The effects of Low-Level Laser Therapy and LED-Mediated

TheeffectsofLow-LevelLaserTherapyandLED-MediatedPhotobiomodulationonthepulpcanalvolumeofhumanpremolarsundergoingorthodontictoothmovement

DRRALUCABALEANU

DISCIPLINEOFORTHODONTICS

FACULTYOFDENTISTRY

UNIVERSITYOFSYDNEY,

AUSTRALIA

A Thesis submitted in conformity with the requirements for the degree of Doctor

of Philosophy, Dentistry (Orthodontics)

Dedication

To my son Rares who support me throughout these years and nevertheless during the

Covid19 pandemic period, my best friends Cosmin G ,Mihaela R, Mary, Anthony and

all the friends that had unconditional love for me during all these years.

To all my supervisors that believed in me. Thank you for being there for me over the

past years.

Candidate Certification

This is to certify that the candidate carried out the work in this thesis in the

Orthodontic Department, University of Sydney, and this work has not been submitted

to any other University or Institution for a higher degree.

………………………………………………….

Dr Raluca Baleanu

Acknowledgments

I would like to express my gratitude to the following people:

Professor M. Ali Darendeliler, Head of Department, Discipline of Orthodontics, University of

Sydney for his supervision and for friendship over these years.

Dr. Oyku Dalci, Senior Lecturer, University of Sydney for being there for me when needed and

for her help in, discussion on study design, and in the editing of the manuscript and thesis.

Dr. Nam(Alice) Wong, Lecturer, University of Sydney for her help in discussion on study

design the editing of the manuscript and thesis.

Dr. Matthew Foley, Australian Centre for Microscopy and Microanalysis, University of Sydney

for his technical expertise and assistance with Avizo.

Associate Professor Peter Petocz, Department of Statistics, Macquarie

University for his assistance with the statistical analysis.

Table of contents

INTRODUCTION 1

ORTHODONTIC TOOTH MOVEMENT 2

RATE OF ORTHODONTIC TOOTH MOVEMENT 4

REPAIR OF ORTHODONTICALLY INDUCED INFLAMMATORY ROOT RESORPTION -OIIRR 6

ACCELERATED TOOTH MOVEMENT 8

BIOLOGICAL METHODS 8

SURGICAL METHODS 9

Corticotomy 10

Piezocision 13

Micro-osteoperforations 15

Periodantal ligament distraction and dentoalveolar distraction 16

Surgery first 17

DEVICE ASSISTED METHODS 18

Electric current 19

Magnetic fields 19

Pulsed electromagnetic fields 20

Vibration 20

Therapeutic whole body vibration 21

Localised therapeutic vibration 22

Vibration and tooth movement 23

Vibration and root resorption 25

LOW INTENSITY PULSED ULTRASOUND-LIPUS 27

LOW LEVEL LASER THERAPY 28

LOW LEVEL LASER THERAPY AND TOOTH MOVEMENT 29

LOW LEVEL LASER THERAPY ROOT RESORPTION 31

LOW LEVEL LASER THERAPY AND PAIN 32

PHOTOBIOMODULATION 33

PHOTOBIOMODULATION USING LASER 33

PHOTOBIOMODULATION USING LED 34

PHOTOBIOMODULATION AND TOOTH MOVEMENT 34

PHOTOBIOMODULATION AND ROOT RESORPTION 35

PHYSIOLOGY OF DENTAL PULP AND FACTORS AFFECTING PULP CANAL VOLUME 35

PULP CANAL VOLUME ALTERATION BY AGE, HORMONES AND NUTRITION 36

THE REACTION OF DENTAL PULP TO ORTHODONTIC FORCE 37

VASCULAR PULP CHANGES 38

FACTORS INFLUENCING THE PULPAL RESPONSE TO ORTHODONTIC FORCES 39

THE RESULTS OF PULPAL MODIFICATIONS 41

VIBRATION AND DENTAL PULP MODIFICATIONS 42

LOW-LEVEL LASER THERAPY AND DENTAL PULP MODIFICATIONS 42

PHOTOBIOMODULATION AND DENTAL PULP MODIFICATIONS 43

RELEVANCE OF THE RESEARCH 45

REFERENCES 46

MANUSCRIPT 63

Effects of Low-Laser Therapy and LED-mediated Photobiomodulation on pulp canal volume of maxillary

first premolars undergoing orthodontic tooth movement.

ABSTRACT 65

INTRODUCTION 67

MATERIALS AND METHODS 70

RESULTS 73

DISCUSSION 74

LIMITATIONS 77

GENERALIZABILITY 77

CONCLUSION 78

REFERENCES 79

LIST OF FIGURES&TABLES 83

MANUSCRIPT 87

Effect of Low-Level Laser Therapy on pulp canal volume of maxillary first premolars undergoing orthodontic

tooth movement and in retention.

ABSTRACT 89

INTRODUCTION 91

MATERIALS AND METHODS 92

RESULTS 95

DISCUSSION 96

LIMITATIONS 98

GENERALIZABILITY 99

CONCLUSION 99

REFERENCES 100

LIST OF FIGURES&TABLES 104

CONCLUSION 108

APPENDIX 119

Abbreviations

2D – Two-dimensional

3D - Three-Dimensional

A – Amplitude

AAC – afibrillar cementum

AL – Loss of anchorage

ANOVA – Analysis of variance

AOO - Accelerated Osteogenic Orthodontics

AST -aspartate aminotransferase

ATP - Adenosine Triphosphate

bFGF - basic fibroblast growth factor

CBCT - Cone beam computed tomography

Cm – Centimeters

CSs– cell sheet

CT - Computed Tomography

ddMSC – dentoalveolar-derived mesenchymal stem cell

ER - Electromagnetic Radiation

ECM –extracellular matrix maturation

F – Frequency

FGF-2 – fibroblast grow factor

g - grams

gm/gms ¬-grams

h - hour

hPDL - human Periodontal Ligament

Hz - Hertz

IGF-1 - insulin derived growth factor

IL-1β-interleukin-1 beta

IR – Infrared light

J - Joulle

LASER - Light Amplification by Stimulated Emission of Radiation

LED - Light Emitting Diode

LIPUS - Low Intensity Pulsed Ultrasound

LLLT - Low Level Laser Therapy

LTV - Localized Therapeutic Vibration

min - minutes

mm – millimeters

MSC - Mesenchymal stem cell

Mw – Mega-watts

N - Newtons

NIR - Near-Infrared Light

Nm – Nano-meters

OIIRR - Orthodontically Induced Inflammatory Root Resorption

OPG - Osteoprotegrin

OTM - Orthodontic Tooth Movement

PAOO - Periodontally Accelerated Osteogenic Orthodontics

PCO – Pulp canal obliteration

PDGF - platelet-derived growth factor

PDL - Periodontal Ligament

PEMF - Pulsed Electromagnetic Fields

PGE2 - Prostaglandin E2

PMB - Photobiomodulation

q-PCR - quantitative Polymerase Chain Reaction

RANKL - Receptor Activator of Nuclear Factor- ĸB ligand

RAP - Regional Acceleratory Phenomenon

RCT – Randomized Controlled Trial

TGF - transforming growth factor

V - Volts

VAS - Visual Analog Scale

VEGF - vascular endothelial growth factor

W - watt

μm - micrometere

Dr Raluca Baleanu 1

Introduction

The Hippocratic Oath includes the promise ‘Primum non nocere’, meaning to abstain from doing harm,

is an important goal when we treat our patients. However, even in the best of hands, and with every

positive intention, it is not always possible.

Since the demand for fast orthodontic treatment has expanded, researchers have focused on

manipulating bone biology to produce accelerated tooth movement.

One of the few pernicious consequences of accelerated tooth movement is described by the

remodeling of dental and paradental tissues as well as causing a response in the pulp.1,34,43

The pulp response to orthodontic force is thought to involve inflammation, wound healing,2 pulp

calcifications/obliterations,3,4 and in the severe cases, permanent pulp destruction with loss of

vitality.5,6 It is postulated that a short duration of orthodontic force causes biochemical and biological

pulp tissue alteration and that orthodontic forces may be less biologically safe for the pulp tissue, as

the age of the patient increases.7

Techniques which have been proposed to accelerate tooth movement include mechanical/physical

methods, biological methods and surgical methods.8,9,10,11 However, some of these methods have

proved to be invasive or impractical to integrate into the orthodontic practice.

Dr Raluca Baleanu 2

Low-level laser therapy (LLLT), also known as soft laser, has been used in accelerating orthodontic

tooth movement by stimulating bone remodeling12 and for pain relief13,14.

Photobiomodulation(PBM) is a current therapeutic approach that uses low level laser (LLL) or light

emitting diode (LED) and has been widely practiced and tested in medicine and dentistry. Research has

shown that photobiomodulation improves wound healing,13,15,17 relieves pain,18 enhance nerve

regeneration19 and stimulates tissue regeneration and tissue growth by promoting fibroblasts,20 and

chondral21 proliferation.

The increased interest into the methods of accelerated tooth movement has paved the way into

numerous studies on its effects on tooth movement. However there are no studies that directly

investigated the effects of those methods when applied during orthodontic tooth movement, on pulpal

volume. Therefore we aimed to study the effects of two popular methods of accelerated tooth

movement that are non-invasive, LLLT and LED PBM, on human pulp canal volume.

Orthodontic tooth movement

Orthodontic tooth movement (OTM) is brought about by reversible injury to the periodontium and

coordinated bone remodeling in response to mechanical strains.22 Pressure-tension, bone bending and

piezoelectric theories are commonly described to explain the biologic processes involved in tooth

movement. Piezoelectric theory postulates that piezoelectric signals generated by bone bending

initiates OTM while the classic pressure-tension theory indicated that it is chemical rather than

electrical signals which initiate cell differentiation and subsequently tooth movement. None of these

Dr Raluca Baleanu 3

theories have been proven,23. though Thilander and colleagues believed that both mechanisms

contribute in the biological control of OTM (Thilander 2011). 24

In general, orthodontic tooth movement depends on the recruitment of precursor cells to facilitate the

biological mechanism required for tooth movement.25 Monocyte precursors form osteoclasts and

osteoprogenitor cells originating in the adjoining periodontal ligament and alveolar bone convert into

osteoblasts and ultimately these osteoblasts, fixed in the bone pattern become osteocytes. To achieve

tooth movement, osteoclasts, osteocytes and osteoblasts, are necessary to produce bone resorption

and apposition.26

Application of light and constant orthodontic force initiates a cascade of events leading to OTM.

Bending of alveolar bone occurs almost immediately due to the incompressibility of periodontal

ligament (PDL) fluid, generating piezoelectric signals. Seconds later, PDL fluid expresses into the

surroundings, causing the alveolar bone to spring back and the tooth moves within the PDL space.

The pressure and tension zone created by constant orthodontic force alters blood flow in the PDL. This

leads to a change in the chemical environment. Specifically, while there is a fall in the oxygen content

and an increase in carbon dioxide levels in the pressure zone, the reverse occurs in the tension zone,

resulting in a release of prostaglandins and cytokines as well as other chemical messengers.27

Approximately four hours following force application, cellular differentiation begins within the PDL with

detectable levels of cyclic adenosine monophosphate (cAMP). Tooth movement begins two days later

as osteoclasts and osteoblasts begin to remodel the bony socket,27,28,29 with bone resorption on the

pressure side and bone deposition on the tension size.30,31

Physiological response to orthodontic force changes as the force level increases. Excessive force

magnitude occludes the blood vessels on the pressure side within three to five seconds following force

Dr Raluca Baleanu 4

application, resulting in pain. This is followed by cessation of blood flow on the pressure side, leading to

cells being distorted mechanically. Subsequently, leakage of cytokines and prostaglandins occur

resulting in sterile necrosis within hours of force application. Hence cells are recruited from adjacent

bone marrow space in about 3-5 days for differentiation into osteoblast and osteoclast. Tooth

movement occurs in 7-14 days following force application as undermining resorption removes lamina

dura adjacent to the compressed PDL. The delay in tooth movement along with other complications

resulted in a consensus that light and constant force is optimal.32

The amount of OTM is driven by mechanical and biological stimulants, as well as magnitude of applied

forces and the rate of bone remodeling.33 Studies have shown that the tissue reactions caused by

orthodontic forces may lead to not only root resorption and iatrogenic damage to the periodontium

but also pulp reaction.1,34,35

The developmental stages of the tooth at the time of stimulation and the magnitude of orthodontic

force used is believed to be related to the response of the pulp. In addition, previous history of pulp

injuries or chronic pulp injuries, such as caries may also alter pulpal response.30

Thus far, numerous published studies have demonstrated that pulpal reactions to the orthodontic

forces include:

● Altered sensation to stimuli36

● Mild hyperemia36

● Pulpal calcifications/obliterations33,37

● In severe cases, permanent pulp damage with loss of vitality23, 28,38

Dr Raluca Baleanu 5

Similar to the process that initiate angiogenesis in wound healing39, OTM generates neurovascular

alterations in the pulp, producing the discharge of certain neurotransmitters that can determine the

cellular metabolism and influence the bloodflow.40

Rate of orthodontic tooth movement

The degree of tooth movement is limited by the rate of physiological change in the paradental tissues,

particularly the alveolar bone and the periodontal ligament.23,40,41,42

Schwarz et. al.43 assumed that the optimal force for tooth movement should be the force required to

produce a change in tissue pressure that approximates 20-26gm/cm2 of capillary pressure. “The

mechanical input that leads to the maximum rate of tooth movement with minimal irreversible damage

to roots, PDL, and alveolar bone” 1 is the current definition of ‘optimal force’. The mechanical input is

seen as a stimulus for a cellular response that aims to restore equilibrium by the remodeling of the

periodontal tissues.

Quinn and Yoshikawa49 originally outlined that there is a correlation between OTM and the extent of

force application.44,45,50 They stated that as the applied force to a tooth is increased, the rate of tooth

movement would also increase up to a certain point. The ‘optimal force’ is also known as the force that

generates the highest level of tooth movement. Optimal forces should not cause any side effects, while

producing the desired tooth movement. Currently there is no consensus on the optimal force levels as

it depends on multiple factors. Some suggested that an increase in force application raised the rate of

canine retraction.44 . However, Boester and Johnston45 found an ‘optimal force’ level for canine

retraction in their study, where forces beyond 140gms did not result in further increases in the rate of

canine retraction. Storey et al.46 studied the effect of various force levels on the rate of canine

retraction and suggested that the ‘optimal force’ value would be 150-200gms. Theoretically, under

Dr Raluca Baleanu 6

ideal conditions, it is possible for osteoclastic resorption to progress at a rate of 100μm/day, translating

to orthodontic tooth movement at a rate of 3mm/month.40

Research47 using a specially designed Drum Spring (without an arch wire) was able to achieve an

average rate of canine distalization of 1.648 +/- 0.576mm per month, with one subject showing

2.755mm/month. This was achieved with only a 50gm force. The level of tooth movement is more

plausible at 0.8-1.2mm/month in clinical practice.48

The relationship between force levels and optimal tooth movement was examined in a systematic

review,51 with no evidence regarding an ‘optimal force’ found. The authors suggested that the reasons

for this finding include:

● The fact that tooth movement can be divided into an Initial phase, Lag phase and Post-Lag

phase; still most studies only have a short experimental period, which includes only the first two

phases;

● The incapacity to calculate the diffusion of stress and strain in the periodontal ligament;

● The failure of studies to control the type of tooth movement performed. Most studies have

tipping tooth movements, which not only result in an uneven distribution of stress and strain,

but also result in an over-estimation of rates of tooth movement when measurements are

made at the crown level;

● Inter-individual variation.

Dr Raluca Baleanu 7

Repair of orthodontically induced inflammatory root resorption -OIIRR

Repair of orthodontically induced inflammatory root resorption appears through new cement

formation and re-organization of periodontal ligament. when active forces are removed.34 Several lines

of evidence established that the rate of root repair appears to increase with time. 52,53,54,55 Owman-Moll

et al.55 compared the reparative potential of OIIRR with the degree of resorption area following

different length of retention, using teeth that were extracted and examined histologically. They found

that following 7 days of retention, repair was 28%, compared to 75% repair following 86 days.

Repair was shown to slow down after day 35 to day 42 and remains at a stable level thereafter.55,56

Cheng et al.56investigated cementum repair following light or heavy orthodontic force application and 4

and 8 weeks of retention and concluded that there was a lot of individual variation in repair response.

The patients that had light forces had steady repair after 4 weeks, whereas in the group that had heavy

forces, the repair only started following 4 weeks of retention.

While Brudvik and Rygh described repair to initiate at the peripheral sites, it was also shown to start at

the base of the crater or from all directions.58,59,60

As signs of repair, at the periphery as well as the lacunae of the resorbed cementum, cementoblasts

with similar characteristics as during cementogenesis and fibroblasts, indicating repair of periodontal

ligament were observed.60,62

Dr Raluca Baleanu 8

It was also shown that the deposit of a fine coating of acellular afibrillar cementum (AAC) induces the

reparative activity. 61,63

Accelerated tooth movement

The 2015 AAO Economics of Orthodontics Survey, revealed a dramatic increase of orthodontic

treatments in adults. On average an orthodontist treated 41 adult patients in 1989 compared to 125

patients in 2014.64 Both adult and adolescent patients can benefit more from accelerated tooth

movement given that with long treatment durations, compliance and oral hygiene declines, creating

more unwanted side effects and . Furthermore, adult patients are more predisposed to periodontal

problems and other time related side effects.

The potential to accelerate OTM is supported by the fact that under ideal conditions, bone can be

resorbed as fast as 100um/day, which translates to orthodontic tooth movement at a rate of

3mm/month. This exceeds the efficiency achievable with any clinical method, indicating a considerable

gap between clinical proficiency and biologic potential (Roberts 1981).40

Clinicians around the globe have been exploring this biological potential aiming to minimize treatment

time, decrease adverse effects such as pain, discomfort, dental caries, and periodontal disease, and

minimize iatrogenic damage such as root resorption and elevated risk of white spot lesions.65,66,67,68

These acceleration methods have been broadly categorized as: Biological, Surgical and Device assisted

methods.29

Dr Raluca Baleanu 9

Biological Methods

There has been large amounts of research performed on the biological methods of accelerated tooth

movement. This was achieved through the use of medications and chemical mediators, manipulation of

selective mediators and hormones that are implicated in bone metabolism via enhanced osteoclastic

action and/or repressing osteoblasts.69

The administration of dihydroxy vitamin D3,70 parathyroid hormone,71 prostaglandin E2(PGE2),72

thyroid hormone,73 and RANKL (Receptor Activator of Nuclear Factor- ĸB ligand),74 osteocalcin,75

relaxin76 have been demonstrated to accelerate the rate of orthodontic tooth movement in animal

studies. It has been proven that other agents, such as fluoride,77 bisphosphonates,78 calcitonin,

corticosteroids,79 interleukin antagonists, anticancer drugs,80 trigger side effects that impact the cells

targeted by orthodontic forces.

Hence, biological approaches are not currently recommended due to their limited applicability and

efficacy, as well as health and safety related concerns.9,69,81

Surgical Methods

Surgically accelerated tooth movement has been practiced for over a century.50 Osteotomy was first

performed in the 1890s.50,53 In 1950, Köle82 described the use of corticotomy for the purpose of

accelerating the rate of tooth movement. Both techniques were not widely practiced owing to their

invasiveness.

Surgical approaches to accelerate OTM are based on the regional acceleratory phenomenon, or RAP

effect and was first proposed by Frost in 1983.83 The phenomenon describes the increased rate of bone

turnover and remodeling in response to localized trauma to bone. Frost also believed that the

Dr Raluca Baleanu 10

magnitude of noxious stimulus was proportional to the intensity of the RAP effect obtained. RAP effect

peaks at 1-2 months, and then reduces after 2-3 months as the density of bone increases. In general,

RAP effect lasts for 4 months duration.83

In 2001 Wilcko et al.51 also described the demineralization and remineralization procedure underlying

surgically assisted accelerated tooth movement as (RAP)-Regional Acceleratory Phenomenon,84 which

consisted of increased bone remodeling. The authors postulated that it is the increased rate of bone

metabolism which elevates the rate of OTM rather than a mere reduction in cortical resistance

following bone injuries.

To date, various surgical approaches of accelerated tooth movement have been proposed, which

include corticotomies, piezocision, micro-osteoperforations, periodontal ligament distraction,

dentoalveolar distraction and the surgery first approach.

Corticotomy

This surgical procedure involves cutting or perforating the cortical layer of bone without the extension

of the cuts into the medullary bone,56,82 to initiate RAP effect.83 Triggering RAP generates an increase

in osteoblastic and osteoclastic activity in the alveolar bone, that could provide up to 4- 6 months of

accelerated tooth movement.85 However, due to the temporary nature of the effect;86 the procedure

needs to be repeated for the continuation of this effect.

Köle82 first described the use of corticotomies for the purpose of accelerating the rate of tooth

movement in 1950. He speculated that the main resistance to tooth movement was the presence of

dense cortical bone around the teeth. The corticotomy procedure involves making full-length

osteotomy cuts on the buccal and lingual alveolar plates of cortical bone and movement of teeth with

the associated ‘blocks’ of bone 86.Cancellous bone was left intact, as this was believed to provide the


necessary nutrients to the bone and teeth to prevent devitalization of teeth, injury to the

periodontium, as well as root resorption.

The method of corticotomy for OTM was refined by the Wilcko brothers applying their certified

‘Accelerated Osteogenic Orthodontics’ (AOO) technique, known later as the ‘Periodontally Accelerated

Osteogenic Orthodontics’ (PAOO), to reduce total treatment time to a quarter to one third that of

common non-extraction and extraction orthodontic therapies.87 The Accelerated Osteogenic

Orthodontics method relies on the development of a transient ‘localized demineralisation-

remineralisation phenomenon in the alveolar bone consistent with the wound healing pattern of the

Regional Acceleratory Phenomenon’.81

Animal88,89,90,91,92,93 and human88,94,95,96,97,9899 studies, consisting of clinical trials, case reports and case

series, have found that there has been an acceleration of tooth movement with corticotomy-facilitated

orthodontic therapy. The degree of acceleration of tooth movement varies widely in the studies, with

up to four times the rate of normal tooth movement in animals92 and two to three times in

humans.88,89,87,94,96,90,97,99,100,101 The variability is likely due to different study design parameters, such as

differences subject species (animals vs humans) and numbers, corticotomy procedure variations, force

magnitudes, types of tooth movement with differing activation/reactivation regimes, and durations of

force activation.

Gantes et al.102 performed a case series that tested the effects of corticotomies on tooth movement.

Five subjects had corticotomy procedures performed and each subject was compared to an age and

malocclusion matched control that, underwent concurrent orthodontic therapy. Treatment time was

reduced on average by 50%. Further, Aboul-Ela et al.96 performed a split mouth study in 13 adult

subjects, the corticotomy side of the mouth had a statistically significant rate of canine distalization


that was double the control side during the first 2 months post-surgery. This reduced to 1.6 times

faster at the 3-month period and only 1.06 times faster by the 4-month period. Similarly, Sanjideh et

al.89 performed a split mouth animal study where the rate of tooth movement peaked at 22-25 days on

the corticotomy side, with the corticotomy peak being 85% greater than the control side. Both sides

then experienced a deceleration in the rate of tooth movement, such that there was no difference

between the corticotomy and control sides at 7-8 weeks. In the same study, the effects of one versus

two corticotomy procedures (separated by a 4-week period) were compared. The side with only one

corticotomy showed a decrease in the amount of tooth movement during the last few weeks of the

experimental period. The side that underwent a double corticotomy showed a steadier rate of tooth

movement throughout the 8-week experimental period. This also translated to maintenance of a

higher rate of tooth movement for a longer period of time. However, the differences between the one

and two corticotomy procedures were small and not considered clinically significant. In addition, Al-

Naoum, Hajeer and Al-Jundi103 found in their randomized clinical trial that the acceleration of tooth

movement varied between 2-4 times that of controls, with the fastest rate in the first 2 weeks post-

corticotomy. The rate of tooth movement decreased over the 12-week experimental period.

In terms of the complications, several pieces of literatures indicate that corticotomy-facilitated

orthodontic tooth movement yet does not increase the risk of unwanted sequelae when compared to

conventional orthodontic treatment. Specifically, there is no increased risk of adverse changes in

periodontal parameters,94,95,100,104,105, root resorption100,99,105,106,112,113 or tooth vitality issues.99,106,107

Further, Düker108 found that corticotomy accelerated tooth movement did not adversely affect the

pulp vitality or marginal periodontium. In addition, in a human split mouth study that aimed to assess

the effect of corticotomies on canine distalization with temporary anchorage devices, Aboul-Ela et al.96


found that corticotomies did not adversely affect periodontal parameters in terms of plaque index,

probing depths, attachment loss or gingival recession.

With regard to root resorption, most studies have found that corticotomy-facilitated orthodontic

procedures do not increase the risk of root resorption when compared to conventional orthodontics.

Interestingly, several studies even found that corticotomy procedures resulted in less root resorption

when compared to conventional orthodontic treatment. Abbas and Hassan109 examined the effect of

corticotomy-facilitated orthodontics on root resorption through the use of Cone Beam-CTs. They found

that for the teeth adjacent to corticotomy, there was on average 0.6mm of root resorption, whilst for

the control teeth, there was 1.5mm of root resorption. This difference was statistically significant.

Another study100 also found that there was a reduction in root resorption in the corticotomy group

when compared to the controls, but the results of this finding were questionable. It is noteworthy that

none of these studies have reported the presence of iatrogenic root damage following corticotomy

surgical procedures.

Despite the promising findings from various studies, a systematic review concluded that “combining

conventional orthodontic treatment with corticotomy reduces the duration of orthodontic treatment

by accelerating tooth movement.110 However, most studies conducted on this topic involved high risk

of bias, therefore, the efficiency/safety ratio is not conclusive.

In addition, drawbacks associated with corticotomy have been reported, including damage to adjacent

vital structures, post-operative pain, swelling, infection and avascular necrosis.111

Piezocision

Piezocision is a variation of the corticotomy technique. 111,112,113 It is a minimal intrusive, flapless

method merging piezo-surgical cortical incisions with selective tunneling to facilitate bone or soft-


tissue grafts. Piezoelectric cuts have a reduced risk of osteonecrosis when compared to corticotomies

as a result of the small size and high precision of the incisions.42

It was suggested that by selective piezocision cuts it is possible to alter the anchorage value of

teeth.101,111,114 The areas not affected by surgery have higher anchorage values because they are

uninfluenced by the demineralization process associated with RAP. A recent article115 described the use

of sequential or segmental piezocision, where selected areas of the arches or segments undergo

piezocision procedures at various planned times during the orthodontic treatment to achieve specific

goals.

The recommended indications for piezocision in terms of case selection include:111

● Rapid adult orthodontic treatment;

● Class I malocclusions with moderate/severe crowding (non-extraction);

● Deep bite correction;

● Some ½ unit Class II malocclusions;

● Rapid intrusion and extrusion of teeth;

● Simultaneous correction of osseous and mucogingival defects;

● Prevention of mucogingival defects.

Dibart et al.112 described the use of the piezocision tool without raising flaps as a more conservative

and less invasive form of corticotomy. The surgical procedure is carried out one week after placing

fixed appliances. Applying anesthesia locally, vertical interdental incisions are created under the

interdental papilla of the jaws, through the periosteum and, preferably, through the attached

gingiva.111,112 Afterwards to produce the cortical alveolar cuts of a 3mm depth to the vertical soft tissue

sections they use a piezo surgical blade. The soft tissue cut ‘tunneled’ is necessary in order to permit


the fixing of bone grafting in areas with thin or no buccal cortical bone. This grafting procedure is said

to allow positive modification of the dental arch that will allow crowding correction without extraction.

While ungrafted areas do not need closure of the sections, the areas that are grafted demand closure

with sutures. Dibart claimed that the active treatment time on the cases was reduced by an average of

3-fold,112 which compared favorably to other forms of corticotomy-facilitated orthodontic

procedures.113

In a recent systematic review it was reported that there is some evidence, although weak, to support

that piezocision is a secure technique to increase OTM in the short term.46 In another recent clinical

trial it was reported that piezocision resulted in a reduction of 43% in treatment time with no

periodontal complications such as recession and increased pocket depth. However, scars were

observed in 50% of patients.44

Apart from scars, root resorption was also mentioned as a piezocision associated complication.

Patterson et al.114 performed a Micro-CT analysis which revealed that the teeth adjacent to the

piezocision cuts had a mean of 0.435 mm³ root resorption versus 0.133 mm³ seen in the control group.

In addition, 5 out of 28 teeth (17%) had iatrogenic root damage due to direct contact with the

piezocision blades. The authors also highlighted that even though the piezocision procedures were

carried out by a highly trained senior periodontic registrar with radiographic aids to assist with the

positioning of the incisions, damage still took place. Further studies focusing on the effects of different

cut lengths and depths should be investigated, given that these techniques were introduced for

patients with crowding.


Micro-osteoperforations

Micro-osteoperforations (MOP) or alveolocentesis is the least extensive with regards to the amount of

local injury amongt the surgical methods of accelerated tooth movement. The technique involves

controlled micro-trauma of the alveolar bone without raising a mucoperiosteal flap. The philosophy in

achieving accelerated tooth movement is again an application of RAP effect, amplifying the expression

of inflammatory mediators that are usually expressed within OTM.116

A human study by Alikhani et al.97 proposed that three perforations by the “Propel” device through

gingiva into maxillary interproximal alveolar bone are enough to generate a regional acceleration of

bone modeling, significantly raising the amount of tooth movement by 2.3-fold. The authors also

observed a raise in the degree of the markers of inflammation in gingival crevicular fluid. However, that

study period lasted only 4 weeks and this research was carried out by the team that invented the

Propel device.

Other more recent studies on humans that showed increase in tooth movement mostly had short

durations of 4 weeks or repeated the procedure during space closure.117,118,119,120 Studies utilizing

different methods for MOP such as Propel and miniscrews, different number and depth/width

perforations with different durations have shown that MOPs did not create a clinically significant

difference in tooth movement.121,122,123,124,125,126

A recent meta-analysis evaluated the effect of micro-osteoperforations (MOPs) performed with Propel

and other mini-screws on the rate of tooth movement, pain, periodontal parameters and root

resorption and concluded that Propel device induced MOP’s do not produce significantly increased

tooth movement.127 Therefore, there is still no consensus regarding the effects of MOP and further

studies are needed.


Periodontal ligament distraction and dentoalveolar distraction

Periodontal ligament distraction was first introduced by Liou and Huang.128 The authors were able to

distalise a canine by 6.5mm in a short period of 3 weeks and they recommend this technique being

utilized particularly in orthodontic treatment that involves first premolar extraction that has anterior

dental crowding. Following the procedure, minimal resorption of canine roots but no significant pain

was reported. An animal study 129 established that the rate of tooth movements is up to 1.2mm/week

when the teeth are moved into the new bone created by distraction osteogenezis.

In 2002 dentoalveolar distraction was described by Kişnişci et al. 130 They have used a rigid distraction

appliance which was applied to retract canines into premolar extraction spaces in a technique similar

to the periodontal ligament technique. They found that canine distraction was completed in a period of

8 to 12 days without anchorage loss (AL) and noticed no deficit in root length or vitality.130

However even though very fast canine distalization is accomplished with dentoalveolar distraction, it is

a very invasive technique and not preferred by clinicians and patients.131

Surgery first

The “surgery-first” theory is a team approach between surgeons and orthodontists for orthognathic

surgery.132 As opposed to conventional surgical approach, surgery first approach manages facial

esthetics before alignment and occlusion. Orthodontics in the surgery first approach is a post-operative

treatment to transfigure the transitional occlusion into a solid final occlusion.

One of the proposed advantages of surgery first approach is that patient’s chief complaint, dental

function and facial esthetics are improved in the beginning of treatment. Another added benefit of


surgery first was shown that it reduced total treatment time to 1-1.5 years depending on the

complexity of the malocclusion.133

In the early days this approach had many issues, such as, postoperative occlusal instability leading to

serious problems in masticatory function and relapse.134 Currently, with the use of three-dimensional

(3D) imaging and simulation, and advances in skeletal anchor plates, an accurate treatment plan and

more predictable outcomes are possible.135

To date, the suggested criteria of malocclusion for surgery first approach includes well-aligned

dentition to mild crowding, flat to mild curve of Spee, normal to mild proclination/retroclination of

incisors, minimal tranverse discrepancy.133 In these patients with RAP, it was shown that during the

period 3-4 months following orthognatic surgery tooth movement is also much faster with the

transient increase in metabolic activity.136 Surgery first approach was also shown to reduce the number

of appointments patients needed to attend.137

Device assisted methods

Device assisted methods, aimed at accelerating tooth movement, trigger activity of the bone cells liable

for bone remodeling during orthodontic tooth movement, therefore enhancing the amount of OTM.138

Device assisted methods include direct electric currents, magnetic fields, pulsed electromagnetic fields,

vibration, low level laser therapy(LLLT), photobiomodulation (PBM), low intensity pulsed

ultrasound(LIPU).


Electric current

The electrical energy has the capability to influence the depository as well as resorptive activities of

bone and cartilage cells by stimulating cell proliferation and metabolism,139,140 applied in the form of

direct currents or pulsed electromagnetic fields.141

The result of these interventions is considered to depend on biological agents, as well as the type and

environment of the cell and the electrical field applied. Davidovitch et al., applied 7V (volts) current to

canine gingiva in cats, and found a notable increase in the rate of OTM43 while others have published

less successful results.142 Pulsed electromagnetic fields

Pulsed electromagnetic fields (PEMF) were previously used to accelerate orthodontic tooth movement

by inducing electricity in bones and tissues. It does not involve using high voltages or implanted

electrodes, to avoid complications related to the use of direct stimulation.141

Bassett et al.141,146 were the first who studied this topic. The authors generated pulsed electromagnetic

fields by placing two coils in parallel manner.141,146 They found that the cellular activities are increased,

lag phase reduced, with increased bone deposition and resorption.144 Darendeliler et al. researched the

outcome of pulsed electromagnetic field vibration on the amount of tooth movement with coils and

magnets in rats as well as guinea pigs, and their results reflected that both intensified the rate of tooth

movement.143 The increase in rate of tooth movement was also demonstrated by several other

studies.144,145

Showkatbakhsh et al. performed a split mouth trial design that used a fixed acrylic device and applied

PEMF for eight hours to the canine tooth. They found that the rate of OTM was over 0.3mm/month for

the experimental group when compared to control. 147 Nevertheless, because of the difficulties in

clinical application, this method did not become popular.


Vibration

Therapeutic vibration (VT) is possible to be conducted locally or on the entire human body and has

been investigated for application in medicine and dentistry approach. Vibration was observed to be an

impulse similar to an oscillatory motion149 that has the frequency-F, magnitude and amplitude-A, which

creates intensity. 150

Magnitude (grams; g): represents the extent of vibration which is conveyed as being a function of the

Terra gravitational force.

Amplitude (millimeters; mm): subject to the extent of the oscillatory motion.

Frequency (Hertz; Hz): subject to the number of oscillation cycles per one second.

It was postulated that the application of decreased magnitude, increased frequency vibration increases

the rate of tooth movement.151 This theory assumes that tooth movement is a consequence of

immediate reaction of the cells of the bone to mechanical stimulation. However, studies on long bones

show osteogenic effects of increased bone density but no resorption outcome.151

This postulation was derived from the Bone Bending hypothesis in which orthodontic forces produce

alteration of the crystalline formations into alveolar bone to generate a piezoelectric current, abetting

osteogenesis.152 Nevertheless, when continuous orthodontic forces are applied, piezoelectric currents

are not created, they are generated when stress is applied or released.153

At a fast rate over a long period, when vibrational forces are used and discharged it is possible for the

vibration to trigger piezoelectric charges caused by stress.153


Therapeutic whole body vibration

Therapeutic whole-body vibration is used in the therapy of a particular condition like diabetes,

osteogenesis imperfecta, osteopenia and fibromyalgia,154,155,156,157,158 but is also recommended for use

in enhancing body function (balance, muscle power).159,160,161

In the 1970’s Nazarov was first to propose therapeutic whole body vibration in astronauts to decrease

the causes of the free gravity bone density and of the loss of muscle mass.162,163

Frost’s mechanostat theory is the basis for using vibration in therapeutic applications through which

the bone adjusts its strength to mechanical forces which are in general inflicted by muscle, in an

exposure-response relationship.164 Extensive research has been carried out on animals and humans to

appraise the outcome of therapeutic whole body vibration on the conservation or development of

muscle and the quality of the bones and strength and aspect for patients who cannot do upper level

exercises.165

Localised therapeutic vibration

The first use of localized therapeutic vibration (LTV) was restricted to application within medicine,

however, afterwards there was some interest in the use of vibration in the alteration of orthodontic

tooth movement.166 Vibration was thought to trigger stress-inducing piezoelectricity signals while

energies are enforced and discharged on a fast rate throughout an extended period.140

In 1979 the application of intermittent forces-induced piezoelectricity to achieve physiologic

orthodontic tooth movement was investigated by Shapiro et al.153

LTV is presumed to encourage the increase and separation of mesenchymal stem cells (MSCs) within

osteogenic cells and chondrocytes.167,168,169 Research done on animals showed the use of low


frequency vibration produced a change in the levels of RANKL and OPG (Osteoprotegrin).169,170,171 The

numbers and the activity of the osteoclast are defined by the balance between OPG and RANKL.170

Studies in vitro showed a reduction in RANKL of up to 55%, suggesting the reduction in osteoclast

thereby impacting the bone and root remodeling cycles and facilitating the anabolic activity, after the

application of LTV.172

Vibration and tooth movement

Extensive studies have been performed on animals which have showed encouraging outcomes

following the application of vibration expressed an increase in the amount of tooth movement.

Darendeliler et al. in 2007 established that OTM, using coils and magnets, can be increased by pulsed

electromagnetic field vibration. The study was conducted on 7-week old Wistar rats, using low-

magnitude vibration, neodymium-iron-boron magnets and sentalloy closed coil springs, evaluating the

effects of high-frequency, low-magnitude vibration on OTM. The study concluded that vibration

stimulated tooth movement by exerting additional mechanical and magnetic forces on teeth.144

Nishimura et al. in 2008 also studied the use of vibration and perpetually changing frequency on root

resorption and amount of tooth movement in Wistar rats.173 It was found that a 15% raise in the

amount of tooth movement resulted when a 60 Hz, 1g vibration stimulus was administered to the

occlusal molar surfaces of rats for 8 minutes, once a week, for 21 days. It was reported that the use of

vibration increases the activation of the RANK-RANKL signaling pathways which intensifies the number

and activity of osteoclasts and stimulates the PDL.173

Kalajzic et al. 2014 used full-body vibration on a rodent model (one-week old Sprague Dawley) and

described the effects of lower frequency (30Hz) and higher magnitude vibration on OTM.174 In

contradiction to the outcomes of Darendeliler et al. and Nashimura et al., they reported that vibration


decreases the amount of OTM and was correlated with elevated numbers among the apoptotic cells.

The differing reported effects of vibration can be linked to the heterogeneity of the study method,

which include the vibration parameter.

Takano-Yamamoto et al. conducted a study on rats (Wistar adults, more than 3 weeks old), evaluated

the outcomes of several vibration regimens on the amount of tooth movement, and they found that

the ideal condition for secondary vibration were 3g/70Hz for 3min., once per week.131 Nevertheless,

the theory concluded that when vibration was combined with static force applied to teeth, it stimulates

the alveolar bone resorption and accelerates the tooth movement via an increase of the osteoclast and

osteoclast genesis function.

Research conducted on rabbits using whole body vibration has revealed anabolic reactions at the

cranial suture following compressed forces at 5N and one cycle per second (1Hz).175

A literature review evaluated the implementation of forces to the upper jaw of ageing rabbits using

histomorphometric analysis.176 They concluded that, after using of each tensile strength and

compression forces, stimulation of the genes and the product of the genes accountable for sutural

increase appeared.

Human studies were also conducted with the first performed by Kriststabet et al. in 1986. The authors

emphasized that vibration at 50Hz for 1-6 min. each 2-3 days minimized the time required for tooth

movement of the teeth with 1.5-to 2-fold.177

OrthoAcel Technologies, the developer of AcceleDent, subsidized a randomized controlled trial that

measured directly in the patients’ mouths the amount of canine’s tooth movement, using calipers, and

found an increase in movement (0.37mm/4 weeks) in the experimental group when compared to the

control. They observed that vibration devices increase the amount of canines distalization throughout


extraction and space closure.150 This publication had some limitations, such as conflict of interest and

potential for bias. Therefore, outcomes should be interpreted with caution.138

Conversely, several other human studies were unable to demonstrate any remarkable differences in

the amount of tooth movement and alignment.178,179 A randomized control trial concluded that

additional vibrational force had no effect on the amount of orthodontic tooth movement and

alignment149,178,179 or the pain experienced.149,179 The initial incisor irregularity was established to be

the determined element that influences the initial and the general amount of alignment.178

DiBiase et al. 2018 studied the results of vibration on orthodontic space closure in terms of treatment

time, in a parallel randomized clinical trial.176 The findings of his research were that there was no

difference in the amount of space closure, final occlusal aftereffect, treatment period or the number of

visits using vibration.

A recent Cochrane review138 evaluated the available literature on vibration and found only two articles

which matched the selection criteria.149,150 The authors emphasized that there was insufficient

evidence supporting the effectiveness of vibration devices to increase OTM movement. Although the

ability of the vibration to accelerate tooth movement was statistically significant, it was not clinically

significant.

Overall, evidence backing up the results of vibration on tooth movement is limited and low-quality.

Vibration and root resorption

Recent studies have proved that localized therapeutic vibration, not only does it increase the amount

of tooth movement,150,151 but also it decreases postoperative pain150,166 and root resorption.169,180


In 2015, Yadav et al. assessed different vibration frequencies (5Hz, 10Hz, 20Hz) on rodent model and

noted different outcomes. They observed an important decline of the Receptor Activator of Nuclear

Factor- ĸB ligand and a consistent growth of the Osteoprotegrin, and the also noticed a reduced root

resorption (not statistically significant) when 20Hz low vibration is applied.169

Grove et al. in a split-mouth RCTs (randomized control trial), used forces (150g) and vibration (50Hz) on

the maxillary first premolars, over 4-week study period, and they found that vibration decreased the

total root resorption volume by 33% when compared to the control group.181

A number of publications on the contrast, found that when therapeutic vibration was applied, the

decrease in OIIRR is not statistically significant.180,182

In 2011, a split mouth experiment was performed to test the outcome of a vibration of 30Hz following

OTM on the maxillary premolars of 15 patients that experienced repair of orthodontically inflammatory

root resorption and no statistically significant differences were found between experimental and

control groups.181

In 2017, Takano-Yamamoto et al. observed no dissimilarities in OIIRR when vibration frequencies (58Hz,

70Hz, 268Hz) were used for 3 min., 6 min., 10 min. and 30 min. for 1-week period.183

DiBiase et al., in a randomized control trial using AcceleDent, found that after an average of 28 weeks

and 5 days, the mean OIIRR is 1.08 (mm).180 That is, the use of vibration did not reduce OIIRR of

maxillary central incisors during alignment phase.


Low intensity pulsed ultrasound-LIPUS

Ultrasound is composed of acoustic pressure waves past the range of human hearing.184 Low intensity

pulsed ultrasound (LIPUS) is widely used in the field of medicine. It is used as a tool in diagnosis and

treatment. At low intensities ((0.5-50Mw/cm2) LIPUS is often used as a diagnostic tool, while an

increased power (0.2-100 W/cm2) produces heat energy, which is favorable for therapeutic purposes.

Animal research has discovered that LIPUS may also stimulate the number of osteoclasts and osteoclast

activity, as well as increasing the numbers of cells in the periodontal ligament, on both the tension and

compression sides.185

Direct comparison between the effects of LIPUS and low level laser therapy (LLLT) on orthodontic tooth

movement was researched by Mohamed M.J.A. et al.,186using gene expressions and histological

evaluation on rats. They divided the animals into four groups, in which the first group had diode laser,

second group LIPUS, third group a combination of LLLT and LIPUS, and the fourth group served as the

control. They highlighted the fact that when LLLT and LIPUS are combined, upregulation of tissue gene

expression was evident and bone remodeling was enhanced, resulting in an increase in the rate of

orthodontic tooth movement.

A meta-analysis concluded that LIPUS also enhanced bony healing, 187 and it was noted that human

periosteal cells can be stimulated to proliferate and evolve into cells with an Osteogenic cell lineage

through the use of LIPUS.187


Low level laser therapy

Since the discovery of the ‘Rubby Laser’ by Maiman,188 studies have been done to introduce laser

technology in the area of dentistry.

Light Amplification by Stimulated Emission of Radiation known as “laser light “is different from normal

sunlight or daylight by its distinctive properties.

Laser light is:

1. synchronized – the light waves travel similar as the surrounding waves in the beam

2. coherent – they circulate in a one-way, well organized, collimated light beam

3. monochromatic – all waves are similar colored189

It has been shown that the use of soft laser synonym with cold laser broadly known as low-level laser

therapy (LLLT) is a non-invasive method with its effects limited to the target tissue.190 LLLT was utilized

in the late 1960s for wound healing and it also caused accelerated hair regrowth.191,192

The technique is based on the semiconductor diode device that uses laser wavelength, which are

assimilated mainly by tissue pigment (melanin) and hemoglobin.197 The suggested process requires the

stimulation of the cell metabolism near-infrared (NIR) or infrared light (IR) at a lower energy density.

The variables such as the quality, quantity, density of the light energy as well as the light source-power,

spot size, number and frequency of administration, administration period, and applied energy create

different effects on the tissues it is applied.194


In the field of dentistry, LLLT has been used in procedures including aesthetic gingival recontouring,

soft tissue crown lengthening, exposure of impacted teeth, removal of inflamed and hypertrophic

tissue, frenectomies, and photo stimulation of aphthous and herpes lesions.195

Low level laser therapy and tooth movement

There are extensive studies on the effects of LLLT on bone remodeling, orthodontic tooth movement as

well as orthodontic pain.196,197,198 Some studies suggested that LLLT is effective in accelerating the rate

of orthodontic tooth movement since it increases the rate of bone remodeling.13,15,17

Application of Low-Level Laser Irradiation in animal198,199 and human clinical research199,200 has

demonstrated the potential for acceleration of tooth movement without increasing the risk of adverse

sequalae such as root resorption and alveolar bone resorption of supporting teeth. The mechanisms

behind Low Level Laser’s stimulatory activity are in the present unknown, but laser is thought to

accelerate metabolism in cells and in bone, increasing the rate of bone resorption as well as new bone

formation.199,200

The first human study done on humans to examine the effect of LLLT on orthodontic tooth movement

using a split-mouth design was performed by Cruz et al,201 in which they showed a 35% rise in the rate

of space closure compared with controls.

Youssef et al. 13 have similar findings in a study which investigated the rate of orthodontic tooth

movement using LLLT in 15 patients. They reported an increase in the tooth movement rate of

experimental LLLT groups compared with the controls. Kreisler et al.,202 in a vitro study, found that the

rate of proliferation of human periodontal ligament (hPDL) fibroblast using low level laser, increased

significantly up to 72 h after 1 dose. However, for the treatment to be effective it needs to be repeated


more than once a week. Both Wu et al.203 and Walsh et al.204 found that the increased rates of hPDL

cells effects of low laser occurred at energy density up to 4j/cm2 whereas the inhibitory effects

occurred at higher energy. Thus, rates increased at day 3 but not observed at day 5 using 2 J/cm2

fluence. Lanzafame et al.,204 using a pressure ulcer model, tested the dose dependent effects of LLLT

on wound healing. Results demonstrated that even when the energy density was maintained (J/cm2),

alteration of the power density and exposure time produced different effects.

LLLT was used by Limpanichicul et al. and Doshi-Metha G. et al. with orthodontic force to accelerate

tooth movement on a human study. However, their findings were not aligned, varying from no

difference to a 30% rate increase.205,206

The use of LLLT to accelerate the amount of tooth movement on animals studies, noted a significant

increase rate between the experimental and control.30,41,43 Nevertheless, it has been suggested that

this was achieved due to accelerated bone remodeling over increased osteoblastic and osteoclastic cell

proliferation and function during tooth movement.

Goulart et al. 207 used LLLT in a dog study and revealed that when the LLLT was applied to the dental

tissue during space closure, it enhances the rate of tooth movement whereas higher rates levels have

been reported to be inhibitory.

Kawasaki et al.,198 showed an increased rate of tooth movement on the experimental rats group

exposed to LLLT versus the controls. Furthermore, they observed, on the tension side, a growth in the

rate of bone formation and cellular proliferation, as well as, on the pressure side an increased number

of osteoclasts on the pressure side.


Low level laser therapy root resorption

Sousa et al. 17 studied the effects of LLLT on root resorption. Canine retraction was performed on 26

canines. Half of them were included in the experimental group and the other half served as controls.

There was no significant difference in root resorption between the experimental and control groups.

Ng et al. (2017)208 studied the effect of LLLT using a split-mouth design comparing the effects of

continuous and pulsed lasers with placebo laser on the contra-lateral sides to investigate differences in

root resorption crater volumes. They found that teeth exposed to LLLT had less total root resorption

than the placebo laser sides.

Low level laser therapy was administered by Vasconcelos et al.209 and in a recent study (2020) and

Yassin et al.,210 to evaluate the effects on root resorption on rats. However, their findings were

different, from no difference, to preventing or reducing orthodontics root resorption in addition to

accelerating the healing and the repair of OIIRR.

Altan et al.211 found that LLLT irradiation applied in combination with orthodontic forces, in rats, had

lower root resorption, as well as a significantly increased osteoblast and fibroblast numbers when

compared with controls group influenced by orthodontic force alone.

Multiple mechanisms behind the association of light energy from LLLT, cellular stimulation, and

negative effects of cells engaged in root repair and resorption, are not well known and may be related

to the relativity of the results. Moreover, there are some difference between rats and human samples

such as tissue density and size which may impact laser energy and penetration to the target tissue, and

nevertheless the cellular metabolism, and the rates of cementum formation may differ.212


Low level laser therapy and pain

The effects of Low-laser therapy on orthodontic pain has also been investigated numerous

studies.196,213 They reported that LLLT relieve discomfort and also delays the pain from orthodontic

adjustment. A research done on 76 patients by Turhani et al.214 found that the pain reduction effect of

a single irradiation of LLLT following orthodontic banding lasted for an average of 30 hours. In a double

blinded randomized study12 on thirty-nine adults that had separators placed between their premolars

pain perception was investigated using Visual Analog Scale (VAS)for 5 successive days. Teeth irradiated

by LLLT had less pain compared to controls. In a meta-analysis that included studies using LLLT after

separator placement215 concluded that the analgesic effect was statistically significant at 6h, 1 day, 2

days and 3 days and not significant at 2h, 4 days and 5 days and suggested further studies are needed

due to the heterogeneity of results.

Photobiomodulation

Another therapy that has been in the focus of accelerated tooth movement studies is

photobiomodulation (PMB). Generally, approved therapeutic tools for photobiomodulation are known

as low level laser (LLL) or light-emitting diode (LED). Light is formation of electromagnetic radiation

(ER), which has a dual nature, viewed as both particles and waves, that exist on the electromagnetic

spectrum and is ranging from gamma rays to radio waves.216 The LED light beams are monochromatic

but not synchronized or coherent like lasers. LED light is not consistent therefore has fewer side effects

and can be safely applied to a large area of the body surface.217


Photobiomodulation using laser

Some animal studies in the field of orthodontics have shown that laser-mediated photo biomodulation

notably raises the amount of orthodontic tooth movement relative to controls,15,198,207,218 due to

accelerate bone remodeling via increased osteoblastic and osteoclastic cell proliferation.

Laser-mediated PBM has also shown the potential to reduce pain related to orthodontic

adjustments.12, 159,176,219 The mechanism is thought to include a stabilization of membrane potential

and release of neurotransmitters associated with analgesia.220,221,222

First time when the photochemical interaction of low powered laser with biological tissues has been

mentioned as photostimulation, photobiomodulation, photobiostimulation and biostimulation, was in

the 1960s.223,224

Endre Mester in 1967 uncovered photobiomodulation, and he found that the irradiation of laser light

to the skin of shaved mice could accelerate hair growth.225 Nevertheless he also found that light

irradiation could enhance non-healing skin ulcers226,227 and stimulate wound healing,217in humans.

Photobiomodulation using LED

Depending on the light parameters used, PMB can generate both stimulatory and inhibitory

responses.213 It has been used for its beneficial effects in clinical practice in orthodontics, distraction

osteogenesis, methicillin-resistant Staphylococcus aureus-induced osteomyelitis, accelerating wound

healing and preventing cell death and tissue damage, improved blood circulation and to reduce pain

and inflammation.14

The biological mechanism that supports the therapeutic effects of PBM are not yet fully understood.190

It is suggested that PBM increases the blood supply through vasodilation, increased production of Nitric


Oxide and other pro-inflammatory mediators and adenosine triphosphate (ATP ) production 228,229,230

and increased cellular metabolism.

Photobiomodulation and tooth movement

Gkantidis et al. in a meta-analysis and systematic review, compared nine trials on photobiomodulation

and laser, and found that there is some evidence that photobiomodulation accelerates tooth

movement.148

Kau et al. investigated the effects of PBM on alignment and showed that the experimental group had

more than double the rate of tooth movement compared with controls, using an extraoral light source

for a period of 20 to 30 minutes daily or 60 minutes a week, on 90 subjects of which 17 were

controls.231 However, there is again conflict of interest in this study as one of the researchers was a

financial stockholder in the company that produced the device and another was an employee,

therefore the results should be interpreted with caution.

Ekizer et al.232 in a randomized control trial found a statistically increased rate of tooth movement of

21% - 58% when canine retraction was performed after the removal of two upper first premolar teeth.

They measured the movement over three time points, one-month apart, utilizing digitized study

models. Another study applying an intraoral LED appliance on the rate of initial alignment, found a 54%

minimization in alignment period and 2.9-fold rise in the rate of tooth movement when compared with

controls (11 subjects were in the experimental group, 8 control).233

Photobiomodulation and root resorption

At present there are a limited number of studies that examining the effects of LED on orthodontic root

resorption.211,234,235


Fonseca et al.235 investigated the effect of LED PBM on OIIRR on 25 Wistar rats over a 7 day period They

divided the rodents into four groups: force and light (1), no force, no light (2), no force (3), light force,

no light (4). Group four showed the highest amount of root resorption among all groups. The

irradiation group displayed a raised number of fibroblasts per unit of the periodontal ligament area, as

well as an increased density of capillaries. Hence there was a decreased amount of inflammation and

root resorption following the application of orthodontic forces, and an enhanced periodontal tissue.

Nimeri et al.236 applied an extraoral LED device on root resorption on 20 orthodontic patients with class

I malocclusion. LED was applied during the initial stage of the orthodontic treatment and all along the

space closure or completion of alignments. Results indicated a wide range of root resorption

determined on CBCT. Therefore, it was concluded that irradiation did not cause root resorption more

than ordinary notice in orthodontic treatments.236,237,238 However, no control group was included in the

study and treatment protocol was not mentioned.

Sambevski et al. in a split mouth randomized controlled trial investigated effects of PBM on maxillary

first premolar root resorption using a sham control device on the contralateral side following 4 weeks

of force application. They did not find any statistically significant difference in the amount of resorption

experienced by the PBM side in comparison to controls.239

More investigations with longer study durations and improved study designs are needed to draw sound

conclusions.


Physiology of dental pulp and factors affecting pulp canal volume

Dental pulp has three main functions, which include maintaining the calcified tissue of the tooth,

responding to external stimuli in form of pain sensation, as well as maintaining the vitality of the

cellular contents.

The odontoblasts and the undifferentiated mesenchymal cells, which can differentiate into dentine-

forming cells, contributes to the repair and maintenance of the hard. As part of the aging process,

secondary dentine is formed by odontoblasts at the periphery of the pulp, at a rate of 0.4µm/day.240 It

has the same tubular arrangement as the primary dentine which was formed prior to tooth eruption.241

In response to irritants such as caries, dental treatment or attrition, reparative, reactionary dentine

sclerotic dentine or are formed, depending on the degree of the damage.241 Reactionary dentine was

formed by primary odontoblast in response to a slowly progressing caries lesion. It has a reduced

number of tubules with a mildly irregular structure. As the carious lesion becomes more noxious,

reparative dentine is deposited by dentine forming cells derived from fibroblasts in the cell-rich zone,

endothelial cells or pericytes of the blood vessels in response to tissue growth factor-b.242,243This layer

of dentine contains tubules that are highly irregular or even absent, reducing the permeability to the

irritants.241,244

The sensory property of the pulp forms a crucial part of the defense mechanism against inflammation,

thermal and mechanical trauma.245 The dental pulp is innervated by the maxillary and mandibular

divisions of the trigeminal nerve. Once the nerve endings are stimulated, all stimuli will be transported


in A-delta, A- beta or C fiber to the brain where they are interpreted as pain regardless of their

nature.246For example, the proprioceptive function protects teeth from excessive occlusal

loading.247,248 In addition, pain sensation from a carious lesion would urge the patient to seek

treatment to prevent further damage of tooth substance. The same would not happen in root-treated

tooth unless the damage is substantial.

The dental pulp is highly vascularized, which is comparable to that of the brain and the tongue.249 The

microcirculation serves to apply oxygen and nutrients to pulpal cells, as well as removing metabolic

wastes. The capillaries in the pulp are innervated by the maxillary artery which branches off from the

external carotid artery, which provides a high blood flow of 40-50ml/min/100g.250The rate of blood

flow to the pulp is higher than the flow to organs such as skin and muscles at rest, indicating the high

metabolic rates of pulp cells and low compliance of pulp.251

Noxious stimulants such as caries, dental procedure or occlusal trauma trigger depending on the

severity of the damage the formation of reparative, reactionary dentine sclerotic dentine which reduce

the pulp canal volume.241

Pulp canal volume alteration by age, hormones and nutrition

• Age:

Secondary dentine accumulation252in the pulp cavity which occurs throughout the life as part of the

aging process resulting in the reduction of the pulp tissue volume 253,254,255,256and is formed by

odontoblasts at the periphery of the pulp, at a rate of 0.4µm/day240.


● Hormones:

Research into estrogen deficiency done one rats conclude that the dentin-pulp of the incisors complex

is reduced compared to sham side. Hence the estrogen plays an important role in dentinogenesis257.

It has been reported that oestrogen deficit can cause menopause 258 also generates mineralization

damage and decrease regeneration function of the dentin-pulp complex.259

● Nutrition:

In a recent study done on tax concluded that the abrasive diet it is related to increase molar pulp

volume.260

The reaction of dental pulp to orthodontic force

Dental pulp is known as the “heart of the teeth“. It is an unmineralized viable tissue.23 If it gets affected

and not treated in time, oral sepsis may occur. The connection between orthodontic force application

and dental pulp tissue has been the subject of research in recent years.3,4,5

Proffit et al.3 described that light continuous forces have little or no effect on dental pulp. However,

several studies postulated that orthodontic tooth movement induce side effects on the dental pulp

tissue.1, 6,46 It creates inflammatory alterations, cellular injuries and circulatory variations to the dental

pulp.261. Clinical signs of pulp modifications include the following:

● pulp obliterations,49,50

● changes in perception to stimuli, mild pulp hyperemia 45


● extreme cases: irreversible destruction with the damage of pulp vitality24, 47,48

Vascular pulp changes

The pulp is one of the most sensitive tissues in the body because to the absence of collateral

circulation. The blood flow and cellular metabolism of the pulp neurovascular system can be affected

by specific neuropeptides that are generated as a result of OTM.46

Rana et al. utilized the dental pulp tissues of rats and applied orthodontic force to establish the point at

which the highest apoptotic activity appears.262 They concluded that apoptotic activity occurred at the

highest rate 3 days following force application.

Another study done on rats evaluated the response of the blood vessels after the application of

orthodontic forces. They concluded that there was an important rise in the density of blood vessels

after six hours of force application in the experimental group compared to controls. Nevertheless, they

found that in day one and day three there was a minimum change and decrease in the density of blood

vessels between the experimental and control groups.263

A research done on rats, which studied polypeptide growth agents involved in the angiogenic response

of the dental pulp after application of orthodontic force, found that VEGF (vascular endothelial growth

factor), FGF-2 (fibroblast growth factor), TGF (beta transforming growth factors), and PDGF (platelet-

derived growth factor) had a major role in the angiogenic reply of the pulp. 52


The connection between vascular modification and increased angiogenesis in the pulp through OTM

have been described in different studies. A study using laser analyzed the blood flow of the human

maxillary canine’s pulp through OTM and found that a decrease in the pulp blood flow started after

about 32 min. of force application, succeeded by a gradual increase of blood flow which lasts for 2

days.264 On average, blood flow returned to normal in 3 days.

A study done on human premolars receiving orthodontic forces assessed the levels of AST (aspartate

aminotransferase), which is an enzymatic marker showing that the cell is dead, and found that after a

week of orthodontic force application, the experimental premolars showed a rise in the level of AST

compared to controls.261 Derringer et al. used the pulp from human teeth extracted after OTM found

an increased number of micro-vessels in the pulp teeth compared to controls.262 Nevertheless,

polypeptide growth factors are able to diffuse and cause angiogenesis in other tissues, using the same

process comparable to wound healing.265 Overall, existing studies concluded that if the mechanical

challenge lies within the biological limits of the pulp’s healing potential, the dental pulp tissue has the

ability to adjust when orthodontic loading is applied.263

Factors influencing the pulpal response to orthodontic forces

The frequency and the amplitude of the dental pulp modifications can be affected if there is history of

trauma or caries. Furthermore, the biological results of orthodontic force on the dental pulp are

considered to have a connection to the developmental status of the tooth. The effect produced was

also proposed to be in proportion to the degree of the applied orthodontic force.46


It has been stated that when force of higher amount and intrusive type is applied the pulp calcification

is increased.49,50

Adults and younger individuals have been observed to have a lag in cellular reactions and changes in

vascularization of the pulp succeeding orthodontic force administration.7 It was stated that when

orthodontic forces are applied the pulp respiratory rate is heightened with age46 an average of 27.4%.7

The increased respiratory rate of pulp cells is linked with higher dentinogenic activity.266 In addition,

teeth with closed apical foramen experience regression and/or swelling reaction of the dental pulp.

Teeth with incomplete apical development, though not protected to unfavorable consequences

experience less severe form of unwanted effects.46

A study analyzed dental pulp reactions of adults and adolescents after 1 month of moderate intrusive

force49. They discovered that, though no major histologic changes were noticed within the

experimental group, following 1 month of intrusive force in adults a severe inflammatory-related

histologic pulpal alteration may appear.

A number of studies have been reviewed to evaluate the different outcomes of tooth vitality with a

history of trauma when distinct orthodontic movements were applied.200,201,202 After receiving

orthodontic treatment, teeth with a record of trauma were at-risk of pulp necrosis (orthodontic

intrusion 10.4% with trauma, 267, 268 orthodontic extrusion 9.1% with trauma269), while on teeth with no

history of trauma, receiving orthodontic force, the risk is 0.5% (no trauma) and 1.6% (trauma with no

orthodontic procedure).


Bauss et al. assessed the effects of trauma during orthodontic treatment and found that pulp necrosis

happened in 18.6% of the teeth if trauma happened during treatment, whereas trauma without

orthodontic treatment triggered tooth necrosis in only 1.6% of patients otherwise.270 Necrosis was

most common with extrusive, intrusive and lateral injuries.270

The results of pulpal modifications

Application of orthodontic force leads to an immediate change in the metabolism of the pulp cell. Over

time, the coronal and radicular pulp experiences a rise in irregular dentine deposition.271 The growth in

dystrophic mineralization could result in total obliteration of pulp canal (PCO).272

In general, inflammatory modifications after orthodontic loading are frequently thought to be

reversible, presuming that the pulp has not encountered prior abuse as well as challenges.46

Several studies have reported decrease in pulp volume preceding orthodontic loading.273,274 However,

other studies found no alterations.51 The first prospective study that evaluated the result of OTM on

the volume of dental pulp tissues, found a statistically important decrease in pulp volume in the

experimental group compared to controls.275 Lazzaretti et al. also discovered a notable rise in the

amount of pulp calcifications after orthodontic movement of the premolars,50 which was also

confirmed in another study.261

Teeth with total PCO is at greater probability of pulp problems throughout orthodontic treatment in

contrast to teeth that has incomplete or no obliterations.268


A systematic review has indicated there is not enough scientific proof that orthodontic loading could

reduce the blood flow and create irreversible modification.277 They concluded that previous dental

trauma might be one of the risk factors to compromise the vitality pulp tissues.277

Overall, up to now there is no consensus regarding the pulp reaction and orthodontic loading;49,50, 210

due to the shortcomings, including short trial duration, paucity of randomization and control group,

usage of 2D radiography scans to investigate the volume of pulp changes.276

Vibration and dental pulp modifications

Thus far, little is known about the outcome of vibration on the dental pulp tissue. There was only one

animal study on this topic. In 1945 a study analyzed the aircraft vibration on animals.278 They applied an

amplitude of 0.05 inches on 2600 cycles/min., 8 hrs./day and for 6 days/week, over a 4-week

investigation. Histological evaluation revealed no difference in the dental pulp tissue, dentine, alveolar

periosteum and the bone of the teeth.

Low-Level Laser therapy and dental pulp modifications

Dominquez et al.263 were the first to investigate the histological changes in the human pulp tissue

during orthodontic tooth movement undergoing low laser therapy. They found an increased

vascularization which accelerates the pulp repair and a widening of the odontoblast layer which was


exposed to LLLT versus the control group. Furthermore, they suggest that the pulp tissue has an

adaptive response and favored the repair from the initial injury produced by the orthodontic force.

Alsulaimani et al.,234 found an increased effect of LLLT on cemental modeling and repair, using

histologic and immunohistochemical methods in rats. They used a laser with a wavelength of 830-nm

on the exposed group for a period of two weeks and the other group served as control, no orthodontic

forces were applied. The volumetric hard tissue changes and root surface were analyzed with Micro-Ct.

Abi-Ramia BP. et al.,280 administered LLLT to study the microscopic pulpal response form

orthodontically induced tooth movement on rats. They use 45 rodents divided in 3 groups (1 – had

orthodontic tooth movement, 2–LLLT and orthodontic tooth movement, 3- served as control). In the

first and second groups, the pulpal response was more intense in the region of application of the force

or laser. The main finding was that LLLT applied currently with orthodontic force to accelerate tooth

movement, showed a quicker repair of the pulp tissue.

Photobiomodulation and dental pulp modifications

Photobiomodulation (PBM) can create both stimulatory and inhibitory responses determined by the

light parameters used.213 Currently, no published information is available in regards to the effects of

LED-PBM on pulp tissue. However, in vitro studies have been done to investigate the effects of laser-

mediated PBM on human dental pulp cells. Garrido et al. 238 investigated the effect of laser-mediated

PBM on extracellular matrix (ECM) of human dental pulp cell sheets (CSs). It was found that laser-

mediated PBM, especially those in lower energy density, improves the characteristics and the ECM in

CSs as well as fibronectin synthesis, but, when utilized at higher energy(5J/cm2) could cause destruction


of some dental pulp steam cells in the cell sheet. Similarly, Ferreira et al. concluded that in the short-

term, PBM stimulates the proliferation of dental pulp stem cells.260 In addition, Vitor et al. suggested

that PBM stimulates the synthesis and secretion of angiogenic proteins by pulp fibroblasts from human

primary teeth.261


Relevance of the research

Published studies investigating the effect of LLLT on tooth movement provided very limited information

on whether they imposed any side effects on dental pulp canal volume.Researchers attempted to

evaluate the impact of age252-256, nutrition260 and hormones257-259 on pulp volume but , there was no

published human study about the effect of LED PBM on pulp canal volume. Therefore, the aim of this

present study was to investigate the effects of LLLT and LED PBM on pulp canal volume of maxillary first

premolars undergoing orthodontic force application for 4 weeks using MicroCT.


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262. Rana M, et al. Detection of apoptosis during orthodontic tooth movement in rats. American Journal of Orthodontics and Dentofacial Orthopaedics, 2001. 119(5): p. 516-521.

263. Santamaria JM, et al. Initial changes in pulpal microvasculature during orthodontic tooth movement: a stereological study. European Journal of Orthodontics, 2006. 28(3): p. 217-220.

264. McDonald FTR, Pitt Ford. Blood flow changes in permanent maxillary canines during retraction. European journal of orthodontics, 1994. 16(1): p. 1-9.

265. Derringer KA, Linden RW. Enhanced angiogenesis induced by diffusible angiogenic growth factors released from human dental pulp explants of orthodontically moved

266. Taintor JF, Shalla CL. Comparison of respiration rates in different zones of rat incisor pulp. Journal of Dentistry, 1978. 6(1): p. 63-70.

267. Brin I, et al. The influence of orthodontic treatment on previously traumatized permanent incisors. European Journal of Orthodontics, 1991. 13(5): p. 372-377.

268. Bauss O,et al. Influence of orthodontic intrusion on pulpal vitality of previously traumatized maxillary permanent incisors. American Journal of Orthodontics and Dentofacial Orthopaedics, 2008. 134(1): p. 12-17.

269. Bauss O,et al. Influence of Orthodontic Extrusion on Pulpal Vitality of Traumatized Maxillary Incisors. Journal of Endodontics, 2010. 36(2): p. 203-207.

270. Bauss O.et al. Pulp vitality in teeth suffering trauma during orthodontic therapy. The Angle Orthodontist, 2009. 79(1): p. 166-171.

271. Seltzer S, Bender IB. The dental pulp: biologic considerations in dental procedures. 3rd ed. 1984, Philadelphia: Lippincott.1984;3rd ed:191.

272. Dougherty HL. The effect of mechanical forces upon the mandibular buccal segments during orthodontic treatment. American Journal of Orthodontics, 1968. 54(1): p. 29-49.

273. Marshall JA. A study of bone and tooth changes incident to experimental tooth movement and its application to orthodontic practice. International Journal of Orthodontia and Dentistry for Children, 1933. 19(1): p. 1-21.

274. Cwyk F. Endodontic implications of orthodontic tooth movement. Journal of Dental Research 1984;63:286.

275. Venkatesh S, Ajmera S, Ganeshkar SV. Volumetric pulp changes after orthodontic treatment determined by cone-beam computed tomography. Journal of Endodontics, 2014.40(11): p. 1758-1763.

276. Bohl MV, et al. Pulpal reactions to orthodontic force application in humans: a systematic review. Journal of Endodontics, 2012. 38(11): p. 1463-1469.


277. Javed F, et al. Influence of orthodontic forces on human dental pulp: a systematic review. Archives of Oral Biology, 2015. 60(2): p. 347-356.

278. Restarski JS. Effect of vibration upon the dental pulp and periosteum of white rats. Journal of Dental Research, 1945. 24(2): p. 205-255.

279. Domínguez, Ballesteros RE, Viáfara JH, Tamayo OM. Effect of low level laser therapy on dental pulp during orthodontic movement. World J Methodol 2013; 3(2): 19-26.

280. Abi-Ramia LB, Stuani AS, Stuani AS, Stuani MB, Mendes Ade M. Effects of low-level laser therapy and orthodontic tooth movement on dental pulps in rats. Angle Orthod. 2010;80(1):116-122.

63

MANUSCRIPT

EffectsofLow-LaserTherapyandLED-

mediatedPhotobiomodulationonpulp

canalvolumeofmaxillaryfirst

premolarsundergoingorthodontictooth

movementDRRALUCABALEANU


FACULTYOFDENTISTRY

UNIVERSITYOFSYDNEY,

AUSTRALIA2020

64

Raluca Baleanu BDSc (Hons)

Post-graduate student

Discipline of Orthodontics

Faculty of Dentistry, University of Sydney

Nam(Alice) Wong Lecturer Discipline of Orthodontics Faculty of Dentistry, University of Sydney Peter Petocz

PhD

Associate Professor

Statistician

Department of Statistics, Macquarie University

Matthew Foley Image Analysis & Computer Tomography Specialist Sydney Microscopy & Microanalysis Faculty of Dentistry, University of Sydney Oyku Dalci DDS, PhD, Spec Orth

Senior Lecturer


Faculty of Dentistry, University of Sydney M. Ali Darendeliler

BDS, PhD, Dip Ortho, Certif. Orth, Priv. Doc

Professor and Chair




Abstract

Background and Objective: Since the demand for fast orthodontic treatment has risen,

different techniques have been proposed and implemented with various success. The use of

Low-Level Laser Therapy (LLLT) and LED-meditated photobiomodulation (LED-PBM) to

accelerate orthodontic tooth movement has been widely studied. However, limited

information is available on the effect of LLLT and LED-PBM on pulp volume. Therefore, the

aim of this study was to assess the effects of Low-Level Laser Therapy (LLLT) and LED-PBM on

pulp volume of maxillary first premolars.

Methods: Micro-CT scans of maxillary first premolars that were collected previously as part

of two different split-mouth randomized clinical trials on orthodontic root resorption were

analysed to determine the volumes of tooth root and pulp canal. All maxillary first premolars

were exposed to 150g buccal tipping force for 4 weeks. Additionally, 20 premolars had

received LLLT and 18 premolars had received LED-PBM while their contra-lateral teeth were

served as controls. Premolars were scanned with MicroCT following extraction. Avizo 3D

software was used to digitally measure the volume of the tooth roots and pulp canal.

Results: The average pulp volume for LLLT premolars and controls were 19.71mm3 and

17.95mm3 respectively. There was an average of 1.76± 5.03 mm3(95% CI: -0.60 mm3 4.11

mm3) more pulpal volume in the experimental group than in the controls. However, this was

not statistically significant (p= 0.135). The average pulpal volume for LED PBM premolars and

controls were 14.11mm3 and 15.65mm3 respectively. The 1.54± 4.81 mm3 (95% CI: -3.93


mm3 - 0.85 mm3) mm3 reduction in pulp canal volume in LED-PBM premolars compared to

controls was not statistically significant (p=0.193) either. Univariate analysis of variance

indicated that application of either LLLT or LED-PBM does not result in statistical difference

changes in pulpal volume. There was no correlation between difference in pulp volume

change and difference in the total amount of root resorption caters developed following the

application of LLLT and PBM.

Conclusion: No significant difference was found on the pulp canal volume of maxillary first

premolars between experimental and control groups when LLLT or LED-PBM was applied for

4 weeks in addition to 150g of orthodontic force.

Key words: Low -Laser Therapy (LLLT), LED-meditated photobiomodulation (LED-PBM,

Orthodontic tooth movement, Pulp volume, Micro-CT.


Introduction

Tooth movement is a process that combines pathological and physiological responses to

externally applied forces1 and the rate of the movement is influenced by bone modelling and

remodelling.2 The rate of alveolar modelling and remodeling is determined by the activity of

bone cells (osteoclast, osteoblast and osteocytes) which are under the influence of

mechanical stimulation and biomechanical factors such as prostaglandins and cytokines.2

Absorption’ as root resorption was first reported by Bates in 1856.3 The board definition of all

forms of root resorption including orthodontic root resorption which refers to the

pathological destruction and subsequent removal of the hard tissues of the tooth root as a

result of clastic activity.4

The demand for fast orthodontic treatment has risen in the last decade. A short treatment

time is beneficial to both patients and clinicians as it minimizes the risk of complication such

as caries, periodontal problems, root resorption and patient burnout.5,6,7 Various methods

ranging from invasive to non-invasive methods have been proposed and implemented to

accelerate orthodontic tooth movement. The non-invasive methods include application of

Low-Level Laser Therapy (LLLT) and LED-mediated photobiomodulation (LED-PBM). From the

discovery of the ‘Rubby Laser’ by Maiman,8 studies have been done to introduce laser

technology in the area of dentistry. Laser can be applied to many fields in dental research and

basic research offers new possibilities by advancing laser technology.

Low-level laser therapy (LLLT), also known as soft laser and cold laser, is a non-invasive

method with its effects limited to the target tissue.9 LLLT is carried out by a the

semiconductor diode device and is using laser wavelength, which is absorbed primarily by


tissue pigment (melanin) and hemoglobin.10 The proposed mechanism of action involves the

stimulation of cellular metabolism near infrared or infrared light at a low energy density.10

Many parameters influence the quality, quantity, and density of the light energy delivered to

the target tissue. Such as light source- power, spot size, number and frequency of

applications, application time, and application energy.9

Clinically, LLLT has been used in procedures including aesthetic gingival re-contouring, soft

tissue crown lengthening, exposure of impacted teeth, removal of inflamed and hypertrophic

tissue, frenotomies, and photo stimulation of the aphthous and herpes lesions.11

Orthodontically, LLLT was widely used in attempts to reduce pain following application of

orthodontic force as well as accelerating orthodontic tooth movement. Some studies

reported controversial results and failed to observe any significant difference between the

rates of orthodontic tooth movement on the irradiated sides versus non irradiated side.12

Ng.et al 13 reported a reduction of the total amount of root resorption on first premolars

compared with sham laser and a minimal difference between pulsed or continuous

irradiation with LLLT after 4 weeks of buccal tipping force application to maxillary first

premolars.

Photobiomodulation is a minimally invasive therapy that has been adopted in modern

medicine and dentistry for pain reduction, promoting wound healing and preventing tissue

damage. It can be performed in the form of low level laser (LLL) or light-emitting diode (LED).

Being a non-coherent light source, LED light produces fewer side effects when compared to

LLL which is coherent. Therefore, it can be safely applied to a large area of the body

surface.14,15



responses 16, therefore it potentially allows a wide range of applications in the field of

orthodontics. Various animal and human studies were conducted in attempt to verify these

applications which include orthodontic pain relief,17 enhancement of mandibular growth as

well as osseointegration of mini-screws, accelerating orthodontic tooth movement and

minimizing orthodontically induced inflammatory root resorption (OIIRR).18,19,20

LED-mediated photobiomodulation has been demonstrated to significantly increases the rate

of orthodontic tooth movement in animal studies.21,22,23,24 by encouraging bone remodeling

via increased osteoblastic and osteoclastic cell proliferation and function during tooth

movement. Laser-mediated PBM has also proposed to potentially reduce orthodontic related

pain. 16,17,25,26 The mechanism is thought to involve stabilization of membrane potential and

release of neurotransmitters which induce analgesic effect.27,28,29 LED- PBM did not produce

a statistically significant difference in the amount of root resorption craters in maxillary first

premolars following buccal tipping force for 28 days. 30 However, the results of this study

should be validated on a patient sample undergoing PBM over the full course of orthodontic

treatment.30

The effect orthodontic force application on dental pulp tissue has long been the subject of

studies in decades with conflicting results.31,32,33 Proffit et al.31 described that light and

continuous force has little or no effect on dental pulp. However, Stevik and Reitan reported

that the reaction to the orthodontic tooth movement varies from mild hyperemia to

complete necrosis.34 Dominquez et al.35 showed a favorable histological result with an

adaptive response in the human pulp following the exposure to LLLT. Similarly, Abi-Ramia et


al.36 also observed an increased rate of pulpal tissue repair in a rodent model after LLLT

application during orthodontic tooth movement.

However, the studies investigating the effect of LLLT and LED PBM on pulpal tissues either did

not involve tooth movement or were carried out on animals. Therefore, the aim of this

present study was to investigate the effects LLLT and LED PBM on pulp volume of maxillary

first premolars undergoing experimental orthodontic tooth movement for 4 weeks.

Material and Methods

76 maxillary first premolars were analysed in this study. The teeth were previously extracted

from 38 patients as part of 2 randomized clinical split-mouth trials investigating the effects of

LLLT and PBM over 4 weeks.13,30 Randomization was generated using a computer random

number generator- www.randomization.com, and allocation was concealed in sequentially

numbered, opaque, sealed envelopes.13,30 All premolars were subjected to 150 g buccal

tipping forces for 4 weeks. One side of the mouth in each patient was randomly allocated to

one of the proposed accelerated tooth movement interventions, while the contralateral

premolars served as control with sham light application/ placebo (Ethics approvals:X14-0200

HREC/14/RPAH/263 and X16-0215 & HREC/16/RPAH/266, Registration: Clinical Trials Registry

-ACTRN12616000829415 and Australian New Zealand Clinical Trials Registry -

ACTRN12616000652471).13,30

Intervention 1: LLLT - 808-nm diode laser (Klas-DX6182, Konftec Corporation, New Taipei City,

Taiwan). 13


There was a total of 20 experimental premolars and 20 controls extracted from 20 patients.

There were 10 girls and 10 boys with a mean age of 16.13

On the experimental side, 10 of the premolars received continuous laser delivery LLLT-A (0.18

W, 9 seconds per point) while 10 received pulsed delivery LLLT-B (0.36 W, pulsed at 20 Hz

with 25 ms on/off, 50% duty cycle, 4.5 second per point). The energy dose was 1.6 J per

point. On the control side the placebo-laser was created by blocking out the laser beam by

covering the beam aperture with black absorbent paper. 13

LLLT application regimen was every day for the first 4 days (days 0, 1, 2, and 3) and then

weekly (days 7, 14, and 21) until 28 days. Application of laser was to the mucosal surface of

the premolar root at four points on the buccal and palatal surfaces at the level of the apex,

middle third, cervical thirds on the mesial and distal sides.13

Intervention 2: LED mediated PBM, applied via the Orthopulse™ device.30

There were a total of 18 experimental premolars and 18 controls extracted from 18 patients.

There were 14 girls and 4 boys with a mean age of 15.30

On the experimental side, the Orthopulse™ applied near-infrared (NIR) light with a

continuous 850nm wavelength from approximately 54 LEDs spaced 5mm apart in two banks

of 3x9 elements embedded within a flexible silicone matrix. The LED array is specified to

produce a power density of 60mW/cm². Following randomization of sides, on the

control/placebo side the light emitted from either the left or right banks of LEDs was

deactivated by physical blocking using a single layer of opaque black obstructive film and a

single layer of medical-grade adhesive tape (Leukoflex, BSN medical GmbH Hamburg,


Germany). The light blocking effect of this material was tested with the use of a laser-check

sensor (Lasercheck®, Edmund Optics Inc, Barrington NJ, USA).30

Patients were instructed to use the device for 5 minutes/day on the maxillary arch for 28

days. Compliance was monitored using a smart-phone app which logged each patient’s use of

the device via bluetooth.

MicroCT Scan

Following 28 days of experimental period the maxillary premolars were extracted and were

scanned using SkyScan 1172 desktop X-ray micro-tomograph system (SkyScan, Aartselaar,

Belgium), from the cementoenamel junction (CEJ) to the root apex, with image resolution set

at 17.6 µm.13,30

Specifically designed software, NRecon (version 1.6.9.18, Skyscan, Aartselaar, Belgium),

reconstructed the image data from SkyScan, utilizing a modified Feldkamp cone-beam

algorithm to produce slice-by-slice axial reconstructions. These were saved in 16-bit tagged

image file format (TIFF) format.13,30

Volumetric Measurements

Avizo 3D software 2019 (Thermo Fisher Science) was used to digitally measure the volume of

the tooth structure. All premolars included in this study were segmented into 3 materials

(Crown, Root, Pulp) using threshold intensity values related to material attenuation. To

minimize crown variability between samples, only the volume from the CEJ to the apical tip of

the tooth was considered for evaluation. The pulp was extracted using Avizo as showed in


Table 6. For the digital conversion a threshold of specific grey values were used, altering the

data into a binary format.

The scale for the datasets was 17.66 micron and was used to analyze the volume of calcified

tooth structure with the section reversed to measure the internal volume of uncalcified

matter outlining the pulp tissue. The ‘Material Analysis’ function of the Avizo was used to

analyze the segmented components. All the pulpal and hard tissue volumetric measurements

were performed by the same operator.

Statistical Analysis

Paired t test was used to assess differences in total pulp canal volume of LLLT or LED PBM

versus control teeth. Statistical significance was set at 0.05. Univariate analysis of variance

was used to compare the difference between the experimental and control sides in between

interventions. Pearson Correlation was used to assess the relationship between difference in

pulp volume and difference in the total amount of root resorption craters between the

intervention and sham among all three groups (Table 5). Error measurements were carried

out ten months after initial measurements, on randomly selected teeth (27%). SPSS (version

25) software, was used to analyze the data.

Results

The average pulp volume following the application of buccal tipping force and LLLT was

19.71mm3 and 17.95mm3 for the controls. There was a mean difference of 1.76 mm3

between the LLLT group and the controls which was not statistically significant (p=0.14,

Table1). Similarly, the average pulp volume for LED-PBM teeth measured 14.11mm3 and


15.65mm3 for the controls. (Table1) The mean difference between the LED-PBM teeth and

the controls was statistically insignificant (-1.54mm3, p=0.19; Table1, Chart1). In addition, the

mean difference between teeth treated with continuous LLLT and the control group was

0.01mm3, while the mean difference between teeth treated with pulsed LLLT and the control

group was 3.5mm3 (Table 2). The difference between intervention and sham in terms of the

ratio between pulp canal and calcified tooth structure in all three groups were shown in

figure 2 and figure 3.

Univariate analysis of variance (ANOVA) demonstrated that the pulp volume changes in LLLT

group and LED-PBM group were not statistically different (Table 3). Similarly, the pulp volume

changes in continuous or pulsed LLLT group were not significantly different to that of LED-

PBM group (Table 4).

There was no correlation between difference in pulp volume and difference in the total

amount of root resorption craters between the intervention and sham sides among all three

groups (Table 5).

The intra-rater reliability test revealed the coefficient of variation was less than 1% for all

measurements.

Discussion

This is the first human study assessing human pulp volume changes after treatment with LLLT

and orthodontic tooth movement with Micro-CT. Further, it is also the first human study to

investigate the effect of photomodulation (LED-PBM) on the dental pulp volume during

orthodontic tooth movement.


It was shown that orthodontic force may generate pulpal changes that range from mild

hyperemia to complete necrosis.34 Furthermore LLLT and LED-PBM have recently been

introduced as methods of accelerated orthodontic tooth movement. However, there are no

studies that investigated the effects of either on humans during orthodontic tooth

movement. Therefore, it is worthwhile to investigate their impacts on pulpal tissues. Most of

previous studies investigating the change in pulp tissue following exposure to LLLT utilized 2D

imaging such as SEM and periapical radiographs.37 These evaluation methods provide limited

information because attempts were made to analyse 3D structure with 2D imaging. In the

present study, analysis was performed with micro-CT which is a non-invasive method used to

visualize dental morphology comprehensively in three dimensions without destroying the

extracted teeth. 13,30,38However, owing to the heterogeneity of study designs and evaluation

methods being used, the results of this study were not comparable to previous studies.

LLLT is used for its stimulatory effects that involves tissue regeneration, wound healing as

well as relieving inflammation and pain.17,25 This study aimed to assess the effects of LLLT and

LED-PBM on pulp volume in human maxillary first premolars that were subjected to 150g of

buccal tipping force for 28 days. No statistically significant reduction on the pulp volume was

detected in the control group when compared to that when LLLT or LED PBM was applied.

Similarly, exposure to both continuous and pulsed LLLT resulted in an increased volume of

pulp chamber when compared to sham, however, these results did not reach statistical

significance. Previous studies showed stimulated adaptive response of the pulpal tissues with

application of LLLT. 35,36



responses.16 It has been demonstrated in several studies that there is a correlation between

energy density and dose.39-41 Currently, no published information is available in regards to

the effect of LED-PBM on pulp tissue. However, several in vitro studies postulated that laser-

mediated PBM with low energy density stimulates the proliferation of dental pulp stem

cells.42,43 In the present study exposure to LED-PBM resulted in a decreased volume of pulp

chamber when compared to sham, although the result was not statistically significant.

Perhaps further prospective studies with larger sample size with the use of lower energy

density PBM source might be helpful in elucidating its effect on pulpal tissues.

Similarly, the current study is the first human study investigating the effect of PBM on dental

pulp tissue. Previous In vitro studies42,43,44 showed positive effects of PBM on pulp cells.

However, these studies had different study designs and evaluation methods therefore direct

comparison is not possible.

The connection between orthodontic force application and dental pulp tissue has been the

subject of studies in recent years.31,32,33 The most pertinent research is linked to metabolic,

biochemical and vascularization reaction of the pulp to orthodontic tooth forces that are the

same as angiogenesis in wound healing.45 The molecular process of PBM for angiogenesis of

pulp healing are still undiscovered.46 Angiogenesis through neovascularization generates new

capillaries.47 However, alteration in the blood supply and angiogenesis have a direct impact

upon pulp metabolism.48-50 Orthodontic forces may encourage vasodilation in dental pulp

tissue46 and causes vascular changes. It is prudent to apply orthodontic loading within the

biological limitations of the pulp.52


Regardless of the significant research on orthodontic movement with LLLT21,53,54 or

PBM21,22,23,24, showing an increase of the rate of orthodontic tooth movement, reports on

pulp volumetric changes are limited. A few authors have reported an increased

vascularization which accelerates pulp repair when irradiated with LLLT or PBM.

Limitations

One of the limitations of this research could be the small sample size.13,30 The current sample

was a convenient sample available from previous randomized controlled trials.

The experimental period applied in this study was short due to ethical reasons.13,30 Since

orthodontic treatment are extended over months to years and different types of tooth

movement are applied to teeth throughout treatment, the current study results may not

reflect the actual clinical situation however could be used as a baseline study.

The confounding factors in this study include factors which may affect pulp canal volume

such as age 55,56,57,58,59, hormones 60,61,62, nutrition 63 and infection64. However, the teeth

involved in the study are caries free, and the rate of dentine deposition brought about by

these factors is rather slow, the effect of these factors on the result should be limited.

Generalizability

The generalizability of these outcomes may be limited since the study was carried out for

only 28 days using buccal tipping forces.13,30 In addition, there is considerable individual

variation in pulp tissue response hence the pulp volume changes in response to orthodontic

force. Pulp tissue response vary between individuals and sometimes within the same


individual on difference teeth due to different exposure to trauma. Therefore, the result may

not be able to apply to the general population.

The results of this study were based on specific LLLT and LED PBM parameters. Therefore,

the results are not applicable to different intervention parameters.

Conclusions

LLLT, continuous or pulsed, or LED PBM, using parameters in this study, when applied on

maxillary first premolars undergoing 150 g buccal tipping forces for 28 days, do not create

significant pulpal canal volume changes. However, additional research should be carried out

with patients receiving LLLT or LED PBM application throughout the entire course of

orthodontic treatment.


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List of tables and figures

Figure 1. AVIZO Workflow

Step Module Sub-settings/steps Justification

1 Extract sub-volume Default To crop each tomogram

down to a minimal size

2 Edit new label field: Crown,

Root, Pulp Canal (1) Threshold tool To select material

(1) Fill volume To fill pores

(1) Assign selection to

label [+]

To tag maaterial for

subsequent label analysis

(1) Circular paint tool

To select tap and bottom of

volume

(1) Interpolate

To select volume between

regions selected at(4)


label [+]

To tag volume for

subsequent label analysis

3 Label analysis Interpretation – 3D Volume

analysis

To calculate the volume of

the tagged regions selected


Figure 2. Box plot depicting the pulp canal to hard tissue volume of LLLT group and LED PBM

group.

Table 1. Average pulp volume of the maxillary first premolars in the intervention and sham group (mm³)

Experimental group Control group

95% CI

Mean t test SE of Lower Upper

N Mean SD Mean SD difference P value mean bound bound

LED PBM 18 14.11 6.89 15.65 6.33 ⁃1.54 0.19 1.13 -3.93 0.85

LLLT 20 19.71 5.50 17.95 4.74 1.76 0.14 1.12 ⁃0.60 4.11

Descriptive statistics (mean, standard deviation), mean differences of pulp volume between intervention and controls, P values, standard error of the mean, and 95% confidence intervals. Non statistically significant differences were noted between intervention and control group.


Table 2. Pulp volume average and difference and root resorption calculation per root surface (mm³)


95% CI

Mean t test SE of Lower Upper N Mean SD Mean SD difference P value mean bound bound

LED PBM 18 14.11 6.89 15.65 6.33 -1.54 0.19 1.13 -3.93 0.85

LLLT-A 10 19,96 4,61 19,95 4,29 -0,01 1.0 1.23 -2.77 2.8

LLLT-B 10 19,46 6,53 15,96 4,51 3.50 0.08 1.77 -0.51 7.51

Descriptive statistics (total, mean, standard deviation), mean differences, average pulp volume (mm3) between laser and the controls, P values, standard error of the mean and 95% confidence intervals. Non statistically significant differences were noted between laser and the controls for all pulp volume.

Figure 3. Box plot depicting the pulp canal to hard tissue volume of LED PBM group, LLLT-A

group and LLLT-B group.


Table 3. Univariate analysis of variance (ANOVA) investigating difference between LLLT group and LED-PBM group

Sum of squares df Mean square F Sig.

6.451 1 6.451 0.711 0.405

Table 4. Univariate analysis of variance (ANOVA) investigating difference between LLLT-A group LLLT-B group and LED-PBM group


16.042 2 8.021 0.885 0.422

Table 5. Correlation between pulp volume changes and difference in total amount of root resorption craters among all three groups

LLLT vs LED-PBM LLLT-A vs LED-PBM LLLT-B vs LED-PBM

Pearson Correlation -0.058 0.152 -0.010

Significance 0.731 0.441 0.959

87

MANUSCRIPT

EffectofLow-LevelLaserTherapyon

pulpcanalvolumeofmaxillaryfirst

premolarsundergoingorthodontictooth

movementandinretention

DRRALUCABALEANU


FACULTYOFDENTISTRY

UNIVERSITYOFSYDNEY,

AUSTRALIA,2020


Raluca Baleanu BDSc (Hons)

Post-graduate student



Nam(Alice) Wong Lecturer Discipline of Orthodontics Faculty of Dentistry, University of Sydney Peter Petocz

PhD

Associate Professor

Statistician

Department of Statistics, Macquarie University

Matthew Foley Image Analysis & Computer Tomography Specialist Australian Centre for Microscopy & Microanalysis Faculty of Dentistry University of Sydney Oyku Dalci DDS, PhD

Senior Lecturer


Faculty of Dentistry, University of Sydney M. Ali Darendeliler

BDS, PhD, Dip Ortho, Certif. Orth, Priv. Doc

Professor and Chair




Abstract

Background and Objective: The outcome of LLLT research on tooth movement has supplied

very limited information about the aftereffects on dental pulp tissue. The purpose of this study

is to investigate the effects of LLLT on the pulp volume in maxillary first premolars subjected

to 150g of buccal tipping force and 6 weeks after the removal of orthodontic force.

Methods: Micro-CT scans of maxillary first premolars that were attained from two published

randomised clinical trials using split-mouth designs on effects of LLLT orthodontically induced

inflammatory root resorption (OIIRR) and repair. All maxillary first premolars were exposed to

150g buccal tipping force for 4 weeks. 20 premolars in the Tooth Movement group (TM

group) had received LLLT during orthodontic tooth movement while the other 20 premolars

in Retention group (Ret group) received LLLT 6 weeks after the removal of orthodontic force.

The premolars on the contralateral side were served as controls. The volume of pulp canal

and calcified tissue were analysed after MicroCT scans digitally using Avizo 3D software.

Results: There were no statistically significant differences between the LLLT teeth and the

controls in terms of pulp canal to hard tissue volume ratio in both TM group and Ret groups.

Univariate analysis of variance demonstrated that there was no differences between TM and

Ret groups either. Further, no correlation was found between the changes in pulp canal to

hard tissue volume ratio and the amount of root resorption craters in both groups.


Conclusion: No significant difference was found on the pulp canal to hard tissue volume ratio

of maxillary first premolars between experimental and control groups when LLLT was applied

during orthodontic tooth movement or in retention.

Key words: Low-laser therapy (LLLT), Orthodontic tooth movement, Pulp volume, Micro-CT

91

Introduction

Orthodontic treatment aims to improve alignment, occlusion and function.1 In response to

orthodontic force, tooth movement occurs following modification to the local environment

including alteration of vascular permeability, recruitment of inflammatory mediators and

alveolar bone remodeling.1 Sometimes, these changes are accompanied by complications

such as orthodontically induced gingival inflammation, caries, orthodontically induced

inflammatory root resorption (OIIRR) and pulpal changes. 2,3,4

Orthodontic force has been considered by some as a form of controlled trauma to the tooth

supporting structures. 5 Histological studies have revealed various degree of pulpal alteration

occurred, they include vascular compression and angiogenesis,6,7,8 deposition of secondary

dentin and alteration of the odontoblastic layer.10,11Depression of respiration rate of pulp

cells was also demonstrated.12

Literature regarding the clinical implications of these histological findings is inconclusive.

Some authors believe that these effects are transient with no long-term damage,12 however,

whilst others suggest that the changes are permanent.6,13 These clinical manifestations

include narrowing of pulp canals,14 presence of pulp stones,15,16 deterioration of long-term

tooth vitality. 17,18 Specifically, a prospective study with CBCT data demonstrated that teeth

which underwent orthodontic treatment experienced a reduction in pulpal volume.19

Low-level laser therapy (LLLT) is used for pain relief20 and accelerating orthodontic tooth

movement by increasing the rate of bone remodeling21. LLLT with wavelength longer than

600nm offers a stimulatory effect on cellular metabolism.22,23,24,25 Animal26,27 and human

clinical research28 has demonstrated the potential for LLLT to accelerate tooth movement

Dr. Raluca Băleanu 92

when compared to controls, whilst not increasing the chance of adverse sequelae such as

root resorption and alveolar bone resorption of supporting teeth.

Ng et. al.29 conducted a randomized controlled trial which demonstrated that LLLT reduced

the total amount of root resorption on maxillary first premolars compared to the sham laser.

The study also found that there was minimal difference between the effect of pulsed or

continuous LLLT on the amount of root resorption craters. However, Chun et. al.30 reported

in another randomized controlled trial that LLLT did not offer statistically significant

improvement in the amount of repair of root resorption craters on maxillary first premolars

when applied 6 weeks after orthodontic tooth movement.

With the increase in knowledge of biological effect of LLLT, the interest in whether it modifies

the pulpal response to orthodontic force has increased. However, to date there is no study

investigating the effect of LLLT on teeth undergoing orthodontic tooth movement and in

retention. Therefore, the purpose of this study is to assess the effects of LLLT on the pulp

volume in maxillary first premolars subjected to 150g of buccal tipping force and 6 weeks

after the removal of orthodontic force.

Material and Methods

Eighty maxillary first premolars included in this study were formerly removed from 40

patients included in two randomized clinical trials with split-mouth designs. 29,30

Randomization was generated using a computer random number generator-

www.randomization.com, and allocation was concealed in sequentially numbered, opaque,

sealed envelopes. The two trials analysed the effect of LLLT on teeth undergoing orthodontic

tooth movement and in retention respectively. One premolar in each patient was randomly


assigned to be exposed to LLLT while the contralateral premolars served as the

controls(Ethics approval:X14-0200 HREC/14/RPAH/263 and X16-0276 &

HREC/16/RPAH/347,Registration: Clinical Trials Registry-ACTRN12616000829415, Australian

New Zealand Clinical Trials Registry-12616000682448).29,30

Intervention 1( TM Group): A total of 20 patients (10 female and 10 male) were included in

this study, providing 20 premolars in the experimental group and 20 in the control group. A

buccal tipping force of 150 g was applied to all teeth for 4 weeks. In each patient, one side of

the maxillary first premolars were randomly assigned to the experimental group and received

808nm AIGaAs diode laser(Klas-DX6182, Konftec Corporation, New Taipei City, Taiwan).29

Out of the 20 premolars in the experimental group, 10 experienced continuous laser delivery

,TM-A (0.18 W, 9 seconds per point) and 10 experienced pulsed delivery ,TM-B (0.36 W,

pulsed at 20 Hz with 25 Ms on/off, 50% duty cycle, 4.5 second per point). In the control

group, the placebo-laser was produced through blocking the output by a sham function of

the device.29

LLLT was applied every day for the first 4 days (days 0,1,2 and 3) and then weekly (day 7, 14

and 21) until 28 days. The teeth were irradiated on the mucosal surface of the premolar root

at four points on the buccal and palatal surfaces at the different levels, including the apex,

middle third, cervical thirds on the mesial and distal sides. 29

Intervention 2 (Ret group): A total of 20 patients (10 female and 10 male) were included in

this study, providing 20 premolars in the experimental group and 20 in the control group. A

buccal tipping force of 150 g was applied to all teeth for 4 weeks. After the active force was

removed, teeth in experimental group were exposed to LLLT for 6 weeks. 30


LLLT regime included weekly application using a continuous beam of 660-nm,75-mW

aluminum-gallium-indium phosphorus laser (Thor Photomedicine, Buckinghamshire, United

Kingdom). Contact application was adopted at 8 points buccally and palatally above the

mucosa over each tooth root for 15s per point. In the control group, the placebo-laser was

produced through blocking the output by a sham function of the device. 30

MicroCT Scan

The samples were scanned with SkyScan 1172 desktop X-ray micro-tomograph system

(SkyScan, Aartselaar, Belgium), from the cementoenamel junction (CEJ) to the root apex, with

image resolution set at 17.6 µm.29,30

NRecon (version 1.6.9.18, SkyScan, Aartselaar, Belgium) software, using a modified Feldkamp

cone-beam algorithm, reconstructed the image data from SkyScan, and produce slice-by-slice

axial reconstructions, which were saved in 16-bit tagged image file format (TIFF). 29,30

Volumetric Measurements

The calcified and non-calcified premolar structure was digitally measured using Avizo 3D

software 2019 (Thermo Fisher Science) to find out the volume of tooth structures. All

samples were segmented into 3 Materials (Crown, Root, Pulp canal) applying threshold

intensity values associated to material attenuation.

‘Material Analysis’ function of the Avizo was utilized to analyse the segmented components.

The volume from CEJ to the apical tip of the premolar was investigated to minimize crown

variability between samples. . The pulp was extracted using Avizo as showed in Table


6.Threshold of specific grey values was utilized, changing the data into a binary format for

digital conversion. The measurements were performed by the same operator.

Statistical Analysis

The SPSS statistics program was utilized for statistical analysis and P >0.05 was considered

statistically significant. Paired t test was used to assess the relationship between the

experimental group and control group in all TM-A, TM-B and Ret groups. Univariate analysis

of variance was used to compare the results between TM and Ret groups. Pearson

Correlation was used to assess the relationship between pulp volume changes and changes in

amount of root resorption craters between the intervention and sham among all three

groups.

After initial measurements (10 months), on randomly selected premolars (27%), error

measurements were carried out. SPSS (version 25) software, was used to analyze the data.

Results

The average pulp canal to hard tissue volume ratio following the application of buccal tipping

force in TM group was 7.86 and 7.31 for the sham. There was a mean difference of 0.54 mm3

between the two groups which was not statistically significant (p=0.37, Table1). Similarly, in

Ret group the average pulp canal to hard tissue volume ratio for experimental teeth and

control teeth measured 8.01 and 6.85 respectively. (Table1) The difference between the

mean pulp canal to hard tissue ratio between the experimental teeth and the controls was

statistically insignificant (1.16, p=0.11; Table1). In addition, the difference of the mean pulp

canal to hard tissue ratio between the continuous LLLT treated teeth and the control group

was -0.14, while that of pulsed LLLT treated teeth was 1.24 (Table 2). The differences


between intervention and sham in terms of the ratio between pulp canal and calcified tooth

structure in all three groups were shown in Figure 2 and Figure 3.

Univariate analysis of variance (ANOVA) demonstrated that the pulp volume changes in TM

group and Ret groups were not statistically different (Table 3). There was no correlation

between differences in pulp to hard tissue volume ratio and difference in the total amount of

root resorption craters between the experimental and control groups among all three

groups. (Table 4)

The intra-rater reliability test revealed the coefficient of variation was less than 1% for all

measurements.

Discussion

This is the first human study using Micro-CT to assess the effect of LLLT on pulp volume

changes in maxillary first premolars undergoing orthodontic treatment and retention.

The premolars used in this study were convenient samples obtained from two previous

randomized controlled trials.29,30 Due to the slightly different micro-CT settings used in two

studies, pulp canal to hard tissue volume ratio was analysed instead of the actual pulp canal

and hard tissue volumes. Results of the current study revealed that LLLT teeth exhibited a

larger pulp canal to hard tissue volume ratio than the controls in both TM group and Ret

groups, however, the findings did not reach statistical significance. Results also indicated that

teeth which received continuous LLLT had a lower pulp to hard tissue volume ratio compared

to the controls, while teeth which received pulsed LLLT had a higher pulp to hard tissue

volume ratio compared to the controls. Again, these outcomes were not statistically

significant. Further, there was no statistically significant correlation between pulp canal to


hard tissue volume ratio and difference in the amount of root resorption craters in all three

groups.

In contrast to the outcomes of this study, literature pertaining to the effects of LLLT on pulp

tissue after the application of orthodontic force were linked to an increase in the repair of

the pulp tissue.31,32 However, it is important to note that due to the difference in study

designs and laser parameters used, as well as the evaluation methods, results of different

studies are not able to be compared directly.

The result of the current study could possibly be explained by the fact that only the portion of

the tooth below the CEJ was analysed as the great individual variation of crown morphology

may have confounded results. Thus, changes such as presence of pulp stones33,34,35 and

deposition of secondary dentin36 occurring at the coronal part of the pulp could not be

detected.

Another possible explanation for the outcome of the current findings could be that the

magnitude and duration of orthodontic force applied in this study did not impose detrimental

effects on pulpal tissue. Pulp tissue has an adaptive response which favors the repair from

the initial injury. 31 A study using laser doppler flowmetry demonstrated that there is an

immediate reduction in blood flow after orthodontic force application. This is followed by a

reactive hyperemia after approximately half an hour. The rate of blood flow was shown to

return to normal within 72 hours. 37

In addition, the extent of immersion and diffraction of LLLT could also be affected by tissue

molecules such as hemoproteins and melanin. 38 Therefore, the amount of light energy

reaching the target tissue also depends on their melanin composition and thickness of


alveolar bone. Conversely, differences may have occurred due to dental structure variability.

even though in both groups the wavelengths at the range of reported penetration depths,

showed no differences between irradiated groups and sham. Further research should be

undertaken on investigating the energy delivered to the periodontal ligament and the use of

greater range wavelengths.

Further, multiple mechanisms regarding the interaction of light energy from LLLT, aiming to

the target tissue are affected through countless variables such as light source- power, spot

size, number and frequency of applicability, application time, and application energy. 39

Further studies, which take these variables into account, will need to be undertaken.

Limitations

The results of this study could be confounded by the different laser parameters applied

throughout TM group and the Ret groups, along with the different types of tooth movement

that are applied to teeth during treatment.29,30 A previous study has revealed that while LLLT

with a wavelength longer than 600nm offers a stimulatory effect on cellular

metabolism,22,23,24,25 wavelengths in the near-infrared range of 810nm-830nm are

demonstrated to have more superior penetration power hence more effective than

wavelengths in the range of 630-680nm in promoting healing, reduction in inflammation and

pain.38 Another limitation of this study is the small sample size.29,30 LLLT teeth in both TM

group and Ret group exhibited a larger pulp to hard tissue volume ratio than the sham, but

the result did not reach statistical significance.

Factors which affects pulp canal volume such as age 40-44, hormones45-47, nutrition48, and

infection49. may potentially confound the result of the study. However, the teeth involved in


the study are caries free, and the rate of dentine deposition brought about by these factors is

rather slow, the effect of these factors on the result should be limited.

Generalizability

The interventions could only be carried out for a relative short duration of time due to ethical

reasons30 therefore the results of the current study may not be able to generalized to the

clinical setting. Ideally, prospective research that follows the participants for the full length of

orthodontic treatments should be conducted, however due to the nature of the study which

uses MicroCT, this was not possible.

Conclusions

No statistically significant difference was found in pulp canal to hard tissue volume ratio

between the LLLT teeth and the control teeth subjected to 150g of buccal tipping force

during force application and after 6 weeks of retention.


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List of tables and figures

Figure 1. AVIZO Workflow

Step Module Sub-settings/steps Justification

1 Extract sub-volume Default To crop each tomogram down to

a minimal size

2 Edit new label field: Crown, Root,

Pulp Canal (2) Threshold tool To select material

(2) Fill volume To fill pores


label [+]

To tag material for subsequent

label analysis

(2) Circular paint tool To select tap and bottom of

volume

(2) Interpolate To select volume between

regions selected at(4)


label [+]

To tag volume for subsequent

label analysis

3 Label analysis Interpretation – 3D Volume

analysis

To calculate the volume of the

tagged regions selected


Table 1. Volume ratio between pulp canal and hard tissue in the experimental group and the control


95% CI

Mean t test SE of Lower Upper

N Mean SD Mean SD difference P value mean bound bound

TM 20 7.86 2.03 7.31 1.97 0.54 0.37 0.59 -0.69 1.77

Ret 20 8.01 2.55 6,85 1.96 1.16 0.11 0.69 ⁃0.28 2.61

Descriptive statistics (mean, standard deviation), mean differences of pulp volume between intervention and controls, P values, standard error of the mean, and 95% confidence intervals. Nonstatistically significant differences were noted between intervention and control group.

Figure 2 Box plot depicting the pulp canal to hard tissue volume ratio of TM group and Ret

group.


Table2.Volumeratiobetweenpulpcanalandhardtissueintheexperimentalgroupandthecontrol


95% CI

Mean t test SE of Lower Upper N Mean SD Mean SD difference P value mean bound bound

TM-A 10 7.93 1.67 8.10 2.01 -0.14 0.82 0.62 -1.55 1.25

TM-B 10 7.78 2.44 6.54 1.69 1.24 1.26 0.99 -1.10 3.46

Ret 20 8.01 2.55 6.85 1.96 1.16 0.11 0.69 ⁃0.28 2.61

Descriptive statistics (total, mean, standard deviation), mean differences, average pulp volume (mm3) between laser and the controls, P values, standard error of the mean and 95% confidence intervals. Nonstatistically significant differences were noted between laser and the controls for all pulp volume.

Figure 3. Box plot depicting the pulp canal to hard tissue volume ratio of TM-A group, TM-B

group and Ret group.


Table 3. Univariate analysis of variance (ANOVA) investigating difference between TM group and Ret group


3.83 1 3.8 0.47 0.50

Table 4. . Correlation between pulp volume changes and difference in total amount of root resorption craters among all three groups

TM vs Ret TM-A vs Ret TM-B vs ret

Pearson Correlation -0.21 -0.14 -0.19

Significance 0.20 0.48 0.31


CONCLUSION

To date, there is no consensus regarding the effects of orthodontic treatment on the dental

pulp. Further, despite being a key component in the anatomy and physiology of the dentition,

pulpal tissue is still not well documented and described, especially in regard to our everyday

orthodontic practice. In addition, the effect of LLLT and PBP on the pulp is poorly understood.

Even though interventions aiming to accelerate orthodontic tooth movement, such as the

use of LLLT and PBM, open up huge possibilities in the quest to shorten orthodontic

treatment and reduce complications, such advances in technology cannot replace the

thorough clinical examination as well as good treatment and biomechanical

planning. However, since those accelerated tooth movement methods are out there and

being promoted by companies, it is our duty as researchers to understand their effects on

oral tissues.

The current study contains numerous confounding factors which may affect the reliability

and applicability of the outcomes. It is obvious that the results of the current study may not

have a significant impact on our current practice in orthodontics. However, previous

randomized control trials on this subject have collected valuable images of maxillary first

premolars which had received LLLT/LED-PBM during orthodontic tooth movement and in

retention. It was worthwhile therefore to analyze this readily available data and in doing so

add to the current best available evidence. We do not know whether the results of the pulp

canal volume reflect what happens to the pulp and this needs to be investigated further.

Furthermore, the results of current study could not be generalized to an entire course of


orthodontic treatment because of the short study duration imposed by ethics criteria and the

singular tooth movement studied.

Despite the shortcomings with this study, it is a pilot study and has provided some framework

on which future studies could be based. For future studies, teeth undergoing longer

durations of these applications as well as different types of orthodontic tooth movement

should be analysed with histology and immunohistochemistry to provide a better

understanding of these interventions on tooth pulp structures.


Appendix: AVIZO Figures

1.Importing the micro-CT scans of teeth into Avizo 3d software. 2.Using ‘Ortho slice’ function to double check if the dataset has imported correctly.

3.Segment the crown of the tooth using the magic wand in 3D.

4.Applying ‘Convex hull’ function do fill the volume from crown to the CEJ the tooth.


5.Verifying if ‘Convex hull’ function work out. 6.Selection of the calcified root structure using Magic Wand tool in 3D with “Same material only” limit applied to avoid the crown volume.

7.Selection of the calcified root structure. 8.Applying ‘Fill all slices’ function to crate the non-calcified internal tooth structure representing the pulp tissue.


9.Digitally extracted pulp canal tissue. 10.Digitally extracted tooth prepared for the volumetric analysis.

11. Applying “Material statistics” function for the volumetric analysis of calcified and non-calcified (pulp canal) tissue.

Documents

The effects of Low-Level Laser Therapy and LED-Mediated