultrasound to stimulate early bone formation in a distraction gap: a double blind randomised clinical pilot trial in the edentulous mandible. , 2005, Archives of oral biology.

[17]  N Matsui,et al.  Low‐intensity pulsed ultrasound initiates bone healing in rat nonunion fracture model. , 2001, Journal of ultrasound in medicine : official journal of the American Institute of Ultrasound in Medicine.

[18]  Win-Li Lin,et al.  Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts. , 2005, Bone.

[19]  Javad Parvizi,et al.  Calcium signaling is required for ultrasound‐stimulated aggrecan synthesis by rat chondrocytes , 2002, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[20]  P. Branemark,et al.  Long-term follow-up study of osseointegrated implants in the treatment of totally edentulous jaws. , 1990, The International journal of oral & maxillofacial implants.

[21]  J. C. Lin,et al.  Study of thermal effects of ultrasound stimulation on fracture healing , 2002, Bioelectromagnetics.

[22]  S. Cook,et al.  Acceleration of Tibia and Distal Radius Fracture Healing in Patients Who Smoke , 1997, Clinical orthopaedics and related research.

[23]  V. Frankel,et al.  Ultrasound – an alternative healing method for nonunions? , 2000, Archives of Orthopaedic and Trauma Surgery.

[24]  T. Takato,et al.  Cyclooxygenase-2 activity is essential for the osseointegration of dental implants. , 2007, International journal of oral and maxillofacial surgery.

[25]  M. Dyson.  Non-thermal cellular effects of ultrasound. , 1982, The British journal of cancer. Supplement.

[26]  A. Banes,et al.  Osteoblasts increase their rate of division and align in response to cyclic, mechanical tension in vitro. , 1988, Bone and mineral.

[27]  G. Du,et al.  Temperature elevation in tissues generated by finite-amplitude tone bursts of ultrasound , 1990 .

[28]  A. Eisen,et al.  Human skin fibroblast collagenase. Assessment of activation energy and deuterium isotope effect with collagenous substrates. , 1981, The Journal of biological chemistry.

[29]  D. Somjen,et al.  Biochemical pathways involved in the translation of physical stimulus into biological message , 2006, Calcified Tissue International.

[30]  T. Kokubu,et al.  Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts. , 1999, Biochemical and biophysical research communications.

[31]  C. Rubin,et al.  Strain Gradients Correlate with Sites of Periosteal Bone Formation , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[32]  K. Lim,et al.  Effects of Electromagnetic Fields on Osteogenesis of Human Alveolar Bone-Derived Mesenchymal Stem Cells , 2013, BioMed research international.

[33]  D. Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton , 1993 .

[34]  Walter H. Chang,et al.  Optimum intensities of ultrasound for PGE(2) secretion and growth of osteoblasts. , 2002, Ultrasound in medicine & biology.

[35]  Ching‐Jen Wang,et al.  Nitric oxide mediates ultrasound-induced hypoxia-inducible factor-1alpha activation and vascular endothelial growth factor-A expression in human osteoblasts. , 2004, Bone.

[36]  L A Crum,et al.  The significance of membrane changes in the safe and effective use of therapeutic and diagnostic ultrasound. , 1989, Physics in medicine and biology.

[37]  D. Jones,et al.  Biochemical signal transduction of mechanical strain in osteoblast-like cells. , 1991, Biomaterials.

[38]  U. Zor,et al.  The transduction of mechanical force into biochemical events in bone cells may involve activation of phospholipase A2 , 1988, Calcified Tissue International.

[39]  Valeria Panebianco,et al.  Supplementary Figure 2 , 2012 .

[40]  W. Marsden.  I and J , 2012 .

[41]  M. Dyson,et al.  The effect of therapeutic ultrasound on calcium uptake in fibroblasts. , 1988, Ultrasound in medicine & biology.

[42]  C. Rubin,et al.  Pressure regulates osteoclast formation and MCSF expression in marrow culture , 1997, Journal of cellular physiology.

[43]  Stricklin,et al.  Human Skin Fibroblast Collagenase Inhibitor , 2022 .

[44]  Javad Parvizi,et al.  Exposure to low‐intensity ultrasound increases aggrecan gene expression in a rat femur fracture model , 1996, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[45]  Niklaus P Lang,et al.  Critical review of immediate implant loading. , 2003, Clinical oral implants research.

[46]  Tomoo Kamakura,et al.  Acoustic streaming induced in focused Gaussian beams , 1995 .

[47]  C. Rubin,et al.  The use of low-intensity ultrasound to accelerate the healing of fractures. , 2001, The Journal of bone and joint surgery. American volume.