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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
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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
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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
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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.
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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
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Table of Contents Dedication………………………………………………………………………………..3 Acknowledgements……………………………………………………………………..4 Declaration……………………………………………………………………………… 5 Table of Contents……………………………………………………………………….6 List of Abbreviations…………………………………………………………………....9 List of Symbols…………………………………………………………………………10 List of Figures…………………………………………………………………………..11 List of Tables…………………………………………………………………………...12 Abstract..………………………………………………………………………………. 13 CHAPTER I. Introduction……………………………………………………………15
1. 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
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2. 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
3. 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
1 Cell Culture……………………………………………………………50
2 Ultrasound Experiment………………………………………………52
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3 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)...55
4 Quantitative Real Time PCR (Q PCR)…………………………......59
5 Immunochemistry…………………………………………………….60
6 Statistical Analysis……………………………………………………61
CHAPTER IV. Results………………………………………………………………62
1. Effect of Ultrasound on osteoprotegerin (OPG) and receptor
activator for NF-қB ligand (RANKL) mRNA and protein
production…………………………………………………………..62
2. Effect of Ultrasound on the mRNA and protein production of TNF-α in human osteoblast like cells…………………………….67 3. 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
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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
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List of Symbols λ Wave length µg Micrograms pg Picograms n Frequency ml Milli-litre µl Micro-litre V Velocity Hz Hertz w Watts
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List of Figures Figure 1 Shape of the Ultrasound Beam…………………………………………29
Figure 2 Rarefaction of the Ultrasound Beam…………………………………..32
Figure 3 The Long Wave Ultrasound Machine………………………………….39 Figure 4 The Ultrasound Transducer…………………………………………….40 Figure 5 An Electric Field re-aligns the dipoles in a Piezo-electric crystal…..41
Figure 6 Current understanding of preosteoblastic / stromal cell regulation of
Osteoclastogenesis……………………………………………………..46
Figure 7 US treatment being performed in a sterile air-flow chamber………..52
Figure 8 The administration of US……………………………………………….53
Figure 9 Schematic of the Ultrasound Experiment……………………………..54
Figure 10 Pictures of gel analysis of RT-PCR products of GAPDH and OPG..62 Figure 11 OPG mRNA expression at various time points………………...…......63 Figure 12 OPG protein levels………………………….……………………………64 Figure 13 RANKL protein levels………………….………………………………...66 Figure 14 TNF-α protein levels.…………………………………………………….67 Figure 15 Pictures of gel analysis of RT-PCR products of GAPDH and OCN..68 Figure 16 OCN protein levels……………………………………………………….69 Figure 17 ALP mRNA expression at various time points………...………………70
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List of Tables Table 1 Approximate Velocities of Ultrasound through Selected Materials…...31
Table 2 Primer Sequences for RT-PCR…………………………………………..58
Table 3 Summary of Results………………………………………………………..71
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Abstract
Osteoradionecrosis (ORN) of the mandible has been a formidable long term
complication of radiotherapy, instituted for the treatment of tumors of the head
and neck region. The un-restorable decayed teeth in the mandible in the path of
radiation have to be removed, but whether before or after radiation, such
extractions can still lead to ORN. Its mainstream prophylaxis and treatment is
Hyperbaric oxygen therapy (HBO), which is expensive 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 factor alpha), receptor
activator of NF-κB Ligand (RANKL) and osteoprotegerin (OPG) have been
shown to act directly or indirectly on osteogenic cells and their precursors
to control differentiation, resorption and bone formation. RANKL and TNF-α
promote osteoclast differentiation and thus bone resorption whereas OPG
promotes osteoblast differentiation and also competes with RANKL for the
receptor activator of NF-κB (RANK) receptor present on the osteoclast. Human
osteoblast cell line (MG63 cells) were treated with long wave (45 KHz ,
intensity-30mW/cm2) continuous ultrasound (US) and incubated for various
time periods following the treatment. The reverse transcriptase polymerase
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chain reaction (RT-PCR) technique was used for observing genetic
expression and real time PCR for quantitative analysis of the genetic
expression of RANKL and OPG along 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 were also very low in both groups and
significantly low in the US group. There was also no TNF-α expression and
the TNFα protein levels were insignificant. ALP and OCN mRNA were
significantly up regulated in the US group.
To my knowledge this is the first study that shows the effect of US on OPG and
RANKL. US appears to up regulate OPG and may down regulate RANKL
production. From these findings, we conclude that therapeutic ultrasound may
increase bone regeneration by altering the OPG/RANKL ratio in the bone
micro-environment.
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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 common long term complication of radiotherapy with a variable
incidence ranging from 2 to 44.2%. With adequate prevention the incidence is
still around 2-5% (Reher et al.)
Osteoradionecrosis (ORN) of the mandible was for a long time considered as an
osteomyelitis of the irradiated bone resulting from a triad of radiation, trauma and
infection. Marx and Klinge redefined this concept and pointed out that
osteoradionecrosis is not a primary infection of the bone; rather it is induced by a
metabolic and tissue haemostatic deficiency due to radiation induced cellular
injury. In 1983, Marx at the University of Miami described a rational foundation
idea, namely radiation tissue hypoxia, hypovascularity and hypocellularity, tissue
breakdown and chronic non-healing wounds as the main pathogenesis for ORN.
Bras et al., have reported that the radiation induced obliteration of the inferior
alveolar artery is the dominant factor in the onset of osteoradionecrosis leading
to an ischaemic necrosis of bone. Marx et al., also showed that micro-organisms
are not the primary causative factor, but play a role only as contaminants in
osteoradionecrosis.
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1.1 Definition
Many definitions have been attributed to ORN over the years but a definition
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 was 2% among 187 cancer patients who underwent 951 dental extractions
with only 7 patients treated with HBO therapy prophylactically.
1.3 Site of Incidence
The mandible is the most common site of incidence of ORN probably because it
is often necessary to deliver a high dose of radiation to tumors of the tongue and
floor of the mouth and also probably as the blood supply is less abundant as
compared to the maxilla. Furthermore most maxillary tumors are treated
surgically before or after radiotherapy.
1.4 Classification of ORN
Coffin in 1983 examined 2853 patients who received radiotherapy for head and
neck malignancies and suggested that ORN develops in two forms a) Minor and
b) Major. The Minor form is considered to be a series of small sequestra which
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separate spontaneously after varying periods of time and can be seen clinically
but not radiologically. The Major form occurs when necrosis involves the entire
thickness of the jaw and a pathological fracture is inevitable. This form is very
obvious radiologically.
1.5 Etiopathogenesis
Many theories have been formulated to explain the etiology of ORN since its first
observation in 1920s but the most accepted one originates from the work done
by Marx in 1983.
The Radiation, Trauma and Infection Theory
In 1970 Meyer named the classical triad of ORN as “radiation, trauma and
infection”. He described the role of trauma as a portal of entry for the oral
bacterial flora into the underlying bone.
The theory of non-healing wound due to Hypoxic-Hypovascular-
Hypocellular tissue
Marx in 1983 examined the traditional concept of the pathophysiology of ORN
described by Meyer questioning the occurrence of ORN without trauma or
infection. He studied 26 cases of ORN from which 12 en bloc resection
specimens were cultured and stained for micro-organisms. The microbiology
reports showed that all specimens were infected superficially but no organisms
could be cultutred from the deep, so called infected bone of ORN. The
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histological findings observed by Marx showed endothelial death, hyalinization
and thrombosis of vessels with a fibrotic periosteum. Osteoblasts and osteocytes
were deficient with fibrosis of the marrow spaces. The overall result was a
composite tissue, which is hypovascular and hypocellular and was proven to be
hypoxic compared with non-irradiated tissue by direct measurement .
Current Concepts
Bilal et al, have found in a histological study of human specimens with ORN as
well as in specimens subjected to just 36 Gy radiation after a few weeks interval,
a loss of vitality of osteocytes. Marx et al and Rosenberg et al 2003 have
reported a type of osteonecrosis occurring in patients using bisphosphonates,
which bears characteristic resemblance to ORN. These osteonecrosis patients
have undergone suppression of osteoclasia with biphosphonates.
Bisphosphonates could reduce osteoclastic bone resorption through: (a) inhibition
of osteoclast recruitment to the bone surface; (b) inhibition of osteoclast activity
on the bone surface; (c) shortening of the osteoclast life span; and (d) alteration
of the bone or bone mineral in ways which reduce, by a pure physicochemical
and not a cellular mechanism, the rate of its dissolution. The first three effects
could be due to direct action on the osteoclast, or indirectly via the cells which
modulate osteoclast activity. So ORN might be the result of early cellular effects
of radiation.
.
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1.6 Risk Factors
• Tumour size and location
• Radiation dose
• Local Trauma
• Dental Extractions
• Infection
• Immune defects
• Malnutrition
Many patients abuse alcohol and tobacco and are in poor general medical
condition, which together with poor nutritional status and lack of oral hygiene may
place them at a higher risk of ORN.
1.7 Time of Development of ORN
The time of development of ORN following radiation can be variable but the
majority of cases occur between 4 months and 2 years. Gowgiel in 1960 noted
that ORN of the mandible develops within 2 years of irradiation. Clayman in 1997
found that the highest risk for ORN was between 4 and 12 months following
irradiation.
1.8 Diagnosis of ORN
The diagnosis of ORN is primarily based on the clinical signs of ulceration of the
mucous membrane with exposure of necrotic bone. The lesion may be
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accompanied by symptoms of pain, dysesthesia, fetor oris, dysguesia and food
impaction in the area (Braumer et al, 1979; Epstein et al, 1987).
Marx and Johnson (1987) found the following diagnostic signs to correlate with
increased signs of radiation tissue injuries:
• Induration of tissue
• Mucosal radiation telangiectasias
• Loss of facial hair growth
• Cutaneous atrophy
• Cutaneous flaking and keratinization
• Profound xerostomia
• Profound taste loss
1.9 Investigation of ORN
ORN can be investigated by many techniques but the ideal investigative tool
according to Hutchinson (1996) should be able to offer the following:
1. Record quantitatively and qualitatively the severity and extent
2. Monitor the progress of treatment
3. Predict patients at risk
4. Predict risk factors more confidently
5. Permit comparisons of treatment regimens
6. Predict the level of bone damage above which surgery is essential
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The following investigations would be useful:
1. Radiography, CT and MRI
2. Nuclear medicine
3. Ultrasonography
4. Transcutaneous and transmucosal oximetry
5. Near Infrared Spectroscopy
1.10 Management of ORN
The aims of the treatment are elimination of pain and associated infections,
achieve vascularisation and mucosal/skin coverage, improvement of mouth
function (opening, speech, mastication) and the elimination of deformity (fistulas,
bone exposures and pathological fractures).
The various treatment options for osteoradionecrosis are as follows –
1. Antibiotics and curettage.
2. Hyperbaric oxygen (HBO) therapy with or without surgery.
3. Debridement and local flaps.
4. Resection and reconstruction.
5. Therapeutic Ultrasound (US) - Most recently described (Harris 1992, Refer et
al, 1997, 1998, Doan et al., 1999)
Dental management of patients who are about to receive therapeutic radiation
involving the jaws, remains a perplexing problem. The unrestorable decayed
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teeth in the mandible which come in the path of radiation have to removed,
whether before or after radiation and can lead to osteoradionecrosis.
Conservative Management
According to Scully and Epstein (1996), conservative management with
medication and local wound care help in resolving 60% of the cases.
Maintenance of good oral hygiene with the use of 0.02% chlorhexidene mouth
washes after meals and constant saline washes are a must. Debris should be
washed/irrigated and sequestra should be allowed to separate spontaneously or
gently removed, since any surgical interference may encourage extension of the
necrotic process because of the lack of normal bone repair. Though ORN is not
primarily an infectious process, tetracyclines have been recommended because
of their selective uptake by bone (Rankow and Weissman, 1971). Penicillin has
also been used, because of the involvement of oral bacteria in the superficial
contamination (Marx et al 1985). Morton and Simpson, 1986 recommend packs
of BIPP (Bismuth and Iodoform Paraffin paste) for covering small areas of
exposed bone and delicate granulation tissue for keeping necrotic bone cavities
clean.
Hyperbaric Oxygen (HBO) therapy
HBO is administered as 20 sessions of 90 minutes each, breathing 100%
humidified oxygen at 2.4 atmospheres absolute pressure before surgery and 10
sessions after surgery. The empirical use of HBO to promote neovascularity and
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neocellularity in irradiated and other sclerotic bone conditions has had success,
though usually as an adjunctive preparation to resection and reconstruction.
Marx has confirmed the value of hyperbaric oxygen as an adjunct to promote
neovascularity and neocellularity but recommends radical excision and
reconstructive surgery for 70% of the cases. Both the adjunctive HBO therapy
and surgery are a formidable experience to the patient who has already had
extensive surgery and other therapies for the malignant neoplasm. The major
disadvantages of HBO are that it is time consuming and expensive and most of
the previous studies done to prove its efficacy are uncontrolled. Also HBO
therapy is hazardous in patients with chronic emphysematous lung disease
which is not uncommonly associated with oral cancer.
According to Clayman it costs 1.5 million dollars to treat 100 patients with HBO.
In Singapore the cost of HBO therapy per person for 30 sessions is 7800 Sing $
at the rate of 260 S$ per session (Tan Tok Seng Hospital). A multicentre
randomized, placebo-controlled, double blind trial done by Annane et al showed
that the patients treated with HBO not only failed to benefit from the treatment but
also they had an unfavourable outcome as compared to the patients treated with
placebo. Similarly Cawood et al have recently shown in 329 patients that HBO
did not improve the integration outcome of titanium implants in irradiated jaws
(unpublished). Coulthard et al, in their review on the therapeutic use of HBO for
irradiated dental implant patients, have clearly mentioned that there is no
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substantial evidence for the use of HBO and that more randomized controlled
trials are required to determine its effectiveness.
Ultrasound (US) Therapy
It is obvious that HBO could not cure many cases of ORN and moreover it is too
expensive to use it as a prophylactic measure in oral cancer patients receiving
radiotherapy. The cost of HBO for a single patient is enough for buying at least 5
US machines which are readily available, economic and easy to apply. US has
been proven to enhance bone formation, angiogenesis and synthesis of growth
factors and cytokines which are essential to bone metabolism.
Surgery
Surgery is very often a treatment option for ORN. Surgical options start with the
removal of small sequestra and increase depending on the case to
sequestrectomy, alveolectomy with primary closure, closure of oro-cutaneous
fistulae and flaps to cover the area. In extreme cases large resections and hemi-
mandibulectomies are performed and the bone should be reconstructed
preferably with a bone source with its own blood supply, like fibula or iliac crest
vascularized flaps.
2. Therapeutic 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
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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 soft tissue disorders by deep heating the tissues using intensities of 0.5
to 3 watts/cm². The intensities used for bone healing are considerably lower than
those used in physiotherapy because of the risk of over-heating the bone.
2.1 Historical
It was in 1880 when Jacques and Pierre Curie first observed that certain crystals
generated electricity when distorted. This observed effect which was called the
piezo-electric effect when reversed 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; fish introduced into a tank with
strong ultrasound field died after a period of violent motions and investigators
experienced pain of considerable severity when they thrust their hands into the
tank.
Pohlman in Germany,1938, was the first to construct an ultrasound device to
treat patients and found that patients with lower back pain, myalgias and
neuralgias responded favorably to treatment of the affected areas for 5 to 10
minutes daily for 10 days using a frequency of 800 kHz and an intensity of 4 to 5
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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 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 old rabbits, a piece
of the radius was resected bilaterally and the ultrasound treatment regimen
involved exposure to different intensities for different time periods. A frequency of
800 kHz was used. The resected sites were examined histologically and
radiologically. Exposures to ultrasound at higher intensities showed a reduction
and arrest of callus formation and detachment of epiphysis. The untreated legs
healed without complications. Interestingly lower doses did cause osteogenesis
at a site distant from the resected site and ultrasound application and also the
simultaneously exposed ulnae showed sub-periosteal osteogenesis. The study
did not show any accelerated healing of bone. However, similar treatment
regimens showed positive effects on callus formation in another study which
involved bilateral femoral fractures in rabbits (De Nunno, 1952). In a controlled
study in rabbits by Carradi and Cozzolino, 1952 continuous wave 800 kHz
ultrasound of 1.5 W/cm² was found to stimulate the formation of callus in radial
fractures. Strauß in 1948 reported an accelerated healing of chronic
osteomyelitis due to gun-shot wounds in human subjects. Two cases of
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osteoradionecrosis, were reported by Halsscheidt et al, 1949, in which
ultrasound treatment led to the covering of the non-healing bone with fresh
granulation tissue, followed by the formation of a sequester and healing of the
defects. Knoch in 1965 successfully treated 31 patients with different types of
fracture non-unions with ultrasound. 27 recalcitrant non-unions were successfully
treated by Xavier and Duarte in 1983 using pulsed ultrasound at an intensity of
30mW/cm². A double blind study done using SAFHS (Sonic Accelerated Fracture
Healing System) showed that ultrasound reduced the time to union by 38% in 61
dorsally angulated fractures (Kristiansen et al., 1997). There was no difference in
the healing rates between treatment and placebo groups during a 75 day course
of SAFHS 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 of compression and
rarefaction. Particles of a material, when exposed to 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 vibration of the tissues appears to
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have effects which are generally considered to be non thermal in nature. As the
US wave passes through a material (the tissues), the energy levels within the
wave will diminish as energy is transferred to the material. The energy absorption
and attenuation characteristics of US waves have been documented for several
types of tissue.
2.3 Terminology:
Frequency - the number of times a particle experiences a complete
compression/rarefaction cycle in 1 second.
Wave Length - the distance between two equivalent points on the waveform in
the particular medium.
Velocity - the velocity at which the wave (disturbance) travels through the
medium. In a saline solution, the velocity of US is approximately 1500 metre sec-1
compared with approximately 350 metre sec-1
in air (sound waves can travel
more rapidly in a denser medium). The velocity of US in most tissues is thought
to be similar to that in saline. These three factors are related, but are not
constant for all types of tissue. Average figures are most commonly used to
represent the passage of US in the tissues. The mathematical representation of
the relationship is V = F. λ where V is velocity, F is frequency and λ is the
wavelength.
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2.4 US Waveform
The US beam is not uniform and changes in its nature with distance from the
transducer. The US beam nearest the treatment head is called the Near field, the
Interference field or the Frenzel zone. The behaviour of US in this field is far from
regular, with areas of significant interference. The US energy in parts of this field
can be many times greater than the output set on the machine (possibly as much
Figure 1 Shape of the Ultrasound Beam
[Stewart C Bushong, Radiological Science for Technologists, 8th Edition, 2004]
as 12 to 15 times greater). The size (length) of the near field can be calculated
using d2
/4λ where d is the diameter of the transducer crystal and λ, the US wave
length according to the frequency being used.
As an example, a crystal with a diameter of 25mm operating at 1 MHz will have a
near field/far field boundary where Boundary = 12.5mm2
/1.5mm ≈ 10cm thus the
near field (with greatest interference) extends for approximately 10 cm from the
treatment head when using a large treatment head and 1 MHz US. When using
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higher frequency US, the boundary distance is even greater. Beyond this
boundary lies the Far Field or the Fraunhofer zone. The US beam in this field is
more uniform and gently divergent. The ‘hot spots’ noted in the near field are not
significant. For the purposes of therapeutic applications, the far field is effectively
out of reach.
One quality indicator for US applicators (transducers) is a value attributed to the
Beam Nonuniformity Ratio (BNR). This gives an indication of this near field
interference. It describes numerically the ratio of the intensity peaks to the mean
intensity. For most applicators, the BNR would be approximately 4 - 6 (i.e. that
the peak intensity will be 4 or 6 times greater than the mean intensity). Because
of the nature of US, the theoretical best value for the BNR is thought to be
around 4.0 though some manufacturers claim to have overcome this limit and
effectively reduced the BNR of their generators to 1.0.
2.5 Ultrasound Transmission through the Tissues
All materials (tissues) will present impedance to the passage of sound waves.
The specific impedance of a tissue will be determined by its density and
elasticity. In order for the maximal transmission of energy from one medium to
another, the impedance of the two media needs to be the same. Clearly in the
case of US passing from the generator to the tissues and then through the
different tissue types, this can not actually be achieved. The greater the
difference in impedance at a boundary, the greater the reflection that will occur,
31
and therefore, the smaller the amount of energy that will be transferred. The
difference in impedance is greatest for the steel/air interface which is the first one
that the US has to overcome in order to reach to body. To minimise this
difference, a suitable coupling medium has to be utilised. If even a small air gap
exists between the transducer and the skin the proportion of US which will be
reflected approaches 99.998% which in effect means that there will be no
transmission.
Table.1 Approximate Velocities of Ultrasound through Selected Materials
The coupling media used in this context include water, various oils, creams and
gels. Ideally, the coupling medium should be fluid so as to fill all available
spaces, relatively viscous so that it stays in place, have impedance appropriate
to the media it connects, and should allow transmission of US with minimal
absorption, attenuation or disturbance. At the present time the gel based media
appear to be preferable to the oils and creams. Water is a good medium and can
be used as an alternative but clearly it fails to meet the above criteria in terms of
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its viscosity. In addition to the reflection that occurs at a boundary due to
differences in impedance, there will also be some refraction if the wave does not
strike the boundary surface at 90°. Essentially, the direction of the US beam
through the second medium will not be the same as its path through the original
medium - its pathway is angled. The critical angle for US at the skin interface
appears to be about 15°. If the treatment head is at an angle of 15° or more to
Figure.2 Rarefaction of the Ultrasound Beam
[Stewart C Bushong, Radiological Science for Technologists, 8th Edition, 2004]
the plane of the skin surface, the majority of the US beam will travel through the
dermal tissues (i.e. parallel to the skin surface) rather than penetrate the tissues
as would be expected.
2.6 Absorption and Attenuation:
The absorption of US energy follows an exponential pattern - i.e. more energy is
absorbed in the superficial tissues than in the deep tissues. In order for energy to
33
have an effect it must be absorbed, and at some point this must be considered in
relation to the US dosages applied to achieve certain effects.
Because the absorption (penetration) is exponential, there is (in theory) no point
at which all the energy has been absorbed, but there is certainly a point at which
the US energy levels are not sufficient to produce a therapeutic effect. As the US
beam penetrates further into the tissues, a greater proportion of the energy will
have been absorbed and therefore there is less energy available to achieve
therapeutic effects. The half value depth is often quoted in relation to US and it
represents the depth in the tissues at which half the surface energy is available.
This will be different for each tissue and also for different US frequencies. To
achieve a particular US intensity at depth, account must be taken of the
proportion of energy which has been absorbed by the tissues in the more
superficial layers.
As the penetration (or transmission) of US is not the same in each tissue type, it
is clear that some tissues are capable of greater absorption of US than others.
Generally, the tissues with the higher protein content will absorb US to a greater
extent, thus tissues with high water content and low protein content absorb little
of the US energy (e.g. blood) whilst those with a lower water content and a
higher protein content will absorb US far more efficiently. It has been suggested
that tissues can therefore be ranked according to their tissue absorption.
34
Although cartilage and bone are at the upper end of this scale, the problems
associated with wave reflection mean that the majority of US energy striking the
surface of either of these tissues is likely to be reflected. The best absorbing
tissues in terms of clinical practice are those with high collagen content –
Ligament, Tendon, Fascia, Joint Capsule, Scar Tissue (Watson 2000, Ter Haar
99, Nussbaum 1998, Frizzel & Dunn 1982)
The application of therapeutic US to tissues with a low energy absorption
capacity is less likely to be effective than the application of the energy into a
more highly absorbing material. Recent evidence of the ineffectiveness of such
an intervention can be found in Wilkin et al (2004) whilst application in tissue that
is a better absorber will, as expected, result in a more effective intervention (e.g.
Leung et al 2004).
2.7 Therapeutic Ultrasound & Tissue Healing
One of the therapeutic effects for which ultrasound has been used is in relation
to tissue 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
35
Thermal Effects:
In thermal mode, it will be most effective in heating the dense collagenous
tissues and will require a relatively high intensity, preferably in continuous mode
to achieve this effect. Both Nussbaum (1998) and Ter Haar (1999) have
provided some useful review material with regards the thermal effects of
ultrasound. Comparative studies on the thermal effects of ultrasound have been
reported by several authors (e.g. Draper et al 1993, 1995) with some
interesting, and potentially useful results. It is too simplistic to assume that with
a particular treatment application there will either be thermal or non thermal
effects. It is almost inevitable that both will occur, but it is furthermore
reasonable to argue that the dominant effect will be influenced by treatment
parameters, especially the mode of application i.e. pulsed or continuous. Baker
et al (2001) have argued the scientific basis for this issue coherently.
US can be used to selectively raise the temperature of particular tissues due to
its mode of action. Among the more effectively heated tissues are periosteum,
collagenous tissues (ligament, tendon & fascia) & fibrotic muscle (Dyson 1983).
If the temperature of the damaged tissues is raised to 40-45°C, then a
hyperaemia will result, the effect of which will be therapeutic. In addition,
temperatures in this range are also thought to help in initiating the resolution of
chronic inflammatory states (Dyson & Suckling 1978). Having made these
comments, most authorities currently attribute a greater importance to the non-
36
thermal effects of U/S as a result of several investigative trials in the last 15
years or so.
Non-Thermal Effects:
The non-thermal effects of US are now attributed primarily to a combination of
Cavitation and Acoustic Streaming (te Haar 99, Baker et al 2001, Williams
1987). There appears to be little by way of convincing evidence to support the
notion of Micromassage though it does sound rather appealing.
Cavitation in its simplest sense relates to the formation of gas filled voids within
the tissues & body fluids. There are 2 types of cavitation - Stable & Unstable
which have very different effects. Stable Cavitation occurs at therapeutic doses
of US. This is the formation & growth of gas bubbles by accumulation of
dissolved gas in the medium. They take approximately 1000 cycles to reach
their maximum size. The cavity acts to enhance the acoustic streaming
phenomena and as such would appear to be beneficial. Unstable (Transient)
Cavitation is the formation of bubbles at the low pressure part of the US cycle.
These bubbles then collapse very quickly releasing a large amount of energy
which is detrimental to tissue viability. There is no evidence at present to
suggest that this phenomenon occurs at therapeutic levels if a good technique
is used.
37
Acoustic Streaming is described as a small scale eddying of fluids near a
vibrating structure such as cell membranes & the surface of stable cavitation
gas bubble (Dyson & Suckling 1978). This phenomenon is known to affect
diffusion rates & membrane permeability. Sodium ion permeability is altered
resulting in changes in the cell membrane potential. Calcium ion transport is
modified which in turn leads to an alteration in the enzyme control mechanisms
of various metabolic processes, especially concerning protein synthesis &
cellular secretions.
The result of the combined effects of stable cavitation and acoustic streaming is
that the cell membrane becomes ‘excited’ i.e, up regulated, thus increasing the
activity levels of the whole cell. The US energy acts as a trigger for this process,
but it is the increased cellular activity which is in effect responsible for the
therapeutic benefits of the modality (Watson 2000, Dinno et al 1989, Leung et al
2004).
Micromassage is a mechanical effect which appears to have been attributed
less importance in recent years. In essence, the sound wave traveling through
the medium will cause the molecules to vibrate, possibly enhancing tissue fluid
interchange & affecting tissue mobility.
2.8 Effect of Ultrasound on Tissue Repair
The application of ultrasound during the inflammatory, proliferative and repair
phases is of value not because it changes the normal sequence of events, but
because it has the capacity to stimulate or enhance these normal events and
38
thus increase the efficiency of the repair phases (ter Haar 1999). It would
appear that if a tissue is repairing in a compromised or inhibited fashion, the
application of therapeutic ultrasound at an appropriate dose will enhance this
activity. If the tissue is healing ‘normally’, the application will, it would appear,
speed the process and thus enable the tissue to reach its endpoint faster than
would otherwise.
Cellular Effects of Ultrasound There is an evidence of increased uptake of calcium ions by fibroblasts exposed
to the therapeutic levels of ultrasound which may be due to the action of shear
forces on the plasma membrane, produced by acoustic streaming in stable
cavities. Temporary increase in the intracellular calcium ions could act as a
signal for changes in the cell activity leading to a cascade of events as a result of
which wound healing is accelerated (Young et al., 1990)
For example in fibroblasts, the protein synthesis is stimulated by ultrasound
therapy, in platelets the release of serotonin and presumably the stimulators of
wound healing such as platelet derived growth factor (PDGF) is induced, in mast
cells the release of histamine is stimulated, and in macrophages growth factor
release is increased. The observed acceleration of wound healing following
exposure to ultrasound therapy could be due to the collective effects of these
cellular events (Doan et al., 1999).
Ultrasound stimulates bone formation It was shown by Doan et al in 1999 that therapeutic ultrasound induces in-vitro
cell proliferation, collagen/non-collagenous protein production, bone formation
39
and angiogenesis. Therapeutic angiogenesis can be used to reduce unfavorable
tissue effects caused by local hypoxia, including osteoradionecrosis and to
enhance tissue repair. Though US enhances bone formation it is not completely
understood as to how it acts at the molecular level.
2.9 Ultrasound Apparatus
The ultrasound apparatus consists of two parts, a generator and a ceramic
transducer mounted in the treatment applicator. The transducer converts the high
Fig.3 The Long Wave Ultrasound Machine; produces intensities from 5 to 50
mW/sq.cm at a 45 KHz frequency; produced and marketed by Orthosonics Ltd,
Devonshire, UK.
40
frequency alternating current from the generator into the ultrasound pressure
waves. The ultrasound machine is calibrated in two criteria – frequency and
intensity. Frequency is measured in mega Hertz (Mhz) where 1 hz is equal to 1
cycle per second. The intensity of the wave energy is measured in Watts per
centimeter square (W/cm2). Recently a new device has been developed that,
instead of using the traditional frequencies of 1 to 3 MHz, uses “long wave”
ultrasound at 45 KHz. This wave penetrates the tissues, reaching areas as deep
as several centimeters, instead of millimeters as with the megahertz machines.
To minimize heating effects it uses low intensities (5 to 50 mW/cm2).
The Ultrasound Transducer
A transducer converts one form of energy to another. The ultrasound transducers
Figure.4 Ultrasound Transducer
41
convert electrical energy to ultrasonic energy. The crystal is the active portion of
the transducer. The backing block is used to dampen the oscillations to form
finite pulses and establish pulse length.
Piezoelectric Crystals
These are materials which change in physical dimension when an electric field is
applied. Most commonly used material for these crystals is Lead Zirconate
Figure.5 An Electric Field re-aligns the dipoles in a Piezo-electric crystal.
A – unaligned crystal, B – Crystal aligned by electricity.
[Christensen’s Physics of Diagnostic Radiology, pg.328]
42
Titanate(PZT). Quartz is a naturally occurring piezoelectric material studied by
the Curies and used commonly in therapeutic ultrasound transducers. The
thickness of the crystal determines the resonant frequency. Transducers are
generally designed to operate at a frequency corresponding to twice the
thickness of the crystal.
3. Bone Remodelling and Tumor necrosis factor alpha receptor (TNFR) superfamily 3.1 Bone remodelling Bone remodeling is a continuous, life long process of bone resorption and bone
formation that renews the skeleton while maintaining its structure. It is a complex
phenomenon which results from the activities of osteogenic cells like osteoblasts
and osteocytes and the osteolytic osteoclasts. Osteoblasts make bone whereas
osteoclasts resorb (Teitelbaum et al., 1997), but the two processes are
inseparable.
Osteoclasts are differentiated cells whose path of development from
monocyte/macrophage precursors to multinucleated cells includes various stages
like proliferation, differentiation, fusion and activation. These events are under
the influence of hormones and cytokines like interleukins, tumor necrosis factor
alpha (TNF-α), transforming growth factor beta (TGF-β) and prostaglandin E2
which act in concert (Teitelbaum et al.,2000). The recently discovered receptor
activator of NF-қβ ligand (RANKL), an essential cytokine for osteoclastogenesis,
has led to a better understanding of osteoclast cell biology and has provided
43
important insights into the pathogenesis of human metabolic bone diseases
(Lacey et al, 1998).
Osteoblasts are bone forming cells derived from bone marrow and are
transformed into osteocytes over a period of time when they get trapped in
mineralized bone. Osteoblasts and osteoclasts are closely related in time and
space. After receiving a stimulus (mechanical load, growth factors, hormones)
osteoclastic cells appear on the bone surface and resorb the mineral
components of bone (Blair et al., 1998). Osteoblasts when stimulated by
osteoclastic contacts and/or osteoclastic soluble factors, deposit osteoid
substance at the resorption site, thereby initiating new bone formation.
Osteoblasts also control mineralization (KT Steeve et al., 2004).
Osteoclastogenic Factors The development of osteoclasts is controlled by osteoclastogenic factors
synthesized by osteoblasts, like RANKL, OPG and TNF-α. RANKL (also known
as TRANCE / OPGL / ODF) which is expressed on the surface of stromal bone
marrow cells and osteoblasts, plays a central role in osteoclastogenesis by
providing an essential signal to OC progenitors through the membrane-anchored
receptor, RANK (Hofbauer et al., 2000). Signal transduction through RANK (Hsu
et al., 1999, Hofbauer et al., 2000) leads to OC differentiation and functional
activation (Yasuda et al., 1998,1999). This signaling pathway can be disrupted by
a naturally occurring decoy receptor for RANKL, termed osteoprotegerin (OPG),
44
which blocks the interaction between RANKL and RANK (Yasuda et al., 1998).
Bone formation in principle can be assessed by the relative ratio of OPG to
RANKL. TNF-α is one of the most potent osteoclastogenic cytokines produced in
inflammation (Kwan Tat S et al., 2004, Warden et al., 2001). TNF-α is
responsible for stimulating osteoclastic resorption both in vitro (Reher et al.,
2002) and in vivo (Warden et al., 2001). TNF-α exerts its osteoclastogenic effect
by activating NF-κB through an intracellular mechanism overlapping that of
RANKL (Lacey et al., 1999). TNF-α potently activates osteoclasts, through a
direct action independent of RANKL but strongly synergistic with RANKL (Lacey
et al., 1999). TNF-α and RANKL, whether acting individually or in combination,
exert a real impact on osteoclast differentiation (Naruse et al., 2000).
3.2 RANKL (receptor activator of NF-қB ligand)
RANKL is a protein with 317 amino-acids. It belongs to the TNF super-family and
is largely expressed in bone, bone marrow and lymphoid tissue. The
predominant role of this cytokine in bone physiology is the stimulation of
osteoclastic differentiation/activation and the inhibition of osteoclast apoptosis
(Khosla et al., 2001). RANKL in association with macrophage-colony stimulating
factor (M-CSF) is necessary and sufficient for the complete differentiation of
osteoclastic precursors into mature osteoclasts. RANKL knock-out mice present
a severe a severe osteopetrosis and a total loss of osteoclasts (Yasuda et al.,
1998). RANKL exists both in soluble and membranous forms (Ikeda et al., 2001).
The soluble form, which corresponds to the c-terminal part of the membranous
45
RANKL, may be produced either directly by the cell through an alternative
splicing followed by an excretion in the extra-cellular medium or by a protelytic
cleavage of membranous RANKL. Different proteases have been proposed to
carry out this cleavage, like, tumor necrosis factor converting enzyme (TACE),
the membranous metalloprotease MT1-MMP and recently the A disintegrin and
metalloprotease 19 (Armstrong et al., 2002). The ectodomain structure of the
protein RANKL is made up of a homotrimer. The structural form of RANKL,
necessary for the interaction between RANKL and its specific receptor RANK,
binds to three RANK molecules (Lam et al., 2001).
The protein kinase C (PKC) pathway plays an important role in the regulation of
RANKL mRNA expression in osteoblasts. The protein kinase A (PKA) pathway
may also have an important involvement since the effects of parathyroid
hormone (PTH) on RANKL mRNA expression in murine bone marrow cultures
depends on the PKA pathway (Lee et al., 2002). It has been proposed that
RANKL would act through phospholipase C to release Ca(2+) from intracellular
stores, thereby accelerating the nuclear translocation of NF-қβ and promoting
osteoclast survival (Komarova et al., 2003).
3.3 RANK (receptor activator of NF-қB ligand)
RANK is a transmembrane protein of 616 amino-acids. It belongs to the TNF
superfamily and is expressed primarily on cells of the monocyte/macrophage
lineage including osteoclast precursors, B and T cells, dendritic cells and
46
fibroblasts (Khosla et al., 2001). It is also present on the surface of matur
osteoclasts. Like other TNFR-related proteins, RANK is known to activate a
cascade of intracellular signaling events, including the recruitment of TNFR-
Figure.6 Current understanding of preosteoblastic/stromal cell regulation of
osteoclastogenesis (S.Khosla, 2001)
associated factor proteins (TRAF), activation of transcription factors (NF-қβ, AP-1
and NFAT2), activation of mitogen activated protein kinases cascades (MAPK-
47
like, ERK, JNK and p38) and induction of Src- and phosphatidylinositol 3-kinase-
dependent Akt activation (Lee et al., 2003).
3.4 OPG (osteoprotegerin)
OPG, which is synthesized as a protein of 401 amino-acids, is cleaved to give a
380 amino-acids mature protein (Khosla et al., 2001). It is released in its soluble
form by stromal cells/osteoblasts after losing its transmembrane and cytoplasmic
domains. Thus OPG differs from other TNFR superfamily members which remain
mainly membrane associated (Tan et al., 1997). In contrast to RANKL, OPG
knock-out mice are osteoporotic (Mizumo et al., 2002). OPG acts as a decoy
receptor for RANKL and down-regulates the RANKL signaling through RANK. It
represents an antagonist endogenous receptor that neutralizes the biological
effects of all forms of RANKL and thus acts as an inhibitor for resorption. It has
also been reported that OPG can directly inhibit osteoclast activity, independently
of RANKL, through interactions with still uncharacterized receptors present on
osteoclasts (Hakeda et al., 1998). The biological effects of OPG on bone cells
include inhibition of terminal stages of osteoclast differentiation, suppression of
mature osteoclast activation and induction of apoptosis.
Bone remodeling appears to be mainly controlled by the OPG/RANKL balance in
the bone micro-environment. Any imbalance in the RANKL to OPG ratio can lead
to an excessive bone resorption or in contrast an excessive bone formation.
48
3.5 TNF-α
TNF-α, a multifunctional cytokine is produced by activated macrophages. The
soluble TNF-α (mature protein) is released, under physiological conditions, by a
proteolytic cleavage of the precursor proTNF-α, a type II transmembrane protein,
at the Ala76-Val77 bond. TACE is the protease responsible for this cleavage.
TNF-α is one of the most potent osteoclastogenic cytokines produced in
inflammation. TNF-α mediates RANKL stimulation of osteoclast differentiation
through an autocrine mechanism (Zou et al., 2001). It has been shown that TNF-
α causes bone loss in periodontitis, orthopaedic implant loosening and other
forms of chronic inflammatory osteolysis (Bingham et al., 2002). TNF-α is
associated with various cell signaling systems via two types of cell surface
receptors, namely TNFR I and TNFR II. Both receptors are expressed on a wide
variety of cell types including bone marrow hematopoietic cells (Sato et al.,
1997). TNFR I is known to mediate most of the biological properties of TNFα
such as programmed cell death and activation of NF-қβ. In contrast TNFR II
lacks a death domain, therefore TNFR II is involved in an antiapoptotic effect of
TNF-α whereas TNFR I is involved in both apoptotic and antiapoptotic signaling.
49
CHAPTER II HYPOTHESIS AND AIMS Null Hypothesis
1. Therapeutic ultrasound does not enhance bone remodeling by enhancing OPG/RANKL ratio in vitro
2. Therapeutic ultrasound does not affect transcription and translation of TNF
alpha in vitro
3. Therapeutic ultrasound does not affect the transcription of bone markers ALP and OCN
Alternate Hypothesis
1. Therapeutic ultrasound enhances bone remodelling by enhancing OPG/RANKL ratio in vitro
2. Therapeutic ultrasound does affect transcription and translation of TNF
alpha in vitro
3. Therapeutic ultrasound does affect the transcription of bone markers ALP and OCN
Specific Aims
To find out whether ultrasound enhances bone remodeling in vitro by
1. enhancing the transcriptional and translational levels of OPG 2. decreasing the transcriptional and translational levels of RANKL
3. decreasing the transcriptional and translational levels of TNF alpha
50
CHAPTER III
MATERIALS AND METHODS 1. Cell culture
MG63 cell line
MG63 (ATCC® Number: CRL-1427 ™) which is a human osteosarcoma cell line
characterized for its osteoblast like properties (Takeuchi et al 1995) was used in
this study. In vitro studies of osteoblast behavior use either primary osteoblasts
or osteogenic osteosarcoma cell lines. Osteosarcoma cell lines have a high turn-
over rate while retaining the basal characteristics of primary osteoblasts. They
can help us in understanding the osteoblast function because they represent
clonal populations derived from specific stages of the osteopath lineage. Clover
et al in 1994 showed that MG63 cells were suitable models for studying integrin
sub-unit expression, cell adhesion and for investigating the regulation and
expression of osteocalcin. They also found that proliferation and alkaline
phosphatase activity exhibited by MG63 were not representative of primary bone
cell cultures.
Bu et al in 2003 studied the expression of TNFs and signaling and non-signaling
TNF receptors in human osteoblasts derived from mesenchymal stem cells and
well characterized osteosarcoma, MG63 with unamplified gene screening. They
found a remarkable similarity in mRNA expression between MG63 and non-
transformed osteoblasts. They suggested that in the un-stimulated state, the
51
overall expression of TNF related proteins (OPG, RANKL, TNFα etc) in primary
osteoblasts overlaps extensively with that in a transformed cell line like MG63.
All cell culture procedures were performed in a sterile air-flow chamber. Cells
were cultured in 75 sqcm tissue culture flasks (Sarstaed) in Dulbecco’s minimum
essential medium (DMEM, Gibco BRL) supplemented with 10% heat inactivated
fetal calf serum (Sigma), 2mmol L-glutamine (Sigma) and 100U/ml each of
penicillin/streptomycin (Gibco BRL) and incubated at 37ºc. The cells reached
confluency usually within 3 days and the culture medium was removed from the
flask and the cell layer was washed using PBS to remove excess culture medium
and dead cells. The cell layer was then trypsinized using 2 ml of trypsin
(Invitrogen) in PBS (Gibco RL), excess trypsin removed and the flasks incubated
at 37ºc for 5 to 10 minutes. Once the cell layer separated 10 ml of culture
medium was added to each flask to stop the action of trypsin. The cell
suspension was then collected in 25 ml plastic tubes (Sarstaed) and subjected to
centrifugation at 1800g for 10 minutes. The supernatant was removed and the
cells resuspended in 25 ml culture medium. The cells were then counted in a
Neubar chamber and distributed at 5 x 105 cells/well in six-well culture plates
(Sarstaed) and incubated for at least 24 hours before being subjected to
ultrasound treatment.
52
2. The Ultrasound Experiment
Ultrasound treatment was done in a sterile airflow chamber. The 45 kHz long
wave ultrasound machine used in this study was a Phys-Assist unit supplied by
Figure.7 US treatment being performed in a sterile air-flow chamber
Orthosonics Ltd (Ashburton, Devon). The six well plates containing the MG63
cells were suspended in a water bath set to a temperature of 37ºc. The water
bath was internally lined with rubber in order to absorb the excess ultrasound
waves and prevent them from reflecting. The ultrasound transducer head was
fixed to a stand which in turn was fixed to a rotator set to 30-40 revs/minute to
prevent standing wave formation (standing waves are formed due to reflection of
ultrasound waves when the transducer head is held stationary and will result in
53
bone resorption in human subjects due to localized concentration of energy). The
transducer head was immersed in the culture medium present in the well. Each
well had 5 ml of culture medium which allowed a distance of 5 mm between the
transducer head and the cells.
An intensity of 30mW/cm² was used for treating the cells. The MG63 cells were
subjected to ultrasound treatment for 5 minutes as previously described by Doan
et al 1999. The cells were incubated at 37ºc for 0*, 3, 6, 12, 18 and 24 hour time
periods after ultrasound treatment (*incase of the 0 hour time point the cultures
were not incubated at all and the cells were harvested or supernatant collected
immediately after ultrasound treatment). The controls were sham-sonicated prior
Figure.8 Administration of US to each cell culture well of a 6 well-plate.
54
to incubation. The wells were sonicated or sham-sonicated (the transducer was
applied to the control wells but with the ultrasound machine switched off) in
triplicates. The culture plates were made to float in a water-bath maintained at
37ºc. The US transducer-head was immersed in the cell culture supernatant and
rotated using a rotator/shaker to distribute the US waves equally. The transducer
head was sterilized with alcohol wipes (Azowipe) and allowed to dry completely
between treatments to make sure there was no introduction of alcohol into the
culture medium.
Figure.9 Schematic of the Ultrasound Experiment (Reher et al., 2002)
55
3. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) RNA
Extraction
RNA extraction from the sonicated and control cultures was done immediately at
the end of the incubation time point using TRIzol reagent (Invitrogen, UK),
according to the manufacturer’s instructions. The supernatant from the treated
and control wells was either collected (for ELISA) or discarded and the cells were
washed with PBS to remove the remaining medium. 500 µl TRIzol was added to
each well and incubated at room temperature for about 5 to 10 minutes. The cell
lysate was collected in 1.5 ml Eppendorf tubes (Sarstaed) and transferred to -
70ºc where it could be stored for up to 1 month. 200µl chloroform was added to
the tubes containing lysate and mixed by inversion of the tubes for 20 seconds
and incubated at room temperature for 3 minutes. The tubes were then
centrifuged at 12500g and 4ºc for 15 minutes. This results in phase separation of
RNA, DNA and protein in the top, middle and bottom layers respectively. The top
aqueous layer containing RNA was carefully pipetted and transferred to a fresh
Eppendorf tube to which 1 ml of isopropanol was added and the tubes were
incubated at -20ºc for at least 10 minutes. Isopropanol helps in precipitating the
RNA. The tubes were then centrifuged at 12500g for 10 minutes to get a pellet of
RNA. The supernatant was carefully discarded from the tube and 1 ml of 75%
ethanol added. The tubes were then centrifuged at 7500g for 10 minutes
following which the supernatant was discarded and the pellets were allowed to
air dry. The pellets once dry were suspended in 50 µl of DEPC-water and
quantified by measuring the absorbance at 260nm and 280nm. A ratio of 1.8 to
56
2.0 between the readings at 260 and 280nm was considered as ideal. At 260 nm,
an absorbance unit of 1 is equivalent to 40ng/ml of RNA.
DNase Treatment
All the samples were DNase treated irrespective of the spectrophotometric
readings. DNase treatment was done using RQ1 DNase (Promega) according to
the manufacturer’s protocol. The reaction was set up as follows:
• Up to 8µl RNA
• 1µl RQ1 DNase 10x reaction buffer
• 1µg RQ1 RNase-free DNase
• Nuclease-free water to a 10µl final volume
The sample was incubated for 30 minutes at 37ºc and 1 µl RQ1 DNase stop
solution added to terminate the reaction, followed by a 10 minute incubation
period at 65ºc to inactivate the DNase. Samples were stored at -70ºc until use.
Reverse Transcription (RT)
It was carried out using SuperScript II RT (Invitrogen) according to the
manufacturer’s instructions. The cDNA synthesis reaction was set up as follows:
• 100ng/reaction RNA template
• 0.1µg oligo (dt) (Promega)
• 0.5mM dNTP mix
• Up to 5 µl of double distilled water
57
The reaction mix was incubated at 65ºc for 5 minutes and quick chilled at 4ºc for
at least 5 minutes. The reaction mixture was then transferred to a tube containing
the following reagents:
• 5x reaction buffer
• o.1M dTT
• 1 µl RNAse OUT
The samples were incubated at 42ºc for 2 minutes. After incubation, 1µl of
SuperScript II Reverse Transcriptase was added to make a final volume of 20 µl
and the samples were incubated at 42ºc for 50 minutes followed by 70ºc for 15
minutes. The cDNA thus obtained was stored at -20ºc until use.
Polymerase Chain Reaction (PCR)
PCR was carried out on cDNA generated from total RNA. Add the following to a
PCR reaction tube for a final reaction volume of 25 microlitre:
• 2.5 µl 10X PCR buffer
• 1.5 µl 50mM MgCl2
• 0.5 µl 10 mM dNTP Mix
• 1 mM amplification primer 1
• 1 mM amplification primer 2
• 0.2 µl Taq DNA polymerase (5U/µl)
• 1 µl cDNA
• Up to 25 µl double distilled water
Amplification was performed in an Eppendorf master cycler gradient PCR
machine programmed for the following cycles:
58
• 94ºc for 2 minutes – 1cycle
• 94ºc for 30 seconds, Annealing temperature for 30 seconds, 72ºc for 30
seconds – 28 to 35 cycles
• 72º for 7 minutes – 1 cycle
RT-PCR was performed to assess the expression of OPG, RANKL, TNF-α and
OCN. The house keeping gene GAPDH was used as the internal control in this
study. The oligonucleotide primers (Sigma) and the annealing temperatures used
to amplify the different genes under study are shown in the following table:
Table.2 Primer Sequences and cycle conditions for RT-PCR
Gene Accession no. Sequence Cycling conditions Amplicon GAPDH BC013310.1 F: GAAGTGGAAGGTCGGAGTC 94˚C 1min 313 bp R: GAAGATGGTGATGGGATTTC 57˚C 1min 72˚C 1min
29 cycles OCN NM_199173 F: GGCCAGGCAGGTGCGAAGC 95˚C 30s 255 bp R: GCCAGGCCAGCAGAGCGACAC 70˚C 30s
72˚C 30s 35 cycles
OPG NM002546 F: AGACTTTCCAGCTGCTGA 94˚C 1min 469 bp R: GGATCTCGCCAATTGTGA 57˚C 1min 72˚C 1min 29 cycles RANKL AF019047 F: CAGGAGACCTAGCTACAGA 94˚C 40s 381 bp R: CAAGGTCAAGAGCATGGA 55˚C 40s 72˚C 40s 29 cycles TNF-α BC000053 F: GTGGCAGTCTCAAACTGA 94˚C 40s 332 bp R: TATGGAAAGGGGCACTGA 55˚C 40s 72˚C 40s 29 cycles ______________________________________________________________________________________
59
Agarose gel electrophoresis of DNA Agarose gels were generally made up as 1.5 % multi purpose agarose (Roche,
Lewes, UK) in TAE buffer (0.04M Tris-acetic acid, 10mM EDTA pH8.0). The
agarose/TAE solution was heated in a microwave on full power (750 W) for 1
minute and cooled to about 50ºc. Then 2 µl of a 10 mg/ml solution of ethidium
bromide was added and the gel solution poured into a gel former and a comb
inserted. When set the gel was placed into an electrophoresis tank, the comb
removed and 1x TAE added. Care was taken to cover the surface of the gel with
the running buffer. Samples to be electrophoresed were mixed with 6x sample
loading buffer (Promega) and loaded into the wells with a micropipette. The gel
was electrophoresed 5-10 volts/cm for approximately ½ hour until the dye had
migrated two thirds of the length of the gel. The PCR products were visualized on
a UV transilluminator.
4. Quantitative Real Time PCR (QPCR)
Real Time PCR was performed for RANKL, OPG and ALP. GAPDH was used
as the endogenous control. TaqMan real-time quantitative PCR was performed
using TaqMan PCR chemistry and the ABI 7300 Real Time PCR system (Applied
Biosystems) according to manufacturer’s instructions. The real-time reactions
were run using the TaqMan Universal PCR Master Mix (Applied Biosystems) with
300 nM of each primer and 200 nM of probe, in a total volume of 25 µl. The real-
time PCR program parameters were as follows: 50 °C for 2 min; 95 °C for
10 min; then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The concentration of
60
each experimental sample was calculated relative to that of the endogenous
control (GAPDH) using ABI software.
5. Immunochemistry
ELISA assays were performed for RANKL (Peprotech, London, UK), TNF-alpha
(Immunotools, Germany), OPG (R&D Systems Europe Ltd) and OCN
(NovoCalcin Immunoassay kit, Metra Biosystems). Microtitre plates (NUNC) were
coated with 100 µl/well of coating antibody at 1µg/ml in PBS (140 mM NaCl, 2.7
mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.2) and incubated overnight at
4˚C or room temperature according to the manufacturer’s instructions. The
plates were washed three times with wash buffer (0.5-0.05% Tween in PBS,
pH 7.2 to7.4) to remove unbound antibody and blocked using blocking
buffer (1% BSA in PBS) for 1 hour . The plates were then washed and
incubated at room temperature with Standards and Samples for 1 hour ,
followed by incubation with detection antibody. The plates were washed
and Avidin peroxidase HRP (Dako Ltd, 1:2000) was added to each well
and the plates incubated for 20 minutes. The plates were then washed and
developed with 100µl/well of colour reagent (0.4 mg OPD, and 0.4 µl of
30% H2O2 in 1 ml of 0.1M citric acid phosphate buffer, pH 5.0). The
reaction was stopped within 15 to 30 minutes with 100 µl of 1 M H2SO4
and the absorbance measured on an MRX II microplate reader (Dynex
Technologies) at a wave length 450 nm.
61
6. Statistical Analysis
The statistical difference between the Ultrasound treated and control groups was
determined using the Student’s T-test for a two-tailed distribution and pooled
variance on SPSS software. This allowed a comparison of the treated and control
groups at individual time points.
62
CHAPTER IV
RESULTS
1. Effect of Ultrasound on osteoprotegerin (OPG) and receptor activator for NF-қB lignand (RANKL) mRNA and protein production Detection of OPG transcription levels by RT-PCR and Q-PCR assays
The RT-PCR assay showed OPG transcription in both ultrasound treated and
control groups but the bands seemed to be more prominent in the former. To
consolidate with those findings the QPCR assay showed an OPG up regulation
at all time points but more significantly at 0h, 12h, 18h and 24h after incubation
when compared to the control group.
_______US Control_____ 0 3 6 12 18 24 0 3 6 12 18 24
a) GAPDH
b) OPG
Figure.10 Pictures of gel analysis of RT-PCR products of a) GAPDH, b) OPG.
US promoted OPG mRNA levels at 0, 6, 18 and 24h prominently while the
control group showed expression at 12, 18 and 24h time points. The MG63 cells
(5x105/well) were treated with 30mW/cm2 intensity from a 45 KHz machine
for 5 minutes. The cells were then incubated for six time periods before RNA
extraction. GAPDH was used as the internal control.
63
Figure.11 US promoted OPG mRNA levels of MG63 significantly at 0h,
12h, 18h and 24h. The MG63 cells (5x105/well) were treated with
30mW/cm2 intensity from a 45 KHz machine for 5 minutes. The cells were
then used for assessment of mRNA levels at 0h, 3h, 6h, 12h, 18h and
24h after US treatment. The graphs were plotted with relative expression of
OPG mRNA with respect to GAPDH, the internal control. (■ ultrasound group, □
control group, * shows significance at p<0.05, **shows significance at p<0.01)
0
0.5
1
1.5
2
2.5
3
3.5
0 3 6 12 18 24
Time (Hours)
OP
G m
RN
A
* * * *
*
*
64
OPG protein levels detected by ELISA assay
OPG protein secretion was observed in both the ultrasound and control groups
but seemed to be significantly up regulated after 3h and 24h as compared to the
control. After 0h and 12h the OPG levels seemed to be lower for the ultrasound
group, though not significantly.
50
150
250
350
450
550
650
750
850
950
0 3 6 12 18 24Time (hours)
OP
G p
g/m
l
Figure.12 US promoted OPG protein levels at 3h and 24h. The MG63 cells
(5x105/well) were treated with 30mW/cm2 intensity from a 45 KHz machine
for 5 minutes. The supernatant was then used for the assessment of
protein levels at 0h, 3h, 6h, 12h, 18h and 24h after US treatment. (■
ultrasound group, □ control group, *shows significance at p<0.05, ** shows
significance at p<0.01)
*
*
65
Detection of RANKL transcription levels by RT-PCR and Q-PCR assays
The effect of ultrasound on RANKL transcriptional levels was investigated using
RT-PCR and Q-PCR. No RANKL expression was observed in untreated MG63
cells, which is consistent with previous studies. It was also observed that
ultrasound did not induce RANKL transcription in MG63 cells (data not shown).
RANKL protein levels detected by ELISA assay
The secretion of RANKL by MG63 cells as a result of ultrasound treatment was
detected using ELISA. It was found that the RANKL protein levels were very low
when compared to OPG. The RANKL levels secreted by ultrasound-treated
MG63 cells appeared to be significantly lower than levels produced by control
cells.
66
0
5
10
15
20
25
30
35
40
0 3 6 12 18 24
Time (Hours)
RA
NK
L p
g/m
l
Figure.13 RANKL protein levels were significantly less at 6h, 12h, 18h and
24h time points in the ultrasound treated group as compared to the controls
which showed no RANKL protein production at all . But in general RANKL levels
were quite low as compared to OPG protein levels. The MG63 cells
(5x105/well) were treated with 30mW/cm2 intensity from a 45 KHz machine
for 5 minutes. The supernatant was then used for the assessment of
protein levels at 0h, 3h, 6h, 12h, 18h and 24h after US treatment. (■
ultrasound group, □ control group, *shows significance at p<0.05, ** shows
significance at p<0.01)
* *
* *
*
*
67
2. Effect of Ultrasound on the mRNA and protein production of TNF-α in human osteoblast like cells TNF-α transcription was not observed in either the ultrasound or control group
(data not shown) and the protein secretion was also very low (0.5-12 pg/ml).
0
5
10
15
20
25
0 3 6 12 18 24
Time (Hours)
TN
F-a
lpha p
g/m
l
Figure 14 - TNF-α levels did not change significantly following ultrasound
treatment. TNF-α levels were very low as compared to OPG. The MG63 cells
(5x105/well) were treated with 30mW/cm2 intensity from a 45 KHz machine
for 5 minutes. The supernatant was then used for the assessment of
protein levels at 0h, 3h, 6h, 12h, 18h and 24h after US treatment. (■
ultrasound group, □ control group, *shows significance at p<0.05, ** shows
significance at p<0.01)
68
3. Effect of Ultrasound on the expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in human osteoblast like cells Differentiated cell function was assessed by the expression of ALP and
Osteocalcin, markers that are representative of early and late stages of
osteoblast maturation and bone matrix production respectively (Malaval et al.,
1994). ALP is expressed by pre-osteoblasts and osteoblasts before the
expression of osteocalcin (Sandberg et al., 1993) whereas osteocalcin binds
hydroxyapaitte and is produced by osteoblasts just before and during matrix
mineralization (Salter et al., 2000)
_______US Control_____ 0 3 6 12 18 24 0 3 6 12 18 24
a) GAPDH
b) OCN
Fig.15 – Pictures of gel analysis of RT-PCR products of a) GAPDH, b) OCN. US
promoted OCN mRNA levels of osteoblasts prominently at all the time points
while there was no expression observed in the control group at all time points.
The MG63 cells (5x105/well) were treated with 30mW/cm2 intensity from a
transcriptionally up regulated in the ultrasound group when initially analyzed by
RT PCR assay but as the bands (not shown) were not clearly demarcated Q
PCR was performed. Q PCR showed that ALP was up regulated by ultrasound at
all time points but significantly after 0h, 6h, 18h and 24h.
69
Osteocalcin was also up-regulated transcriptionally by ultrasound at all time
points (fig.15). To complement the mRNA levels OCN protein levels were
significantly elevated at 3, 6, 12, 18 and 24 time points in US treated cultures as
compared to controls (fig.16). OCN protein levels in the treatment group
gradually increased from 0 to 18 hours and appeared to stabilize at 24 hours.
0
20
40
60
80
100
0 3 6 12 18 24Time (Hours)
OC
N p
g/m
l
Figure.16 US promoted OCN protein levels at 3h, 6h, 12h, 18h and 24h time
points. MG63 cells (5x105/well) were treated with 30mW/cm2 intensity from
a 45 KHz machine for 5 minutes. The supernatant from the cultures was
then used for assessment of protein levels at 0h, 3h, 6h, 12h, 18h and
24h after US treatment. (■ ultrasound group, □ control group, * shows
significance at p<0.05, **shows significance at p<0.01)
* *
* *
* *
* * * *
70
ALP seemed to be transcriptionally upregulated in the ultrasound group at all
time points but significantly after 0h, 6h, 18h and 24h time points.
0
0.5
1
1.5
2
2.5
0 3 6 12 18 24Time (Hours)
AL
P m
RN
A
Figure.17 US promoted ALP mRNA levels at 0h, 6h, 18h and 24h time points.
MG63 cells (5x105/well) were treated with 30mW/cm2 intensity from a 45
KHz machine for 5 minutes. The cells were then used for assessment of
mRNA at 0h, 3h, 6h, 12h, 18h and 24h after US treatment. The graphs
were plotted with relative expression of ALP mRNA with respect to GAPDH, the
internal control. (■ ultrasound group, □ control group, * shows significance at
p<0.05, **shows significance at p<0.01)
* * * *
*
*
*
71
Table.3 Summary of Results
Ultrasound Treated Control Target Factor/Marker
mRNA Protein mRNA Protein
ALP OCN OPG RANKL TNFα
Elevated significantly at 0,6,12,18 and 24 hours Elevated significantly at 0, 3, 6, 12, 18 and 24 hours Elevated significantly at 0, 12, 18 and 24 hours Not Detected Not Detected
--------- Elevated significantly at 3, 6, 12, 18 and 24 hours Elevated significantly at 3 and 24 hours Detected at 6 hours Detected but Inconclusive
Less than US cultures at all time points Not detected Less than US cultures at all time points Not Detected Not detected
--------- Detected at 0, 3, 6, 12, 18 and 24 hours but not significant Detected at 0 and 12 hours but not significant Elevated at 0, 3, 6, 12, 18 and 24 hour time points significantly Detected but Inconclusive
72
CHAPTER V
DISCUSSION
1. Introduction
One of the aims of this study is to explore why US has proved to be empirically
invaluable for the treatment of conditions like fracture non-unions,
osteoradionecrosis (ORN) etc. ORN of the mandible is the most common long
term complication of radiation treatment commonly administered in oral and head
and neck malignant neoplasms. Dental management of patients who are about to
receive therapeutic radiation involving the jaws, remains a problem. The non-
restorable decayed teeth in the mandible which come in the path of radiation
have to be removed, whether before or after radiation and can lead to
osteoradionecrosis. Marx et al., in 1982 had suggested that hypocellularity,
hypoxia and hypovascularization resulting in non-healing wound are the
etiopathogenesis for ORN. There is a sequestration of the cortical bone of the
mandible eventually leading to pathological fracture if left untreated. Sometimes
the bone is sequestrated externally. The patient suffers from mucositis, difficulty
in eating solid food, pain due to sequestration, difficulty in speech and other
symptoms. The empirical use of HBO to promote neovascularity and
neocellularity in irradiated and other sclerotic bone conditions has had
questionable success, though usually as an adjunctive preparation to resection
and reconstruction. Unfortunately few centers have access to hyperbaric oxygen
and the treatment is time consuming and not without hazards for the patient. For
73
example, it is impossible to employ HBO therapy in patients with chronic
emphysematous lung disease which is not uncommonly associated with oral
cancer. Moreover it is expensive.
Subsequently to this Dyson (1982) introduced the use of ultrasound as a simple
means of promoting neovascularity and neocellularity in ischemic tissues (Young
and Dyson 1990). Ultrasound along with other physical modalities of generating
energy like pulsed high frequency electromagnetic stimulation have been
employed to facilitate healing in intractable conditions varying from varicose
ulcers to fracture non-unions.
Therapeutic US was shown by Harris et al, 1992 to be effective in treating
osteoradionecrosis of the jaw. It has two types of effects on tissues, thermal and
non-thermal. The thermal effects of US are commonly used in physiotherapy for
the treatment of acute injuries, strains and pain relief where as the non-thermal
effects are useful in the stimulation of tissue regeneration, healing of varicose
ulcers, and protein synthesis in human fibroblasts. It also induces bone formation
in vitro, by stimulating protein synthesis and cytokine production in human
osteoblasts, fibroblasts and monocytes. It induces in vitro cell proliferation,
collagen and non-collagen protein production and angiogenesis. It stimulates
bone repair and enhances fracture healing in animals. It also enhances fracture
healing in humans. Most recently US has been successfully used for enhancing
healing following distraction osteogenesis in humans.
74
Though the therapeutic value of US in promoting bone formation has already
been established, the mechanism at the molecular level is not completely
understood. In this study we investigated the in vitro release of growth factors in
MG63 (osteoblasts-like cells from an osteosarcoma cell line) following ultrasound
treatment. The main objective of this study is to explain why US can effectively
enhance bone healing clinically and to provide evidence which will establish it as
a conservative and economic mode of treatment for ORN.
Long wave US therapy is mechanical energy administered as acoustical
pressure waves. Mechanical stimulation has been found to raise the adaptive
modeling response of the bone micro-environment by the induction of anabolic or
cytokine molecules. Previous studies have demonstrated that US can raise the
osteogenic response of OBs through the induction of nitric oxide, prostaglandin
E2 and growth factors.
Bone remodeling is regulated by the receptor activator of nuclear factor-kappa B
ligand (RANKL) which is a transmembrane ligand expressed in osteoblasts. It
binds to RANK, which is expressed in osteoclast progenitor cells, and induces
osteoclastogenesis. OPG, a decoy receptor for RANKL, also binds to RANKL,
and competitive binding of RANKL with RANK or OPG. The OPG/RANKL/RANK
cytokine system is essential for bone biology and alterations of this system can
lead to excess bone resorption or formation.
75
The importance of OPG and RANKL as regulators of osteoclastogenesis is well
recognised. Injections of exogenous RANKL induce osteoporosis with increased
serum calcium levels in mice and targeted ablation of RANKL in mice leads to
osteopetrosis. The inhibition of osteoclastogenesis by OPG, the decoy receptor
for RANKL, has also been demonstrated and elevated OPG levels in transgenic
mice cause increased bone mineral density whereas targeted ablation of OPG
results in mice developing early onset osteoporosis. The expression of OPG and
RANKL are developmentally regulated. OPG increases during osteoblast
differentiation whereas RANKL expression is inversely related to the degree of
osteoblast differentiation. TNF-α is a well established resorptive factor which
induces osteoclast differentiation directly or in synergy with RANKL.
OCN and ALP are markers for bone mineralization in osteoblasts. OCN is a
highly specific osteoblast marker produced during bone formation and its primary
structure is highly conserved in vertebrates.
The molecular mechanisms of long wave US on bone remodeling are still not
completely understood. In the present study we examined the effect of long wave
US on MG63 cells for the expression and release of the cytokines; OPG, RANKL
and TNF-α which control bone formation and bone resorption.
2. Discussion of Methodology MG63 cells which are human osteoblast like cells belonging to a human
osteosarcoma cell line were used in this study. Cells were cultured in 75 sqcm
76
tissue culture flasks and incubated at 37ºc till they reached confluency. Once
confluent the cells were passaged and cultured in new flasks. These cells
reached confluency in 3 days time. The cells were then distributed into 6 well
culture plates at 5x105 cells per well and incubated for 24 hours before
ultrasound treatment. The ultrasound experiment was performed in a sterile air-
flow chamber in order to prevent contamination of the cell culture.
The tissue culture flasks were suspended in water contained in a water bath
maintained at 37ºc and restrained from movement by using a sterile glass rod.
The water bath was internally lined with rubber to prevent the ultrasound waves
from reflecting back to the well plates. The ultrasound tranducer head which was
suspended from a stand was immersed in the culture medium to a point from
where it was 5 mm from the cells. The stand holding the transducer head was
placed on a rotator working at 30 rotations per minute to prevent standing wave
formation in the culture. The transducer head was wiped with alcohol before
every immersion.
The cells were treated with 30 mW/cm2 intensity for 5 minutes and incubated for
0, 3, 6, 12, 18 and 24 hour time periods. At the end of these time periods the cell
culture supernatant was collected for analysis of protein production and the cells
were used to extract RNA and used in RT-PCR and real Time PCR analysis. We
wanted to see if ultrasound had an early effect on the transcription and
translation of OPG, RANKL, TNF-alpha and bone markers, so we chose the first
77
24 hour time period after ultrasound treatment. The cultures were treated with
ultrasound in triplicates for each time point and the experiment was repeated at
least twice. Both ELISA and real Time PCR techniques were performed and
analysed under expert guidance.
This study began by examining the transcriptional expression of the cytokines
OPG, RANKL and TNF-α and the late bone marker, OCN in MG63 cells following
ultrasound treatment by RT-PCR assay. The RT-PCR method only allowed us to
conclude that the cytokines and bone markers were expressed or not expressed.
The levels of expression could not be estimated. So QPCR assay was
performed to quantify the transcriptional levels of the genes of interest. ELISA
was also performed in order to analyze the amount of cytokine secreted into the
supernatant from the cell cultures treated with US.
3. Discussion of Results
QPCR results showed that OPG mRNA production increased immediately (0 hr)
after ultrasound treatment as compared to control. The mRNA levels at
subsequent time points of 3, 6, 12 and 18 hrs remained more or less the same
for the treatment group. Only the 24 hr mRNA levels in the treatment group were
considerably lower. The control OPG mRNA levels which were very low at time
0hr rose to a high at 3 hrs and started falling gradually at the subsequent time
points till 18hrs and rose again at 24 hrs. But the treatment group had
significantly higher levels of OPG mRNA at 0, 12, 18 and 24 hrs following
78
treatment. Ultrasound appears to have an immediate and nearly sustained effect
on OPG transcription which dropped only at 24 hrs suggesting that 24 hourly
treatment with ultrasound could be highly effective.
Both ELISA and QPCR confirmed the up regulation of OPG by MG63 cells during
the first 24 hours following ultrasound treatment. RANKL transcription was not
affected by ultrasound and the protein levels were low as compared to OPG but
significantly low in the ultrasound group.
TNF-α is a potent osteoclastogenic factor but there was no TNF-α expression
observed in MG63 as a result of ultrasound treatment and the TNF-α protein
secretion was very low (0.5 – 12pg/ml) and the standard error was high for all
time points suggesting non-specific binding of the HRP to the elisa plate, due to
low target protein, leading to a high variability. Looking at whole protein using
western blotting or flow cytometric analysis for bound protein might be useful in
predicting whether ultrasound affects TNF-α.
Both ALP and OCN mRNA were expressed significantly at various time points
during the 24 hours in the ultrasound group as compared to the controls. OCN
protein levels were elevated in a similar fashion to the mRNA levels in the US
treated samples at all time points except the 0 hour; this could be due to the time
difference between transcription and translation. Elevated levels of OCN and
ALP are an indication that ultrasound promotes bone mineralization.
79
CHAPTER VI CONCLUSION
Based on the results from the present study we concluded that therapeutic
ultrasound may enhance bone formation by up-regulating OPG mRNA and
protein and possibly by preventing RANKL release in osteoblasts. By controlling
osteoclast differentiation ultrasound affects bone remodeling in favor of bone
formation which is observed as an up regulation of the bone markers ALP and
OCN.
80
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Paper Publication from this thesis
1. 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: 1. Gordon Research Conference, “Bones and Teeth”, July10-15 2005,
University of New England, Biddeford, Maine, USA.
2. 2nd Joint Conference of ECTS (European Calcified Tissue Society) and IBMS
(International Bone and Mineral Society), Geneva, Switzerland June 2005.