Long wave ultra sound may enhance bone remodelling by altering the opg rankl ratio in human osteoblast like cells

LONG WAVE ULTRASOUND MAY ENHANCE BONE REMODELLING BY ALTERING THE OPG/RANKL RATIO IN HUMAN OSTEOBLAST-LIKE CELLS ABHIRAM MADDI (BDS, Manipal Academy of Higher Education, India) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ORAL AND MAXILLOFACIAL SURGERY FACULTY OF DENTISTRY NATIONAL UNIVERSITY OF SINGAPORE 2005 Supervisor: A/P Ho Kee Hai BDS (Singapore) FDSRCPS (Glasgow) FAMS (Singapore) Consultant Dept of Oral and Maxillofacial Surgery National University of Singapore Co-Supervisors: Dr Sajeda Meghji BSc, MPhil, PhD (London) Reader in Oral Biology Eastman Dental Institute for Oral Health Care Sciences University College London Professor Malcolm Harris DSc, MD, FDSRCS, FRCS (Edin) Dept of Oral and Maxillofacial Surgery Barts and The London School of Medicine and Dentistry Dedication I would like to dedicate this thesis to my family, my friends and most importantly to my mentors whose constant support and motivation made this work possible Acknowledgements I would like to thank my supervisor A/P Ho Kee Hai for his constant help, guidance and enthusiasm throughout my candidature I am grateful to him and the Faculty of Dentistry for sponsoring my training at the Eastman Dental Institute, UCL, London where the in vitro work was done I warmly acknowledge Dr Sajeda Meghji, my co-supervisor, under whose guidance I had received training for molecular research techniques like ELISA and Real Time PCR and the experience gained is an asset for my future research career I cannot thank enough Professor Malcolm Harris also a co-supervisor who has been a constant motivating factor apart from being a great teacher to me; he has helped me evolve in my research thinking I also acknowledge the help and guidance of my fellow researchers at the Eastman, Dr Ali Reza, Dr Rachel Williams, Dr Lindsay Sharp, Dr Hesham Khalil and Dr Wendy Heywood for teaching me cell culture and laboratory techniques I would also like to thank my colleagues and support staff at the Dentistry research labs, DSO for their constant help I would finally like to acknowledge the National University of Singapore for endowing me with the NUS Research Scholarship and the President’s Graduate Fellowship and would like to congratulate it for impartially selecting foreign students and grooming their talents Declaration I hereby declare that this dissertation is original to the best of my knowledge and does not contain any material, which has been submitted previously for any other degree or qualification Abhiram Maddi Table of Contents Dedication……………………………………………………………………………… Acknowledgements…………………………………………………………………… Declaration……………………………………………………………………………… Table of Contents……………………………………………………………………….6 List of Abbreviations………………………………………………………………… List of Symbols…………………………………………………………………………10 List of Figures………………………………………………………………………… 11 List of Tables………………………………………………………………………… 12 Abstract ……………………………………………………………………………… 13 CHAPTER I Introduction……………………………………………………………15 Osteoradionecrosis 1.1 Definition…………………………………………………………….16 1.2 Incidence……………………………………………………………16 1.3 Site of Incidence………………………………………………… 16 1.4 Classification of osteoradionecrosis…………………………… 16 1.5 Etiopathogenesis………………………………………………… 17 1.6 Risk Factors……………………………………………………… 19 1.7 Time of Development of osteoradionecrosis………………… 19 1.8 Diagnosis of osteoradionecrosis…………………………………19 1.9 Investigation of osteoradionecrosis………………………………20 1.10 Management of ORN…………………………………………….21 Therapeutic Ultrasound…………………………………………………….24 2.1 Historical…………………………………………………………….25 2.2 Physical Characteristics………………………………………… 27 2.3 Terminology…………………………………………………………28 2.4 Ultrasound Wave Form……………………………………………29 2.5 Ultrasound Transmission through tissues……………………….30 2.6 Absorption and Attenuation……………………………………….32 2.7 Therapeutic Ultrasound and Tissue Healing……………………34 2.8 Effect of Ultrasound on Tissue repair…………………………….37 2.9 The Ultrasound Apparatus……………………………………… 39 Bone Remodeling and TNFR Superfamily……………………………………42 3.1 Bone Remodelling………………………………………………….42 3.2 RANKL…………………………………………………………… 44 3.3 RANK……………………………………………………………… 45 3.4 OPG………………………………………………………………….47 3.5 TNF-α……………………………………………………………… 48 CHAPTER II Hypothesis and Aims……………………………………………….49 CHAPTER III Materials and Methods…………………………………………….50 Cell Culture……………………………………………………………50 Ultrasound Experiment………………………………………………52 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 55 Quantitative Real Time PCR (Q PCR)………………………… 59 Immunochemistry…………………………………………………….60 Statistical Analysis……………………………………………………61 CHAPTER IV Results………………………………………………………………62 Effect of Ultrasound on osteoprotegerin (OPG) and receptor activator for NF-қB ligand (RANKL) mRNA and protein production………………………………………………………… 62 Effect of Ultrasound on the mRNA and protein production of TNF-α in human osteoblast like cells…………………………….67 Effect of Ultrasound on the expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in human osteoblast like cells….68 CHAPTER V Discussion……………………………………………………………72 8.1 Introduction…………………………………………………………72 8.2 Discussion of Methodology……………………………………….75 8.3 Discussion of Results…………………………………………… 77 CHAPTER VI Conclusion 79 Bibliography .80 Paper Publications from this thesis………………………………………………98 List of Abbreviations ALP Alkaline Phosphatase DNA Deoxyribo Nucleic Acid ELISA Enzyme Linked Immunosorbant Assay GAPDH Glyceraldehyde Phosphate DeHydrogenase RNA Ribo Nucleic Acid mRNA Messenger RNA NCP Non collagenous protein OB Osteoblast OC Osteoclast OCN Osteocalcin OPG Osteoprotegerin ORN Osteoradionecrosis PCR Polymerase Chain Reaction Q-PCR Quantitative Polymerase Chain reaction RANK Receptor Activator of NF-қB RANKL Receptor Activator of NF-қB Ligand RT-PCR Reverse Transcriptase Polymerase Chain Reaction TNF-α Tumor Necrosis Factor alpha US Ultrasound List of Symbols λ Wave length µg Micrograms pg Picograms n Frequency ml Milli-litre µl Micro-litre V Velocity Hz Hertz w Watts 10 Fyfe MC, Chahl LA Mast cell degranulation: A possible mechanism of therapeutic ultrasound Ultrasound Med Biol 1982; (suppl.1):62-66 Gori F, Hofbauer LC, Dunstan CR, Spelsberg TC, Khosla S, Riggs BL.The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells are developmentally regulated Endocrinology 2000; 141: 4768-4776 Hakeda Y, Kobayashi Y, Yamaguchi K, Yasuda H, Tsuda E, Higashio K, Miyata T, Kumegawa M.Osteoclastogenesis inhibitory factor (OCIF) directly inhibits bone-resorbing activity of isolated mature osteoclasts Biochem Biophys Res Commun 1998 Oct 29;251(3):796-801 Harris M The conservative management of osteoradionecrosis of the mandible with ultrasound therapy Br J Oral Maxillofac Surg 1992; 30; 313 Harvey W, Dyson M, Pond JB, Grahame R The stimulation of protein synthesis in human fibroblasts by therapeutic ultrasound Rheumatol Rehab 1975; 14: 237 Heckman JD, Ryaby JP, McCabe J, Frey JJ, Kilcoyne RF Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound J Bone Joint Surg Am 1994; 76:26-34 84 Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption J Bone Miner Res 2000; 15 (1):2-12 Hofbauer LC, Heufelder The role of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in the pathogenesis and treatment of metabolic bone diseases J Clin Endocrinol Metab 2000; 85; 2355-2363 Hogan RDB, Burke KM, Franklin TD The effect of ultrasound on the micovascular hemo dynamics in skeletal muscle: Effects during ischaemia Microvasc Res 1982; 23;370-379 Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, Tan HL, Elliott G, Kelley MJ, Sarosi I, Wang L, Xia XZ, Elliott R, Chiu L, Black T, Scully S, Capparelli C, Morony S, Shimamoto G, Bass MB, Boyle WJ Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand Proc Natl Acad Sci USA, 1999; 96: 3540-3545 Ikeda T, Utsuyama M, Hirokawa K Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappaB, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones J Bone Miner Res 2001 Aug;16(8):1416-25 85 Kalle Aitasalo A modified protocol for early treatment of osteomyelitis and osteoradionecrosis of the mandible Head & Neck 1998 August; 411-417 Khosla S Minireview: the OPG/RANKL/RANK system Endocrinology 2001 Dec;142(12):5050-5 Review Klug V W, W G Franke, H G Knoch Scintigraphic control of bone fracture healing under ultrasonic stimulation: An experimental study Eur J Nucl Med 11: 494497, 1986 Komarova SV, Pilkington MF, Weidema AF, Dixon SJ, Sims SM RANK ligandinduced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts J Biol Chem 2003 Mar 7;278(10):8286-93 Epub 2002 Dec 20 Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis Nature 1999 28; 397 (6717):315-323 86 Konig A, Mulhbauer RC, Fleisch H Tumor necrosis factor alpha and interleukin stimulate bone resorption in vivo as measured by [³H] urinary tetracycline excretion from prelabeled mice J Bone Mineral Res 1988; 3: 621-627 Krembau FW: Physical considerations In: Biological Effects of Ultraound, ed by WL Nyborg and MC Ziskin, New York, Churchill Livingston, 1985, pp 14-16 Kwan Tat S, Padrines M, Theoleyre S, Heymann D, Fortun Y IL-6, RANKL, TNFα/IL-1: interrelations in bone resorption pathology Cytokine and Growth Factor Reviews 2004; 15; 49-60 Lam J, Nelson CA, Ross FP, Teitelbaum SL, Fremont DH Crystal structure of the TRANCE/RANKL cytokine reveals determinants of receptor-ligand specificity J Clin Invest 2001 Oct;108(7):971-9 Lanyon LE Osteocytes, strain detection, bone modeling and remodeling Calcif Tissue Int 1993; 53(Suppl 1):102-107 Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ Osteoprotegerin ligand is a cytokine that regulates osteoclasts differentiation and activation.Cell 1998 17; 93:165-176 87 Lai CF, Feng X, Nishimura R, Teitelbaum SL, Avioli LV, Ross FP, Cheng SL Transforming growth factor-beta up-regulates the beta integrin subunit expression via Sp1 and Smad signaling J Biol Chem 2000 Nov 17;275(46):36400-6 Lee SK, Lorenzo JA Regulation of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin mRNA expression by parathyroid hormone is predominantly mediated by the protein kinase a pathway in murine bone marrow cultures Bone 2002 Jul;31(1):252-9 Lee ZH, Kim HH Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts Biochem Biophys Res Commun 2003 May 30;305(2):211-4 Review Leung KS, Cheung WH, Zhang C, Lee KM and Lo HK Low Intensity pulsed ultrasound stimulates osteogenic activity of human periosteal cells Clin Orthop 2004; 418: 253-259 Lotsova E I, Amelia A Z: An experimental study of ultrasonic influence upon the healing of bone lesions Kuroortologia I Fizioterapiia 14: 20-22, 1977 88 Maier A gaggi Akieman H Santler G Anneg U Fell B karcher H Smolle – Juttner FM Freihs GB Review of severe osteoradionecrosis treated by surgery alone and with post operative hyperbaric oxygenation British Journal of Oral and Maxillofacial Surgery.2000, 38 (3) 173-6 Malaval L, Modrowski D, Gupta A K, Aubin J E Cellular expression of bonerelated proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures J Cell Physiol 1994 Mar;158(3):555-72 Marx RE, Ames JR The use of hyperbaric oxygen therapy in bony reconstruction of the irradiated and tissue deficient patient Journal of Oral and Maxillofacial Surgery 1982; 40, 412-420 Marx RE A new concept in the treatment of osteoradionecrosis Journal of Oral and Maxillofacial Surger 1983, 41, 351-357 Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin Biochem Biophys Res Commun 1998 29; 247: 610-615 89 Mizuno A, Kanno T, Hoshi M, Shibata O, Yano K, Fujise N, Kinosaki M, Yamaguchi K, Tsuda E, Murakami A, Yasuda H, Higashio K Transgenic mice overexpressing soluble osteoclast differentiation factor (sODF) exhibit severe osteoporosis J Bone Miner Metab 2002;20(6):337-44 Mont MA, Pilla AA, Tenreiro RA, et al.: The effects of ultrasonic stimuation on fresh fracture repair in rabbits Trans Orthop Res Soc 12: 97, 1987 Mortimer A J, Dyson M The effect of therapeutic ultrasound on calcium uptake in fibroblasts Ultrasound Med Biol 1988, 14, 499-506 Naruse K, Mikuni-Takagaki Y, Azuma Y, Ito M, Oota T, Kameyama K, Itoman M Anabolic response of mouse bone-marrow-derived stromal cell clone ST2 cells to low-intensity pulsed ultrasound Biochem Biophys Res Commun 2000 5; 268:216-220 Nolte PA, Klein-Nulend J, Albers GHR, Marti RK, Semeins CM, Goei SW, Burger EH Low intensity ultrasound stimulates endochondral ossification in vitro J Orthop Res 19: 301-307; 2001 Nolte PA, Van der krans A, Patka P Low intensity ultrasound in the treatment of of non-unions J Trauma 2001; 51:693-703 90 Nussbaum E The influence of ultrasound on healing tissues J Hand Ther 1998 Apr-Jun;11(2):140-7 Review Nyborg W L Ultrasonic microstreaming and related phenomenon Br J Cancer 45 (suppl): 156-160, 1982 Pilla AA, Mont MA, Nasser PR, et al Non-invasive low intensity pulsed ultrasound accelerates bone healing in the rabbit Journal of Orthopaedic Trauma 1990;4: 246-253 Paul M Lambert, Nancy Intriere and Ralph Eichstaedt Management of extractions in irradiated jaws: A protocol with Hyperbaric oxygen therapy Journal of Oral Maxillofacial Surgery 1997, 55; 268-274 Reher P, Elbeshir el-NI, Harvey W, Meghji S, Harris M The stimulation of bone formation in vitro by therapeutic ultrasound Ultrasound Med Biol 1997; 23:12518 Reher P, Doan N, Bradnock B, Meghji S, Harris M Effect of ultrasound on the production of IL-8, basic FGF and VEGF Cytokine 1999; 11: 416-23 91 Reher P, Harris M, Whiteman M, Hai HK, Meghji S.Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts Bone 2002; 31:236-241 Robertson VJ, Baker KG A review of therapeutic ultrasound: effectiveness studies Phys Ther 2001 Jul;81(7):1339-50 Review Ryaby JT, Bachner EJ, Bendo JA, Dalton PE, Tannebaum S, Pilla AA Low intensity pulsed ultrasound increases calcium incorporation in both differentiating cartilage and bone cell cultures Trans Orthop Res Soc 1989; 14-15 Salter DM, Wallace WH, Robb JE, Caldwell H, Wright MO Human bone cell hyperpolarization response to cyclical mechanical strain is mediated by an interleukin-1beta autocrine/paracrine loop J Bone Miner Res 2000; 15 :17461755 Sandberg MM, Aro HT, Vuorio EI Gene expression during bone repair Clin Orthop 1993 Apr; (289):292-312 Sato T, Selleri C, Anderson S, Young NS, Maciejewski JP Expression and modulation of cellular receptors for interferon-gamma, tumour necrosis factor, and Fas on human bone marrow CD34+ cells Br J Haematol 1997 May;97(2):356-65 92 Shedden R, Dunham J, Bitensky L, et al Changes in alkaline phosphatase activity in periosteal cells in healing fractures Calcif Tissue Res 1976; 22:19-25 Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Boyle WJ Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell 1997 18; 89:309319 Stewart C Bushong Radiological Science for Technologists, 8th Edition, 2004 Sun JS, Hong RC, Chang WH et al In vitro effects of low-intensity ultrasound stimulation on the bone cells J Biomed Mater Res 2001; 57: 449-456 Takeuchi Y, Fukumoto S, Matsumoto T Relationship between actions of transforming growth factor (TGF)-beta and cell surface expression of its receptors in clonal osteoblastic cells J Cell Physiol 1995 Mar;162(3):315-21 93 Tan KB, Harrop J, Reddy M, Young P, Terrett J, Emery J, Moore G, Truneh A Characterization of a novel TNF-like ligand and recently described TNF ligand and TNF receptor superfamily genes and their constitutive and inducible expression in hematopoietic and non-hematopoietic cells Gene 1997 Dec 19;204(1-2):35-46 Teitelbaum SL, Tondravi MM, Ross FP Osteoclasts, macrophages and the molecular mechanism of bone resorption J Leuk Biol 1997; 61; 381-388 Ter Haar G Therapeutic ultrasound Eur J Ultrasound 1999 Mar;9(1):3-9 Review Thomson BM, Mundy GR, Chambers TJ Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption J Immunol 1987 1; 138 (3):775-779 Tsai CL, Chang WH, Liu TK Preliminary studies of duration and intensity of ultrasonic treatments on fracture repair.Chin J Physiol 1992;35:21-26 Udagawa N, Takahashi N, Yasuda H, Mizuno A, Itoh K, Ueno Y, Shinki T, Gillespie MT, Martin TJ, Higashio K, Suda T Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function Endocrinology 2000;141:3478-3484 94 Wang SJ, Lewallen DG, Bolander ME, Chao EY, Ilstrup DM, Greenleaf JF Low intensity ultrasound treatment increases strength in a rat femoral fracture model J Orthop Res 1994;12:40-47 Warden SJ, Bennell KL, Matthews B, Brown DJ, McMeeken JM, Wark JD Efficacy of low-intensity pulsed ultrasound in the prevention of osteoporosis following spinal cord injury Bone 2001; 29:431-436 Watson JT, Moed BR, Karges DE, Cramer KE Pilon fractures Treatment protocol based on severity of soft tissue injury Clin Orthop Relat Res 2000 Jun;375:78-90 Webster D F, Harvey W, Dyson M, Pond JB The role of ultrasound-induced cavitation in the in vitro stimulation of collagen synthesis in human fibroblasts Ultrasonics 1980, 18, 33-37 Webster DF, Pond JB, Dyson M, Harvey W The role of cavitation in the in vitro stimulation of protein synthesis in human fibroblasts by ultrasound Ultrasound Med Biol 1978; 4: 343-351 95 Wilkin LD, Merrick MA, Kirby TE, Devor ST Influence of therapeutic ultrasound on skeletal muscle regeneration following blunt contusion Int J Sports Med 2004 Jan;25(1):73-7 Williams A R Production and transmission of ultrasound Physiotherapy 73(3): 113-116; 1987 Yang KH, Parvizi J, Wang SJ, Lewallen DG, Kinnick RR, Greenleaf JF, Bolander ME Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model J Orthop Res 1996;14:802-809 Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL Proc Natl Acad Sci USA 1998; 95; 3597-3602 Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio K Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro Endocrinology 1998; 39: 1329-1337 96 Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Goto M, Mochizuki SI, Tsuda E, Morinaga T, Udagawa N, Takahashi N, Suda T, Higashio K A novel molecular mechanism modulating osteoclast differentiation and function Bone 1999; 25; 109-113 Young S R The effect of therapeutic ultrasound on the biological mechanisms involved in dermal repair London: University of London; 1988 PhD Thesis Young S R, Dyson M Effect of therapeutic ultrasound on the healing of fullthickness excised skin lesions Ultrasonics 1990 May;28(3):175-80 Zou W, Hakim I, Tschoep K, Endres S, Bar-Shavit Z Tumor necrosis factoralpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism J Cell Biochem 2001 Jun 26-Jul 25;83(1):70-83 97 Paper Publication from this thesis Maddi A, Kee Hai H, Ong S T, Sharp L, Harris M, Meghji S Long wave ultrasound may enhance bone regeneration by altering the OPG/RANKL ratio in human osteoblast -like cells (accepted by BONE, e-pub ahead of print) Research Conferences Work from this thesis has been presented at the following conferences: Gordon Research Conference, “Bones and Teeth”, July10-15 2005, University of New England, Biddeford, Maine, USA 2nd Joint Conference of ECTS (European Calcified Tissue Society) and IBMS (International Bone and Mineral Society), Geneva, Switzerland June 2005 98 [...]... a sound wave will oscillate about a fixed point rather than move with the wave itself As the energy within the sound wave is passed to the material, it will cause oscillation of the particles of that material An increase in the molecular vibration in the tissue can result in heat generation, and ultrasound (US) can be used to produce thermal changes in the tissues In addition to thermal changes, the. .. treatment in tibial fractures fixed with a locked intramedullary nail (Emami et al., 1999) The presence of the intramedullary nail may be the reason for failure of ultrasound in this study because metals in the path of the beam increase the local temperatures resulting in thermal effects coming into play 2.2 Physical Characteristics of Ultrasound Sound waves are longitudinal waves consisting of areas... therapeutic ultrasound may increase bone regeneration by altering the OPG/ RANKL ratio in the bone micro-environment 14 CHAPTER I INTRODUCTION 1 Osteoradionecrosis (ORN) Radiotherapy is an essential treatment modality for oral and head and neck malignant neoplasms Unfortunately it induces alterations in the normal tissues, resulting in early and long term complications Mandibular osteoradionecrosis is the most... resulted in the emission of a high frequency sound wave which we call the ultrasonic wave or precisely as ultrasound The crystals when subjected to an alternating current, expand and contract resulting in the production of ultrasound Paul Langevin (France, 1926), during the First World War, used this effect in the detection of submarines He was also the first to observe the biological effects of ultrasound;... Disturbed bone healing may occur as a side-effect of various therapies like osteotomies, bone grafting, distraction osteogenesis and radiation therapy Over the years various interventions have been used to stimulate the healing process 24 and among them ultrasound remains distinguished by being non-invasive and easy to apply Ultrasound has been traditionally used in the field of physiotherapy to treat... to 10 minutes daily for 10 days using a frequency of 800 kHz and an intensity of 4 to 5 25 mW/cm² The first reported successes to ultrasound treatment lead to the widespread medical use of ultrasound, however, the influence of ultrasound on bone received little attention at first as bone was considered a limitation to the use of ultrasound and studies concerning the influence of ultrasound on bone focused... given by Hutchinson in 1996 seems to be the most sensible, which states that “ORN is an area of exposed bone in the mouth or on the face for more than two months in a previously irradiated field in the absence of recurrent tumour” 1.2 Incidence There is a great variance in the incidence rates of ORN in the literature According to the most recent study done by Sulaiman et al, 2003 the incidence of ORN... with alkaline phosphatase (ALP), an early bone marker and osteocalcin (OCN), a late marker ELISA was performed to estimate the amount of the cytokine released into the culture media, following US treatment The osteoblasts responded to US by significantly upregulating both the OPG mRNA and protein levels There was no RANKL mRNA expression observed in both the US and control groups and the protein levels... and not accessible to all the patients Therapeutic Ultrasound (US) has been shown to promote repair in bone fractures and soft tissue injuries in in vivo studies in rats and humans In vitro studies have shown that long wave US treated osteoblasts, fibroblasts and macrophages increased synthesis of growth factors which play prominent roles in angiogenesis and healing The cytokines, TNF-α (tumor necrosis... healing It is suggested that the application of US to injured tissues will, amongst other things, speed the rate of healing & enhance the quality of the repair The therapeutic effects of US are generally divided into: • Thermal Effects • Non-Thermal Effects 34 Thermal Effects: In thermal mode, it will be most effective in heating the dense collagenous tissues and will require a relatively high intensity, ... Hypothesis Therapeutic ultrasound enhances bone remodelling by enhancing OPG/RANKL ratio in vitro Therapeutic ultrasound does affect transcription and translation of TNF alpha in vitro Therapeutic ultrasound... of ultrasound in this study because metals in the path of the beam increase the local temperatures resulting in thermal effects coming into play 2.2 Physical Characteristics of Ultrasound Sound. .. influence of ultrasound on bone focused mainly on bone damage In 1950, Maintz published the first study in which the relationship between ultrasound and bone healing was investigated In three month