THE INFLUENCE OF GENETIC AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT AND POSITION OF THIRD MOLARS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Giedr

ė Trakinienė

THE INFLUENCE OF GENETIC

AND ENVIRONMENTAL FACTORS

ON THE DEVELOPMENT

AND POSITION

OF THIRD MOLARS

Doctoral Dissertation Medical and Health Sciences,

Odontology (M 002)

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The dissertation was prepared at the Department of Orthodontics of Medical Academy of Lithuanian University of Health Sciences during the period of 2015–2019.

Scientific Supervisor

Prof. Dr. Antanas Šidlauskas (Lithuanian University of Health Sciences, Medical and Health Sciences, Odontology – M 002).

Dissertation is defended at the Odontology Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Vita Mačiulskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Odontology – M 002).

Members:

Prof. Dr. Jurgina Sakalauskienė (Lithuanian University of Health Scien-ces, Medical and Health ScienScien-ces, Odontology – M 002);

Prof. Dr. Gediminas Žekonis (Lithuanian University of Health Sciences, Medical and Health Sciences, Odontology – M 002);

Prof. Dr. Vytautė Pečiulienė (Vilnius University, Medical and Health Sciences, Odontology – M 002);

Prof. Dr. Triin Jagomägi (University of Tartu, Medical and Health Sciences, Odontology – M 002).

Dissertation will be defended at the open session of Lithuanian University of Health Sciences on the 24th of September 2019, at 13:00 in the Museum of History of Lithuanian Medicine and Pharmacy of Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Giedr

ė Trakinienė

GENETINIŲ IR APLINKOS

VEIKSNIŲ ĮTAKA TREČIŲJŲ

KRŪMINIŲ DANTŲ

VYSTYMUISI IR PADĖČIAI

Daktaro disertacija Medicinos ir sveikatos mokslai,

odontologija (M 002)

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Disertacija rengta Lietuvos sveikatos mokslų universiteto Medicinos aka-demijos Ortodontijos klinikoje 2015–2019 m.

Mokslinis vadovas

prof. dr. Antanas Šidlauskas (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, odontologija – M 002).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos odontologijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Vita Mačiulskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, odontologija – M 002).

Nariai:

prof. dr. Jurgina Sakalauskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, odontologija – M 002);

prof. dr. Gediminas Žekonis (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, odontologija – M 002);

prof. dr. Vytautė Pečiulienė (Vilniaus universitetas, medicinos ir svei-katos mokslai, odontologija – M 002);

prof. dr. Triin Jagomägi (Tartu universitetas, medicinos ir sveikatos mokslai, odontologija – M 002).

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Medi-cinos akademijos Odontologijos mokslo krypties tarybos posėdyje 2019 m. rugsėjo 24 d. 13 val. Lietuvos sveikatos mokslų universiteto Lietuvos me-dicinos ir farmacijos istorijos muziejuje.

Disertacijos gynimo vietos adresas: Rotušės a. 28, LT-44279 Kaunas, Lietuva.

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ABBREVIATIONS

a2 – additive genetic factors

ACE – additive genes, common and specific environment AIC – Akaike information criterion

ALARA – As Low As Reasonably Achievable BIF – bifurcation

c2 – common environmental factors CVM – cervical vertebral maturation CS – cervical stage

CT – computed tomography d2 – dominant genetic factors

DLL8 – distance for the eruption of lower left third molar DLR8 – distance for the eruption of lower right third molar

DNA – deoxyribonucleic acid

DUR8 – eruption level of upper right third molar DUL8 – eruption level of upper left third molar

DZ – dizygotic twin

e2 – specific environmental factors ICC – interclass correlation coefficient

LM2 – lower second molar LM3 – lower third molar

LL6 – lower left first molar

LL7 – lower left second molar LL8 – lower left third molar

LR6 – lower right first molar

LR7 – lower left third molar LR8 – lower right third molar

LES-UL – eruption space for upper left third molar LES-UR – eruption space for upper right third molar MAX – maxillary plane

HP – horizontal plane

M3 – third molar

MCGs – magnification coefficient using metal gauge MCC – magnification coefficient using dental casts MND-L – left mandibular plane

MND-R – right mandibular plane MxP – maxillary plane

MZ – monozygotic twin

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9 OCL-L – left occlusal plane OCL-R – right occlusal plane OPG – panoramic radiograph QTL – quantitative trait loci SE – standard error

STR – short tandem repeats

U3 – upper canine upper canine

UL7 – upper left second molar

UL8 – upper left third molar UR7 – upper right second molar

UR8 – upper right second molar χ2

– Chi square criterion

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PREFACE

“I encourage all of us, whatever our beliefs, to question the basic narratives of our world, to connect past developments with present concerns, and not to be afraid of controversial issues”

Dr. Yuval Noah Harari “Sapiens”

Everything started when I read the publication of Samir Bishara “Third molars: A dillema! Or is it?” in the American Journal of Orthodontics and Dentofacial Orthopedics (1999). Do we really need to extract the third molars? Can they cause relapse of the anterior crowding? Can we do so-mething to manage these problems? These were the questions disturbing me and this was only the beginning of the way where I am still looking for the answers...

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INTRODUCTION

Third molars (M3) are a common source of pain in the dentofacial system and they can induce serious, even life-threatening infections, perio-dontal breakdown, and caries of the adjacent teeth [1, 2]. Consequently, understanding the regulatory mechanisms behind the variation in M3 de-velopment is of great clinical importance.

Third molars exhibit marked variability in the timing of formation, mineralization and eruption, but the mechanisms controlling this variability are not clear [3, 4]. There is substantial evidence that the dental develop-ment is affected by a complex interaction of genetic and environdevelop-mental, or non-genetic factors [5–10]. However, previous dental genetic studies mostly did not include third molars, leaving our understanding of the development of the permanent dentition incomplete, which has an impact on clinical dentistry.

Furthermore, previous dental development studies mostly analyzed data from growing subjects prior to or immediately after the peak of the pubertal growth spurt [9–11]. Therefore, to assess the role played by genetics and the environment in M3 root formation, the study sample should include in-dividuals at both before and after the pubertal growth spurt. Nevertheless, the evaluation of the third molar position requires the study sample with the late growth spurt deceleration as these teeth have a tendency to change their position.

Historically, twin studies have played an essential role in estimating genetic impact on tooth size, morphology, and development [6, 12, 13]. The structural equation modeling applied in twin studies can provide estimates of the proportion of the total phenotypic variation attributable to additive genetic, shared environmental, and unique environmental components [14, 15].

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AIM, OBJECTIVES AND HYPOTHESIS

OF THE STUDY

AIM OF THE STUDY

The aim of this study was to assess additive genetic and environmental influences on the variance of the third molar development, mineralization and position by comparing the twins of the same sex.

OBJECTIVES OF THE STUDY

1. To evaluate the relationship of the growth phase with the mineralization stage of the third molar.

2. To validate reliability of the linear measurements on panoramic radio-grams at the third molar region.

3. To assess additive genetic and environmental influences on the agenesis of the third molar.

4. To determine factors affecting the third molar’s root mineralization va-riability.

5. To estimate the importance of genetic and environmental factors on the third molar position.

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SCIENTIFIC NOVELTY

This is the first study which analyzes the impact of genetics and environmental factors on the development and position of M3 with the highest precision, by the comparison of MZ and DZ twins employing the statistics of path analysis with the best fitting model and DNA zygosity determination based on 15 highly polymorphic DNA regions and Amel fragment of amelogenin gene.

One of the novelties in these methodological studies was that a new validation method of the measurements on the panoramic radiographs was created. First of all, the magnification coefficients in the panoramic radio-grams were estimated and compared using two different methods. Conse-quently, this led to the establishment of a new method for the evaluation of the geometric distortions on the panoramic radiograms.

Another novelty was that a new method was established for the invest-tigation of the human growth stage using the developmental stage of the third molar. This allowed us to assess the growth stage by the use of the commonly used panoramic radiogram.

Moreover, in the genetic studies the main novelty was that the study sample reached even 284 twins who had diagnostic models and panoramic radiograms.

In addition, the zigosity of twins was determined with the accuracy of 99.99%, which is the highest according to the recent literature.

The advanced genetic analysis is another new aspect which allowed distinguishing the effect of four factors: additive genetic, dominant genetic, common environmental and specific environmental factors. This enabled us to understand more clearly the effect of different factors.

Furthermore, the novelty of the study about the influence of genetic and environmental factors on agenesis of the third molars was that the study sample consisted of the twins with the age at minimum twelve years for the assurance that the third molar agenesis is not overestimated if the third molar germ starts its formation later than normal age.

The study about the third molar root formation is new as it investigated the twins with the formation of the third molar roots from the beginning to the end of the root formation and excluded the twins who had all the roots of the third molar already formed. This allowed us to make very accurate evaluation of root developmental stages and to make conclusions.

Finally, the analysis of the third molar position was done using several reference planes, thus the measurements were very precise.

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1. LITERATURE REVIEW

1.1. The third molar formation, development, mineralization and position in the dental arch

Third molars, or wisdom teeth, are the last teeth that erupt into the oral cavity and their eruption time may vary from 17 to 24 years, depending on the ethnographic region and race [16]. Recently, discussions regarding the influence of the third molars on dentition have become highly important from several aspects, including their development, formation, retention and their effect on teeth crowding [17, 19]. Consequently, understanding the regulatory mechanisms of third molar development variability is of great clinical importance for making decisions on the timing of the third molar surgical removal, autologous transplantation, orthodontic treatment planning and chronological age estimation for medico-legal purposes [20].

1.1.1. Formation of the third molar

In human embryos, a group of cells, called neural crest (NC) cells, sepa-rates from the neural tube and migsepa-rates away from their parental epithelium to reaggregate with other cells as epithelial–mesenchymal interactions. Tooth initiation and morphogenesis occur by cooperation of numerous ge-netic and epigege-netic factors [21, 22]. At the same time, most of the deve-lopmental defects in teeth usually occur as a result of mutations in genes encoding signaling molecules and transcription factors such as mutations in the PAX9 gene resulting in partial or total anadontia and mutations in RUNX2 causing supernumerary teeth [23]. Each tooth passes through four morphological stages: initiation, bud, cap and bell stages [24].

The initiation of the tooth begins with a localized thickening within the primary epithelial bands which forms the dental lamina. The epithelial grows into the ectomesenchyme by the formation of the epithelial invagina-tion, called bud [24]. The internal part of the tooth bud contains star-like shaped glycosaminoglycan synthesizing stellate reticulum cells [25]. During the bud stage odontogenic potential is switched from the epithelium to ectomesenchyme.

During the cap stage the tooth bud transforms into a cap by differential proliferation and in-folding of the epithelium [22]. As the epithelial bud cells proliferate, ectomesenchymal cells condense and morphological differ-rences between tooth germs begin during the cap stage. Histo-differentiation begins late in the cap stage and in the next, bell stage, the cells of the crown

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ameloblasts and odontoblasts are differentiated. A single layer of columnar cells, which borders the dental papilla and resides inside the cap, is called inner dental epithelium. The outer part of the cap is covered by the outer dental epithelium [26]. While the cap-shaped epithelial growth is widely referred to as enamel organ, the condensed ectomesenchymal cells are referred as dental papilla. The dental follicle covers the outside of these two substances. The enamel organ, dental papilla and dental follicle constitute the tooth germ. The dental papilla is separated from the enamel organ by a basal lamina and is located between inner dental epithelium and undiffe-rentiated mesenchymal cells of the papila.

During the bell stage the terminal differentiation of ameloblasts from inner dental epithelium and odontoblasts from mesenchymal cells of the dental papilla proceeds with the formation of enamel and dentin. Ameloblast and odontoblast differentiation is regulated by interactions between the epithelium and mesenchyme [27, 28]. While dental papilla is the origin of the future dental pulp, dental follicles give rise to cementoblasts, osteoblasts and fibroblasts. In conclusion, neural crest cells give rise to dentin-produc-ing cells, odontoblasts; cementoblasts, which produce root dentin coverdentin-produc-ing; osteoblasts, which participate in the formation of dental alveolus; and fibroblasts, which synthesize collagen for periodontal ligaments.

Simultaneously to odontoblast differentiation, inner dental epithelium differentiates to ameloblasts that secrete enamel. It has been thought that the proteins or growth factors secreted by ameloblasts have some effects on the terminal differentiation of odontoblasts, possibly by interacting with compo-nents of basement membrane [27]. After dentin has formed, the enamel producing cells assemble as a layer. Then, ameloblasts move away from the dentin leaving secreted enamel behind. Differentiating odontoblasts need signals from differentiating ameloblasts and vice versa, meaning that tooth development needs reciprocal, complex epithelial-mesenchymal interactions [29].

When the first calcified matrix appears at the tip of the principal cusp, the dental papilla is referred as the tooth pulp. The cells of the pulp in this stage are undifferentiated mesenchymal cells and a few collagen fibrils are seen in the extracellular matrix [27]. The blood vessels in the dental papilla form clusters, whose position coincides with the root formation positioning. Unfortunately, there is no detailed information about angiogenesis during tooth development. On the other hand, the first nerve fibrils, as well as the vessels, approach the developing tooth during the bud-to-cap transition stage. Nerve fibrils penetrate the papilla when dentinogenesis begins. It has been assumed that the initial innervations are involved in the sensory inner-vations of future periodontal ligament and pulp [27].

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The root is formed via Hertwig’s epithelial root sheet, which consists of epithelial cells. This sheet extends around the dental pulp and is almost closed except for the little opening, apical foramen, in the apical portion of the root. As root formation proceeds, epithelial cells influence the differ-rentiation of odontoblasts from the ectomesenchymal cells at the periphery of the dental papilla as well as cementoblasts from follicle mesenchyme. This leads to the deposition of root dentin and cementum, respectively. Although this describes the formation of a single root, multi-rooted teeth are formed in the same manner [27, 28]. While roots are forming, the sup-porting tissues of the tooth from the dental follicle also develop. The dental follicle gives rise to various components of the periodontal ligament fib-roblasts, the alveolar bone of the tooth socket and the cementum. These structures also play a role during tooth eruption, which marks the end phase of odontogenesis [28].

The upper third molars start their development at the cervical neck of the second molar and with the occlusal surface facing distally. Later, these teeth gradually upright during the formation of the crown and roots, and become positioned vertically. These teeth have to adapt to the adjacent structures in the maxilla such as the second molar, maxillary sinus and pterygoid fossa. If there is not enough space for the eruption of the upper third molar, its crown may remain severely constricted mesio-distaly and the roots become diver-gent as the third molar adapts to the sinus and second molar.

The formation of the lower third molar starts during the mineralization of the permanent second molar. A posterior extension of the dental lamina is seen distally to the bud of the permanent second molar and bone resorption occurs where the bud of the third molar will develop. This results in the formation of the groove in the bone which becomes the initial tooth socket. The germ of the lower third molar becomes located in the ramus of the mandible with the occlusal surface at the angle to the mandibular plane [30]. Later, the tooth germ has to undergo sagittal uprighting to erupt into the normal position [31]. During the crown formation the third molar germ remains between the mandibular canal and crestal bone. Its eruption begins when the cervical part of the root complex is formed. After that, the germ moves occlusally from the mandibular canal, assuming a distally curved eruption path. If the resistance is met during the eruption, the germ will grow in close relationship to the mandibular canal and it can make the deformation of the third molar roots [32]. The root can become deflected due to changes in the direction of the eruption whereas the divergences of the roots are usually the result of intrusive growth.

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17 1.1.2. Eruption of the third molar

Tooth eruption is defined as the movement of the tooth from its site of development within the alveolar process to its functional position in the oral cavity. In general there are 5 eruption stages: preeruptive movement, intra-osseus movement, mucosal penetration, preocclusal movement, occlusal function. Initially a tooth develops in the alveolar bone. After the crown formation is completed and roots begin their formation, active formation moves the tooth towards its functional position.

During the intraosseus stage, the tooth moves through the bone. When the occlusal plane is approached, the rapid eruption phase starts. Later, when the occlusal plane is reached, the consolidation of the periodontal supports begins. Finally, the eruption and alveolar growth in height proceeds slowly for the compensation for cuspal attrition. In contrast, if the contacts are lost in the opposite arch, the growth and eruption rate increase again.

Anatomic structures participating in the tooth eruption process are the tooth germ, the dental sac, the dental crypt and the gubernacular cord.

The true dental follicle is a marginal condensation of ectomesenchyme surrounding the enamel organ and the dental papilla. The dental sac consists of an outer layer associated with the surface of the surrounding bone, an intervening layer of loose connective tissue, and an inner layer associated with the tooth. In the eruption stage the dental follicle is invaded by mononuclear cells, which subsequently fuse and form the coronal osteclast – resorbing front [33].

The dental crypt is formed from a thin layer of a compact bone and at the fundus of this crypt there is a foramen for the neurovascular supply to the tooth germ. In the coronal part of the follicle there is the entrance to the gubernacular cord which is a strand of fibrous connective tissue with the remnants of dental lamina epithelium. This structure provides guidance for the permanent tooth germ in its intraosseus stage [34].

The bone resorption and formation is polarized around the erupting teeth and is dependent to the dental follicle and structures adjacent to it. The fact that active eruption begins only after crown formation is completed, sug-gests the role of the enamel organ and its proteases in the early signaling of eruption [35].

Usually the third molars erupt at the age between 18 and 20 [36]. Mo-reover, these teeth can erupt even 1.5 year earlier, when the teeth anterior to the third molar are extracted [37]. The eruption process of the upper third molar begins with their inclination distally, but later they upright in a more vertical position. After the emergence these teeth usually erupt from 5 to 10 micrometers per day [38].

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Lower third molars were found to erupt at around 38 degrees of incli-nation to the mandibular plane, but it can range from 11 to 83 degrees [39]. However, even then these teeth with severe tilting may spontaneosly upright [39, 40]. Tait found that the angulation of the third molar reflects the curve of the overlying bone: the third molar will become more mesially tilted when the curvature is small [41]. In case of extraction of premolars, the third molars will be allowed to tip mesially 3 degrees for each 1mm of space. A lack of space for eruption of the third molar enables the third molar to become impacted. The space for eruption of the third molar changes 0.9mm per year during the period from 8 to 16 due to ramus resorption and mesial movement of the first molar. Obstruction of the eruption pathway may be accomplished by the non-extraction orthodontic treatment with the distalisation of the lateral segments [42].

Prediction of the third molar impaction is important for the prognosis of its eruption and depends on the patient’s age. Prediction at a very early age (8–9 years) implies that the third molar removal can be confined to ablation of a superficially placed tooth germ, a rather simple surgical procedure, although the early age may result in some psychological problems. At the age of 10–15, prediction is relevant when the replacement of the first or second molar by the third molar can be a treatment option. Finally, at the age of 16–20, prediction is essential because removal of the third molar at this age is optimal for the prevention of inflammatory episodes due to the start of the tooth emergence and periodontal healing distal to the second molar.

1.1.3. Agenesis of the third molar

In general, agenesis is considered to be the result of disturbances during the early stages of tooth development [20]. The prevalence of third molars agenesis was found to be considerably higher than the agenesis of other teeth, varying from 10% to 41% among different countries [43–45]. The lowest values for the agenesis of these molars were found in black Africans and Indians, exhibiting a prevalence of 10%–11% of the population, while in the Iranian population, the prevalence rates for the third molar absence approached 34.8% [46–48]. The highest values of the third molar absence were reported in Koreans (41%) [49]. These large differences could be explained by different methodologies and different ethnical backgrounds [50].

Previous studies showed that genetics played a crucial role in the age-nesis of lateral incisors or second bicuspids using dental casts [51, 52]. However, these studies did not clearly reveal the influence of heredity on

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the formation of the third molars, which could be highly different from the other teeth because of their unique development.

First, these teeth begin their development later. The formation of the third molar follicle begins approximately at the age of 7, exhibiting an unusually long developmental period, and these teeth do not have a primary counter-part. Second, evaluation of third molar agenesis needs a radiological evalua-tion of tooth germs, as they must attain a certain level of mineralizaevalua-tion to be visible in the radiographs. Consequently, Rakhshan recommended that only individuals over 12 years old could be used to analyze the formation of these teeth and for the precise determination of their mineralization stages [53].

1.1.4. Mineralization of the third molar

Mineralization of upper and lower third molar is highly correlated. Levesque et al. and Pelsmaekers et al. showed through genetic modeling of twins that variation in tooth mineralization was almost equally dependent on additive genetic influence and common environmental factors [9, 54]. In contrast, Hughes et al., utilizing the same methodology, demonstrated that emergence of human primary incisors was under strong genetic control. Hence, the genetic and environmental influences on dental maturation need more detailed studies [55].

The role of sex on dental maturation is also not completely understood. Sexual dimorphism in tooth development and morphology has been reported in some studies [14, 55, 56]. Levesque et al. found girls were ahead of boys by stages of crown formation, but this difference disappeared at the first stage of root formation and root development was found to proceed faster in males than in females [54]. But another group of researchers reported that there were no differences in the mineralization rate between males and females [57–59]. Ethnicity may have an impact on sexual dimorphism with respect to dental maturation. Prieto et al. studied Spanish individuals and found significant sexual dimorphism in stages E to G with males reaching these stages earlier [60]. In line with these are findings from Japanese [61], Austrian and French-Canadian samples [54, 62, 63]. Data from African American are different, with most stages showing faster molar mineralize-tion among females relative to males [64]. It is also the case that the denti-tion in African American tends to mature faster than American whites and this presumably reflects differences of ancestry [65]. To date, there are no studies of sexual influence on tooth mineralization among members of the Lithuanian population and the neighboring Baltic countries.

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The second lacuna in previous studies concerns the chronological age of the study subjects. Previous dental mineralization studies analyzed data from growing subjects prior to or just after the peak of the pubertal growth spurt [9, 10, 65, 67]. As such, the results of such studies for estimated genetic influence on molar maturation should be interpreted with caution. This is because exclusive genetic predisposition of molar mineralization can only be detected if the data include both growing and adult subjects appro-aching the completion of molar root formation. Thus, to assess the roles played by genetics and the environment on M3 root formation, the study sample should include individuals who both predate and postdate the pubertal growth spurt.

1.1.5. Position of upper third molars

Third molars are the last to erupt into the oral cavity, which mostly affects their tendency to become impacted. Impaction may be defined as the failure of a tooth to erupt completely into a normal, functional position in a normal time [67]. The main factors associated with the retention of these teeth are the disproportionate size of the wisdom teeth and jaw, lack of space for the eruption of the tooth and unfavorable angulations of these teeth [68].

The incidence of impacted upper third molars ranges from 9.5% to 25% in different populations [69]. The main cause of upper third molar impaction is lack of retromolar space, which depends on the growth of the maxillary tuberosity. However, due to the prolonged growth of the maxillary tuberosity and the greater mesialization of maxillary dentition, there is more space for the buccal eruption of the upper third molars [70]. This finding explains why retention in the maxilla is less commonly observed than in the mandible.

Impacted maxillary third molars can also be mesioangular, distoangular, vertical or horizontal in position. The extraction of impacted third molars can prevent the occurrence of pathologies, such as pericoronitis, odonto-genic cysts and tumors, root resorption of the second molars and others [71]. Although complications with the surgical removal of upper wisdom teeth appear in up to 6% of patients, they are not observed as often as in the mandible [72, 73]. This outcome might be explained by the minimal risk of nerve damage, better blood supply or less frequent retention of saliva and food particles in the maxilla than in the mandible [74, 75]. However, sur-gical extraction of an upper third molar can cause serious complications, such as displacement of the tooth into the adjacent anatomic spaces. The level of the occlusal plane, contact with the second molar and the

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ship of the molar to the maxillary sinus have been found to be significant predictors of surgical difficulty [76].

Hence, the current literature does not answer questions about the in-fluence of genetic factors on the position of the teeth, especially upper third molar position using the twin-study model with precise zygosity determi-nation.

1.1.6. Position of lower third molars

The impaction of lower third molars ranges from 25% to 50% and is higher in females than in males [67, 77]. Impacted mandibular third molars are traditionally classified according to position following the method of Winter [78]. Matzen found that the most common angulation of the lower third molar was mesioangulated (53%), followed by horizontal (23%), disto-angulated (15%), and vertical (9%) [79]. Mesioangulary impacted mandibu-lar third momandibu-lars lie obliquely in bone with the crowns slanted in a mesial direction. Panoramic radiograms clearly demonstrate the mesiodistal and vertical position of the impacted tooth, but do not provide details of the buccolingual positioning or buccolingual angulation.

Although the main treatment option of this pathology is extraction of these problematic teeth, numerous studies reported that the complications of such treatment were really high and in some studies even reached 33% [80]. Thus, the knowledge about the developmental mechanisms of the lower third molars could help minimize the number of these complications.

As it was found with upper third molars, the most common reasons for M3s impaction are the late formation of third molars, large crowns of M3s, lack of space for the eruption and changes in the inclination of the wisdom teeth during the eruption process [81, 82].

Usually, third molars begin their formation at the age of 7–9, although it could begin at the age of 5. Even when they start their development earlier it does not mean that they will erupt faster. Richardson found that retained lower third molars were slightly larger than those which germinated [83].

Furthermore, Olive and Basford found that the space for M3 eruption should be equal or higher than the width of lower third molar. It was be-lieved that until 15 years this area could grow 11.4 mm and later the growth was negligible [84]. Moreover, Haavikko found that the angle between the third and second molars’ longitudinal axes should be less than 10 degrees for the proper eruption of the third molar [85]. Venta I. found that if lower third molars roots were not fully formed and the crown of these teeth were at the height of the second molar neck at the age of 20, then they were more

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likely to stay retained [86]. However, the genetic environmental interplay for the lower third molar positional polymorphism still is not clear.

1.2. Methodological issues related to the studies of controlling mechanisms of the development of the third molar 1.2.1. The role of twin studies in dental genetics

The major sources of human dental variability are genes, environment and epigenetic mechanisms regulating gene expression [12]. The genetic mechanisms, specific genes leading to a particular dental variability are not completely understood and clear yet. Technological advances have now made association analysis possible on a genome-wide level, but usually before starting to look for quantitative trait loci for complex traits it is cru-cial to know that there is a significant component of genetic variation pre-sent.

Historically, twin studies have played an essential role in estimating ge-netic impact on tooth size, morphology and development [3, 7, 12]. A la-cuna in previous dental genetic studies is that the majority of studies do not include third molars. The exclusion of M3s from such studies not only renders our knowledge of the development of the permanent dentition in-complete, but this exclusion also has impacts on clinical dentistry and related disciplines.

Another reservation that may be raised with earlier twin studies is the reliability of twin zygosity determination. For many years, determination of zygosity was based on assessment of anthropological similarity including tooth anatomy [87]. Although a comparison of physical appearance can provide a reasonably reliable means of determining zygosity, errors occur in up to 15–20% of cases [88]. The situation changed when serum and enzyme polymorphism analyses were introduced [14]. However, even then, this method did not yield very high accuracy rates. In subsequent years, another method, which uses the highly polymorphic regions of DNA obtained from blood or buccal cells, has been used to determine the zygosity of twins with an accuracy rate greater than 95% [89]. Ming-Jie Yang found that with 15 specific DNA markers, the determination of the zygosity is not only cost and time saving, but also shows greater sensitivity and precision than conventional methods [91].

The present study overcame the mentioned limitations by studying third molars and using most advanced zygosity determination methods.

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1.2.2. Advancement of statistical methods in twin dental genetics There have been two main quantitative genetic approaches used to clarify causes of observed variation in the human dentition: classical correlation analysis and multiple abstract variance analysis. The classical correlation approach compares the degree of association for selected traits between pairs of related individuals, with maximum correlation values that are assumed to be 1.0 for MZ co-twins and 0.5 for DZ co-twins. Estimates of heritability (h2) can be derived according to the formula: h2 = 2 (rmz–rdz), where rmz and rdz are the values of correlation coefficients between sam-ples of MZ and DZ co-twins for the feature under investigation. Variance analysis is a more systematic and comprehensive approach that compares within and between-family variances, leading to estimates of both genetic and environmental contributions to observed variation. A third method for analyzing quantitative data is Fisher’s biometrical approach that has several advantages over those previously described. Fisher’s approach represented a major breakthrough in genetic analyses, as it enabled testing for components of variation, such as shared or common environmental factors, that were previously assumed to be absent or undetectable.

The development of more sophisticated model-fitting methods for ana-lysis of twin data made it possible to estimate the strength of genetic and environmental contributions within calculable confidence intervals [90].The structural equation modeling applied in twin studies can provide estimates of the proportion of the total phenotypic variation attributable to the additive genetic factor (A), the shared environment (C), the non-additive genetic fac-tor (D) and the unique environment (E) [14, 15].

The Akaike information criterion (AIC) statistics and the difference in the chi-square (χ2) value relative to the change in degrees of freedom provided an indication of the models’ goodness of fit [16]. The most parsi-monious model (lowest AIC value) to explain the observed variance was se-lected. Additive genetic factors (A) refer to the deviation from the mean phenotype due to the inheritance of a particular allele and this allele's re-lative (to the mean phenotype of the population) effect on phenotype. Dominant genetic variance (D) involves deviation due to interactions bet-ween alternative alleles at a specific locus. The general environmental va-riance (C) is attributed to the non-genetic sources of variation between individuals that are experienced by multiple individuals in a population. This variance is typically the largest component of variance in populations in natural conditions. While specific environmental variance (E) is deter-mined by the variation within replicated genetic lines, to obtain an estimate of general environmental variance, replicates of each of the genetic lines

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need to be assessed in each natural or experimental environment of interest [17].

This study for the assessment of contribution of genetic and environ-mental influences on the individual differences in the observed M3 variables was estimated by the most modern method – genetic model – fitting and AIC statistics was used to indicate model’s goodness to fit.

1.2.3. The relationship of the third molar development stage with the growth phase

Considerable variations in the development among individuals of the same chronological age have led to the concept of assessing biological or physiological maturity. The concept of physiological age is based upon the maturation degree of different tissue systems [92]. The most commonly used methods for growth evaluation are: the somatic (based on the general body changes along with the development of the secondary sex character-ristics) and the radiological ones (assessment of the hand-wrist radiographs or serial lateral cephalometric radiographs).

Cervical vertebral maturation (CVM) method has been proved to be effective for the estimating the growth phase according to the morphological characteristics of the second, third, and fourth cervical vertebrae in the lateral cephalometric radiographs [93]. When specific training is provided along with precise guidelines in assessing visually each stage, CVM method proves to be accurate and repeatable to a satisfactory level [93]. This me-thod has advantages over the hand-wrist meme-thod, which additionally re-quires hand-wrist radiograph and experience of the observer to evaluate growth indicators in it.

Moreover, most of the bones of the body are preformed in the cartilage and later are developed by endochondral ossification, while the facial bones are formed by intramembranous ossification. Therefore, the growth of the face may be regulated by other factors than those responsible for the growth of the long bones [95]. Even though CVM method is a reliable method for the evaluation of the growth phase, it requires a lateral cephalometric ra-diograph, which is not always compulsory pre-treatment record for every patient. An increasing awareness of the risks associated with X-rays has led clinicians to re-evaluate the indications for taking a lateral cephalometric radiograph. Although the majority of orthodontists judge that lateral cepha-lometric radiograph is important for producing a treatment plan, it still does not seem to have an influence on orthodontic treatment planning [96]. Re-cently, McCabe and Rinchuse in a survey of orthodontic practitioners re-garding the routine use of lateral cephalometric radiographs established that

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in orthodontic treatment 60.34% orthodontists reported always taking pre-treatment lateral cephalometric radiographs and only 38.53% reported always performing a cephalometric analysis on pre-treatment lateral cepha-lometric radiographs. They concluded that there is a current trend toward the decrease in the amount of practitioners routinely tracing lateral cephalo-metric radiographs [97].

Furthermore, in some cases, optimal treatment timing is delayed after the diagnosis, making a later re-evaluation of the growth phase necessary. The-refore, lateral cephalometric radiographs are not taken routinely, whereas panoramic radiographs are routinely available in orthodontic practice and are useful to assess dental maturity. So, as an alternative to CVM method, dental development has also been widely investigated as a potential predict-tor of the growth phase [98–103]. Generally, dental development can be assessed either by the phase of tooth eruption, or the stage of tooth cal-cification, with the latter being more reliable [104]. However, Krailassiri et al. suggested that the interpretation of the relationship between skeletal development and mineralization of the third molar at the early stages of skeletal maturity was not meaningful [105].

The aim of this investigation was to evaluate whether the calcification of mandibular third molar is useful to determine the growth phase.

1.2.4. Reliability of the linear measurements on the panoramic radiographs

The crown widths of erupted and unerupted teeth in the molar region can be measured using different radiographic techniques [106]. Computed tomography (CT) is considered to be the gold standard for the bone thick-ness and dental crown measurements but involves the highest radiation exposure, which should be avoided, especially in the growing individuals due to the increased risk of developing cancer [107–109]. In comparison with CT and other expensive precision tests, panoramic radiography is rapid and inexpensive, and its radiation dose is low. Furthermore, if metal prostheses, posts or pins are present, CT may generate streak artifacts [110].

Digital panoramic radiography, nowadays, is a common imaging tech-nique in the dental, periodontal, surgical or orthodontic practice as it pro-vides a general view of the teeth and the surrounding structures [111, 112]. However, for the measurements or relative comparisons, panoramic radio-graphy should be used with caution due to image distortion and magni-fication [113–116]. There are many recommendations and studies done on how to calculate the magnification in digital panoramic radiographs. Start-ing from mathematical, theoretical analysis of dental panoramic imagStart-ing

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and ending with the use of the different calibration objects (steel ball bear-ings, implants, metal balls, or even gutta-percha as opaque markers) on dry skulls and live patients [117–123]. However, majority of the studies have been concentrated on anterior, premolar, or first molar region of the lower jaw and vertical measurements. There were no studies on the assessment of horizontal linear measurement accuracy in the third and second molar region in the digital panoramic radiographs.

One of the major necessities for precise measurements of the lower molar crowns width is prediction of the third molar impactions and assessment of the oral health problems related with this pathology. Today, however, in the 21st century, the routine removal of asymptomatic pathology-free third mo-lars has become a dated practice that is rapidly running out of valid excuses, and it has no justification in contemporary dentistry and medicine. Despite the various guidelines, reviews, and risks associated with these extractions, many clinicians continue routinely remove pathology-free third molars just by their subjective judgment on the lack of space for these teeth to erupt [124, 125]. The simple, accurate, and reliable method to measure molar crown widths and space available in the molar region could be useful for dental radiology practice and help define indications for the removal of the third molars.

Thus, the aim of this study was to determine the accuracy of linear mea-surements of the lower third and second molar crowns in the digital pa-noramic radiographs and to compare them with plaster models as the cali-bration standard. Our hypothesis was that magnification in the lower molar region was the same, and calculation of the magnification coefficient using dental plaster models is an adequate method for precise measurements in the digital panoramic radiographs in the lower molar region.

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2. MATERIALS AND METHODS

The approval for the study was obtained from the Regional Biomedical Research Committee (No. BE-2-12). The study was conducted in accor-dance with relevant guidelines and regulations.

2.1. Study sample

The informed consents were obtained from each participant and from the parents/guardians of any participant younger than 18 years old. Each par-ticipant was individually informed regarding the project upon the human subject information consent. Subjects were randomly selected from the Lithuanian university of health sciences (LUHS) Twin Center and stratified according to zygosity of the same sex twin pairs with normal growth and development according to human growth curves using anthropometric mea-sures, such as height and weight.

The aim and objectives of the study were two folded. The main objective was to assess additive genetic and environment influences on phenotypic M3 variability, but for the completion of this task some methodological issues requested detailed analysis. It was necessary to validate the growth phase and reliability of the linear measurements assessment using pano-ramic radiograms. For these purposes 5 groups of study samples (first – to assess ability to determine growth phase on panoramic radiogram, second – to validate reliability of the linear measurements on panoramic radiogram and three separate groups to assess additive genetic and environment influences on phenotypic M3 variability) were collected.

2.1.1. Study sample size calculation

The sample size calculation was based on the following formula: 𝑛 =(s1

2_{+ s}

22) × �z1−α₂+ z1−β� 2

∆2

Where n – the minimum sample size for each sample; z1–α/2=1.96 and z1–β=0.84 if α=0.05 and β=0.2;

s1, s2 – the standard deviations of pilot samples;

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The minimal calculated sample size for the study to assess relationships of M3 development stage with skeletal maturity was 212 and to validate reliability of the measurements on panoramic radiogram was 32. To assess additive genetic and environmental influences on phenotypic M3 variability the minimal study sample was as follow: for M3 agenesis – 224 twins (112DZ, 112 MZ), for M3 root mineralization variability – 120 twins (60 DZ, 60 MZ) and for M3 position in the alveolar bone variance – 160 twins (80 DZ, 80 MZ).

2.1.2. Study sample inclusion and exclusion criteria

Study sample to assess relationships of third molar development stage with growth phase

The inclusion criteria: (1) Caucasian descent, (2) chronological age from 7 to 19 years, (3) good quality panoramic and lateral cephalometric radio-graphs, taken at the same day.

The exclusion criteria: (1) previous orthodontic treatment, (2) permanent teeth extractions, (3) congenital anomalies or syndromes, (4) congenitally missing teeth, (5) any facial trauma that could have resulted in bony fracture or soft tissue scarring.

Study sample to validate reliability of the linear measurements on the panoramic radiograms

The inclusion criteria: (1) Caucasian descent, (2) erupted and good-posi-tioned lower third, second and first molars, (3) good quality panoramic radiographs and plaster models.

The exclusion criteria: (1) extracted at least one lower molar, (2) rotated or displaced lower molars, (3) restorations in the mesio-distal surfaces of the lower molars, (4) congenital anomalies or syndromes (5) any facial trauma that could have resulted in bony fracture or soft tissue scarring.

Study sample for third molar agenesis study

The inclusion criteria: (1) Caucasian descent, (2) twin pairs of the same sex, (3) chronological age over 12 years, (4) good quality panoramic radio-graphs.

The exclusion criteria: (1) teeth extractions, (2) missing teeth other than M3, (3) previous orthodontic treatment, (4) congenital anomalies or syn-dromes, (5) any facial trauma that could have resulted in bony fracture or soft tissue scarring.

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Study sample for third molar root mineralization assessment

The inclusion criteria: (1) Caucasian descent, (2) twin pairs of the same sex, (3) good quality panoramic radiographs.

The exclusion criteria: (1) teeth extractions, (2) missing teeth, (3) pre-vious orthodontic treatment, (4) twin pairs with completed mineralization of all four M3s for at least in one sibling, (5) twins whose M3 formation have not reached stage D at the time of examination, (6) congenital anomalies or syndromes, (7) any facial trauma that could have resulted in bony fracture or soft tissue scarring.

Study sample for third molar position evaluation

The inclusion criteria: (1) Caucasian descent, (2) twin pairs of the same sex, (3) at least No.4 Gleiser and Hunt stage in the panoramic radiograms (according to the study No.1) and after that the evaluation of the cephalo-grams, (4) cervical vertebral maturity stage 5 and 6 (according to Baccetti), (5) good quality panoramic (according to the study No.2) and lateral cepha-lograms, (6) good quality plaster models.

The exclusion criteria: (1) teeth extractions, (2) missing teeth, (3) pre-vious orthodontic treatment, (4) congenital anomalies or syndromes, (5) any facial trauma that could have resulted in bony fracture or soft tissue scarring (Table 2.1.2.1).

Table 2.1.2.1. Study sample

Study name Minimal study _sample Collected study _sample

Methodological studies Relation of M3 with skeletal maturity 212 274 Magnification in M2, M3 region 32 41 Genetic studies M3 agenesis 224 284 M3 mineralization 120 162 M3 position 160 212

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Fig. 2.1.2.1. Flowchart of selection of study groups

The study sample descriptive characteristics of age, sex and zygosity are shown in Table 2.1.2.2.

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Table 2.1.2.2. The age, sex and zygosity of the study sample

Study sample N Age

Mean SD Min Max Methodological studies

Relationship of M3 development stage with the growth phase

274

12.3 2.7 7.5 19.8

Male 120 12.6 3.4 8.9 19.8

Female 154 12.0 2.9 7.5 17.7

Reliability of the linear

measurements on OPG 41 18.5 2.3 16.3 21.5 Genetic studies M3 agenesis 284 19.3 4.6 11.9 35.9 MZ 172 19.7 4.3 11.9 38.1 Male 62 19.5 3.6 12.1 29.5 Female 110 19.9 4.9 11.9 38.1 DZ 112 18.9 4.8 12.3 35.9 Male 58 19.6 4.4 12.9 35.9 Female 54 18.9 5.1 12.3 27.6 M3 root mineralization assessment 166 17.9 2.5 12.8 24.9 MZ 96 18.0 2.7 12.8 24.9 Male 30 18.5 2.9 13.9 24.9 Female 66 17.8 2.5 12.8 24.4 DZ 66 17.7 2.1 13.0 21.9 Male 36 17.6 2.4 13.0 20.8 Female 30 17.9 1.6 14.9 21.9 M3 position evaluation 212 18.6 0.65 16.8 38.1 MZ 132 19.1 0.74 17.2 38.1 Male 78 19.4 0.85 17.9 38.1 Female 54 18.9 0.63 17.2 24.3 DZ 80 18.1 0.56 16.8 33.8 Male 42 18.7 0.78 17.1 33.8 Female 38 17.4 0.35 16.8 22.5

DZ – dizygotic twin pairs; MZ – monozygotic twin pairs; N – number of participants; SD – standard deviation in years; Min – minimum age in years; Max – maximum age in years.

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32 2.2. Methods 2.2.1. Zygosity determination of twins

Zygosity determination was carried out at the certified laboratory of the enterprise “UAB Synlab Lietuva“. The DNA based tests were performed using venous blood. The procedure was started with DNA isolation from blood and purification. Then polymerase chain reaction set AmpFℓSTR® Identifiler®(Applied biosystems, USA) was used to amplify short tandem repeats (STR) and 15 specific DNA markers (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TROX, D18S51, D5S818, FGA) and Amel fragment of amelogenin gene were used for comparison of genetic profiles (Fig. 2.2.1.1).

Fig. 2.2.1.1. Graphic representation of DNA markers used for zygosity

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Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different that it is possible to distinguish one indi-vidual from another, unless they are monozygotic twins. The true power of STR analysis is in its statistical power of discrimination. Because the 15 loci and Amel fragment of amelogenin gene used for discrimination are inde-pendently assorted, the product rule for probabilities was applied. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1×1018) or more. Therefore, the accuracy for zygosity determination was at the level of 99.99%.

2.2.2. Radiographic safety and quality control of panoramic radiograph

To minimize radiation doze digital panoramic systems were used and ALARA (As Low As Reasonably Achievable) radiation safety principle was followed. The radiographs were assigned a unique code number thereby masking the identity of the individual to the observer.

While analyzing the panoramic radiograms it is essential to evaluate the possibility of the appearance of geometric distortions. Asymmetric patient positioning and differences in the patients' orientation in the vertical plane can cause distortion on panoramic radiographs and consequently affect measurements [128]. To avoid this issue, in the present study all panoramic radiograms were performed by the same operator for all study participants, using standard protocols of patient positioning. The patient’s mid-sagittal line was centered in the machine for the prevention of midline horizontal shift or sideways tilting of the head. The Frankfort plane of the patient was used to prevent the patient’s head tilt in the vertical plane. The forward and backward positioning of the patient’s head was determined using the canine-corner base of the nose alignment line. The head was moved forward or backward and adjusted until the maxillary canine was bisected with the alignment line [129].

2.2.3. Assessment of the relationship between third molar developmental stage and the growth phase

The calcification stage of lower third molar was assessed on the digital panoramic radiographs according to the modified method of Gleiser and Hunt, which was simplified by authors from 10 to 6 calcification stages (Fig. 2.2.3.1) [130].

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I II III IV V VI

Fig. 2.2.3.1. Representation of the calcification stages for molars [4]

I – enamel formation is complete at the occlusal surface; dentinal deposition has commenced; II – crown formation is complete to the cementoenamel junction; III – walls of the pulp chamber are straight and the pulp horns are more differentiated; the root length is less than the crown height; radicular bifurcation is visible. IV – root length is equal to or greater than the crown height; bifurcation is developed sufficiently to give roots a distinct outline with funnel shaped endings; V – the walls of the root canal are parallel and apical

end is still partially open; VI – the apical end of root canal is completely closed; the periodontal membrane has an uniform width around the root and the apex.

The growth phase was assessed using the CVM method modified by Baccetti et al. [4]. Modification is based on visual assessment of the size and shape of the reduced number of cervical vertebrae. The morphology of the bodies of the second (C2), third (C3), and fourth (C4) cervical vertebrae was analyzed and one of six CVM stages was established (Fig. 2.2.3.3).

CS1 CS2 CS3 CS4 CS5 CS6

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35 The six stages are defined as follows:

Cervical stage 1 (CS1). The lower borders of all the three vertebrae (C2-C4) are flat. The bodies of both C3 and C4 are trapezoid in shape (the superior border of the vertebral body is tapered from posterior to anterior). The peak in mandibular growth will occur on average 2 years after this stage.

Cervical stage 2 (CS2). A concavity is present at the lower border of C2 (in four of five cases, with the remaining subjects still showing a cervical stage 1). The bodies of both C3 and C4 are still trapezoid in shape. The peak in mandibular growth will occur on average 1 year after this stage.

Cervical stage 3 (CS3). Concavities at the lower border of both C2 and C3 are present. The bodies of C3 and C4 may be either trapezoid or rectangular horizontal in shape. The peak in mandibular growth will occur during the year after this stage.

Cervical stage 4 (CS4). Concavities at the lower border of C2, C3, and C4 now are present. The bodies of both C3 and C4 are rectangular hori-zontal in shape. The peak in mandibular growth has occurred within 1 or 2 years before this stage.

Cervical stage 5 (CS5).The concavities at the lower borders of C2, C3, and C4 still are present. At least one of the bodies of C3 and C4 is squared in shape. If not squared, the body of the other cervical vertebra still is rectangular horizontal. The peak in mandibular growth has ended at least 1 year before this stage.

Cervical stage 6 (CS6). The concavities at the lower borders of C2, C3, and C4 still are evident. At least one of the bodies of C3 and C4 is rec-tangular vertical in shape. If not recrec-tangular vertical, the body of the other cervical vertebra is squared. The peak in mandibular growth ended at least 2 years before this stage.

Teeth calcification stages and cervical vertebrae maturation were esti-mated by two trained orthodontists separately and blindly. Examiners were calibrated for inter-examiner reliability by means of Kappa statistics. The kappa values for inter-observer agreement of the teeth calcification stages (0.81–0.85) and CVM stages (0.82–0.85) showed almost perfect agreement. As there were no statistically significant differences between teeth calcifi-cation stages on the right and left sides, only mandibular third molar (LM3) was used as a sample.

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2.2.4. Assessment of the reliability of linear measurements on the panoramic radiographs

The orthodontic patients scheduled for panoramic radiographs and fabrication of plaster models for diagnostic and treatment planning purposes were examined. Just before taking panoramic radiograph, standardized 9 mm stainless steel gauges were bonded to the buccal surfaces of the lower third, second, and first molar on the right and left sides with a drop of flow composite (Filtek Ultimate, 3M). The metal calibration gauge (MCGs) was attached 2 mm lower and parallel to the occlusal surface using the ortho-dontic positioning gauge Rocky Mountains Orthoortho-dontics with accuracy 0.5 mm. Later, MCGs were removed and the teeth surfaces were polished.

The diagnostic impressions were taken using “Prestige” (Vannini Dental Industry, Italy) silicon material and dental casts produced from stone “Mar-morock N” class IV (Siladent, Germany).

The crown widths were measured from the most prominent mesial contact surface point to the most prominent distal contact surface point of the lower first, second, and third molars. All measurements were done twice by the same orthodontist with 2 weeks interval on the dental casts. The same crowns widths and 9 mm stainless steel gauge lengths were also measured in the panoramic radiographs (Fig. 2.2.4.1).

Fig. 2.2.4.1. The position of metal calibration gauges on the lower molars

The measurements on the dental casts were done using digital caliper with the tips sharpened to a point and 0.01 mm accuracy (Dentaurum, Ger-many). The measurements in the digital panoramic radiographs were done using commercially available software (Dolphin Imaging 11.8 Premium). All image measurements were compared with the physical measurements on the plaster casts or actual length of stainless steel gauge. The coefficients of image magnifications were calculated by dividing the width of the molar crown in the panoramic radiograph by the actual molar width on the plaster

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casts or by dividing the length of the stainless steel gauge in the panoramic radiograph by 9 mm( actual length of gauge). The magnification coefficients of 9 mm stainless steel gauge were used as a standard (control group) and magnification coefficients calculated using plaster casts (MCC) comprised study group. Each group included 6 subgroups: lower right third, second, first molar and left third, second, first molar group magnification coeffi-cients.

2.2.5. Analysis of panoramic radiographs for the third molar agenesis

Every panoramic radiograph was evaluated by one orthodontist. The investigations of all the panoramic radiographs were conducted twice in a 2-week interval for the evaluator's calibration. The post hoc analysis of the power of the study showed a level of 0.8.

2.2.6. Assessment of the third molar root mineralization stage

M3 root mineralization was assessed in accordance with the teeth mine-ralization scheme (Fig. 2.2.6.1) proposed by Demirjian et al. (1973) as it distinguishes the initiation of the root formation in comparison with other schemes [131]. The assessment of mineralization stages in all panoramic radiographs was performed by the same observer within a two-week inter-val. Intra-examiner reliability was assessed with ICC coefficient.

A B C D

E F G H

Fig. 2.2.6.1. Teeth mineralization stages according to Demirjian et al. (1973) Stage A – calcification of the occlusal surface occurs without fusion; Stage B – formation of occlusal surface by the fusion of the mineralization points; Stage C – enamel is formed and the formation of dentin has started with no pulp horns in the pulp chamber; Stage D – crown is formed until the cement enamel junction with the commence of the root formation;

Stage E – the roots are shorter than the crown height and the radicular bifurcation is calcified; Stage F – the roots are equal or longer than the crown height; Stage G – the walls of the root canal are parallel with the apical ends opened; Stage H – the apexes of

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2.2.7. Assessment of the upper third molar position

Digital panoramic radiographs were analyzed for the positions of the maxillary third, second and first molars in the right and left sides.

Previous studies showed that significant changes in the inclination of the occlusal plane take place during growth; with no significant differences between the mean values of all investigated variables according to sex [132]. Thus, we used additional planes for the measurements to avoid inter-preting mistakes. The positions of these teeth were described by 10 angular and 8 linear parameters using the maxillary plane (MxP) and occlusal plane (OCL) [133–139] (Fig. 2.2.7.1).

Fig. 2.2.7.1. The description of variables for estimating the positions

of upper third molars

UR8 – longitudinal axis of the upper right third molar is traced from the midocclusal point to the midpoint of the root bifurcation; UL8 – longitudinal axis of the upper left third molar

is traced from the midocclusal point to the midpoint of the root bifurcation; UR7 – longitudinal axis of the upper right second molar is traced from the midocclusal

point to the midpoint of the root bifurcation; UL7 – longitudinal axis of the upper left second molar is traced from the midocclusal point to the midpoint of the root bifurcation; UR6 - longitudinal axis of the upper right first molar is traced from the midocclusal point to the midpoint of the root bifurcation; UL6 – longitudinal axis of the upper left first molar is traced from the midocclusal point to the midpoint of the root bifurcation; Max-R – right

maxillary plane is drawn through the spina nasalis anterior to the spina nasalis posterior on the right; Max-L – left maxillary plane is drawn through the spina nasalis anterior to

the spina nasalis posterior on the left; OCL-UR – upper right occlusal plane is drawn through the incisal edge of the upper right lateral incisor to the distal cusp of the upper right second molar; OCL-UL – upper left occlusal plane is drawn through incisal edge

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The eruption levels of the upper M3 were assessed according to their relationship to the occlusal plane of the adjacent second molars. The distance between the occlusal surface of the third and second molars (DUR8/UR7, DUL8/UL7) was measured as the distance from the mesial cusp of the maxillary third molar tangentially to the line that extended over the occlusal surface of the second molar on the right and left side (Fig. 2.2.7.2).

Fig. 2.2.7.2. The description of variables for estimating eruption levels

of upper third molars

DUR8/UR7 – the distance between the occlusal surfaces of the upper right third molar to the upper right second molar; DUL8/UL7 – the distance between the occlusal surfaces

of the upper left third molar to the upper left second molar.

Later, the available retromolar space (LES-UR, LES-UL) was measured as the distance between the distal surface of the second molar crown and to the tangent line to the cortex of the maxillary tuberosity on the right and left sides(Fig. 2.2.7.3).

Fig. 2.2.7.3. The description of variables for estimating the eruption space

for upper third molars

LES-UR – eruption space for the upper right third molar, LES-UL – eruption space for the upper left third molar.

THE INFLUENCE OF GENETIC AND ENVIRONMENTAL FACTORS ON THE DEVELOPMENT AND POSITION OF THIRD MOLARS

Giedr

ė Trakinienė

THE INFLUENCE OF GENETIC

AND ENVIRONMENTAL FACTORS

ON THE DEVELOPMENT

AND POSITION

OF THIRD MOLARS

Giedr

ė Trakinienė

GENETINIŲ IR APLINKOS

VEIKSNIŲ ĮTAKA TREČIŲJŲ

KRŪMINIŲ DANTŲ

VYSTYMUISI IR PADĖČIAI

CONTENTS

ABBREVIATIONS

PREFACE

INTRODUCTION

AIM, OBJECTIVES AND HYPOTHESIS

OF THE STUDY

SCIENTIFIC NOVELTY

1.

LITERATURE REVIEW

2.

MATERIALS AND METHODS