Thesis Final

THESIS/DISSERTATION APPROVED BY

4-24-2020 Barbara J. O’Kane

Date Barbara J. O’Kane, MS, Ph.D, Chair

Margaret Jergenson

Margret A. Jergenson, DDS

Neil Norton

Neil S. Norton, BA, Ph.D.

Gail M. Jensen, Ph.D., Dean

COMPARISON OF PERIODONTIUM AMONG SUBJECTS TREATED WITH CLEAR ALIGNERS AND CONVENTIONAL ORTHODONTICS

By:

Mark S. Jones

A THESIS Presented to the Faculty of The Graduate College at Creighton University In Partial Fulfillment of Requirements For the Degree of Master of Science in the Department of Oral Biology

Under the Supervision of Dr. Marcelo Mattos Advising from: Dr. Margaret Jergenson, Dr. Neil S. Norton, and Dr. Barbara O’Kane

Omaha, Nebraska 2020 i

iii

Abstract

INTRODUCTION: With the wider therapeutic use of clear aligners the need to investigate the periodontal health status and microbiome of clear aligners’ patients in comparison with users of fixed orthodontic has arisen and is the objective of this thesis.

METHODS: A clinical periodontal evaluation was performed, followed by professional oral hygiene treatment on a patient under clear aligner treatment, another under fixed orthodontics and two controls that never received any orthodontic therapy. One week after, supragingival plaque, swabs from the orthodontic devices, and saliva samples were collected from each volunteer for further 16s sequencing and microbiome analysis.

RESULTS: All participants have overall good oral hygiene. However, our results showed increases in supragingival plaque, higher number of probing depths greater than 3mm, higher number of bleeding sites on probing, and a higher amount of gingival recession in the subject treated with fixed orthodontics. A lower bacterial count was observed colonizing the clear aligners, with less diversity than the other samples analyzed. Clear aligners exhibited a higher proportion of genus Porphyromona, which has a well-known periodontal pathogen, P. gingivalis. The genus

Aggregatibacter had higher proportions on the subject with fixed orthodontics and the control with higher attachment loss. The species Aggregatibacter actinomycetemcomitans has been linked to aggressive forms of periodontal disease.

CONCLUSION: Clear aligners showed improved periodontal status indices when compared to fixed orthodontics. Overall, fewer bacteria were found on the clear aligner when compared to fixed orthodontics. Genera with known periodontal pathogens were found on both orthodontics devices.

Acknowledgements

I would like to thank Dr. Marcelo Mattos for being my mentor throughout this process.

The countless number of times I dropped by to ask questions or clarification he always welcomed me with open arms, thank you. I would like to thank Dr. Barbara O’Kane, Dr. Margaret Jergenson,

Dr. Neil S. Norton, Dr. Sonia Sanchez, and Dr. Laura Brown for their advice and expertise throughout the project. I would also like to express my gratitude to Umesh Pyakuel for his technical assistance with the DNA extraction and interfacing with the UNMC sequencing core. Additionally,

I would like to thank Jenifer Bushing and the UNMC sequencing core for their assistance. I would like to express my gratitude to Irina M. Velsko for her willingness to assist with the microbiome analysis. I would also like to thank Adrian Hankewycz and Saif Shah for their assistance with the clinical and data collection. Lastly, I would like to thank my parents for their never-ending support and encouragement. v

Table of Contents

Abstract ...... iii

Acknowledgments ...... iv

Table of Content ...... v

Tables and Graphs ...... vii

Chapter 1: Introduction ...... 1

Chapter 2: Literature Review ...... 7

Chapter 3: Objectives ...... 14

Chapter 4: Materials and Methods ...... 15

i. Subject Selection ...... 15

ii. Clinical Evaluation of Periodontium ...... 15

a. Supragingival Plaque ...... 15

b. Probing Depth ...... 16

c. Bleeding on Probing ...... 16

d. Clinical Attachment Level ...... 16

e. Gingival Recession ...... 16

iii. Oral Hygiene Treatment ...... 16

iv. Samples Collection ...... 17

v. Laboratory Analysis ...... 17

a. DNA Extraction ...... 17

vi. 16S Sequencing ...... 19

a. PCR Amplification ...... 19

b. PRC Clean-up 1 ...... 20

vii

c. PCR Index ...... 20

d. PCR Clean-up 2 ...... 21

e. Library Denaturing and MiSeq Sample Loading ...... 22

vii. Microbiome Analysis ...... 23

Chapter 5: Results ...... 24 i. Demographic Data ...... 24

ii. Clinical Evaluation of Periodontium ...... 24

a. Supragingival Plaque ...... 24

b. Probing Depth ...... 25

c. Bleeding on Probing ...... 28

d. Clinical Attachment Level ...... 29

e. Gingival Recession ...... 30 iii. Microbiome Analysis ...... 31

a. Quantitative Analysis ...... 31

b. Qualitative Analysis ...... 36

Chapter 6: Discussion ...... 45

Chapter 7: Conclusion ...... 59

Chapter 8: Bibliography ...... 61

viii

Tables and Figures Tables Table 1...... 24

1. Demographic information on study subjects

Table 2 ...... 32

1. The total number of OTUs and total number of bacterial species found in each sample

Figures Figure 1a ...... 25

1. Absolute number of surfaces with supragingival plaque.

Figure 1b ...... 25

1. Percentage of surfaces with supra gingival plaque.

Figure 2 ...... 26

1. Average probing depth in mm.

Figure 3a ...... 27

1. The absolute number of probing depths greater than 3mm.

Figure 3b ...... 27

1. Percentage of probing depths greater than 3mm.

Figure 4a ...... 28

1. The absolute number of bleedings on probing.

Figure 4b ...... 29

1. The percentage of bleedings on probing.

Figure 5 ...... 30

1. The average clinical attachment level.

Figure 6a ...... 30

1. The number of sites with gingival recession.

Figure 6b ...... 31

1. The percentage of sites with gingival recession.

Figure 7a ...... 33

1. The total number of bacteria and species for all samples.

Figure 7b ...... 34

1. The pooled bacteria and species number in the subgingival plaque sample.

Figure 7c ...... 35

1. The bacteria and species number between clear aligners and fixed orthodontics with swabs.

Figure 7d ...... 35

1. The bacteria and species number between clear aligners and fixed orthodontics in saliva.

Figure 8a ...... 36

1. Faith’s phylogenetic distance between all samples.

Figure 8b ...... 37

1. Unweighted Unifrac PCoA of all samples.

Figure 8c ...... 38

1. Weighted Unifrac PCoA of all samples.

Figure 8d ...... 39

1. Shannon Index for all samples.

Figure 9a ...... 40

1. The top 10 taxonomic species between all samples.

Figure 9b ...... 41

1. The top 10 taxonomic species between the samples for the subject with clear aligners.

Figure 9c ...... 42

1. The top 10 taxonomic species between the samples for the subject with fixed orthodontics.

Figure 9d ...... 43

1. The top 10 taxonomic species found colonizing orthodontic devices.

Figure 9e ...... 44

1. The top 10 taxonomic species between subgingival plaque samples.

Chapter 1: Introduction

Orthodontic treatment is the third most prevalent dental treatment after diagnostic and preventative procedures with an estimated value of 9.7 billion dollars in 2012 in the United States

(Laniado et al., 2017). Roughly four million people have one form or another of braces and one- in-three of those individuals are adults (“Why the Number of Adults Seeing an Orthodontist Is at an All-Time High,” 2020). While the adult population is the fastest growing sector of individuals seeking orthodontic treatment, children between the ages of 9 and 14 still make up the majority of patients with braces (“Dental braces,” n.d.). As of 2016, roughly 1.7 million adults have undergone orthodontic treatment, dramatically increasing the adult patient population (“Moving Adult Teeth

| Adults’ Guide to Orthodontics,” n.d.). Braces, as we know them today, date back to the early

1900s, but had a much earlier beginning (Phulari, 2013).

As early as 1723, Pierre Fauchard, who is referred to as the founder of modern dentistry references what is thought to be the first orthodontic appliance in “The Surgeon Dentist, A Treatise on the Teeth” (Asbell, n.d.). The appliance described was called a bandolet and used to expand that arch of anterior teeth. Several other clinicians worked to advance the field of orthodontics, but it was not until 1875 when John Farrar came along and accelerated the understanding of orthodontics. He is commonly referred to as the father of American Orthodontics (Asbell, n.d.).

Since then, our knowledge of orthodontics has grown exponentially. Subsequently, a variety of orthodontic appliances have been developed.

Fixed orthodontics come in a variety of different types. The most familiar type consists of traditional stainless-steel wires anchored to the teeth by a bracket and rubber bands (“Orthodontic

Treatment Options | American Association of Orthodontists,” n.d.). The brackets are usually

bonded to the teeth thus fixing them in place. They can be bonded on the facial side or lingual side of the tooth, depending on the type of treatment.

Removable orthodontic appliances sometimes, referred to as retainers, are another commonly used form of orthodontic treatment (“Orthodontic Treatment Options | American

Association of Orthodontists,” n.d.). As the name suggests, these appliances can be easily removed by the patient. They consist of an acrylic base plate used as a point of attachment for all the functional or active units. The retentive components extend off of the base plate and securely attaches the entire appliance on the designated position. Lastly, the active components can consist of a screw, wires, and springs that facilitate tooth movements (Fricker et al., 2013).

As early as 1946, thermoplastic aligners, or clear aligners, have been used as retainers and later to treat malocclusion in patient (Kesling, 1946). Several other clinicians made valuable contributions to further the use of clear aligners as an orthodontic treatment (Nahoum 1959, Ponitz

1971, McNamara 1985, and Sheridan 1993). Thermoplastic aligners and the rapid advances in digital treatment planning laid the groundwork for clear aligners treatment as we know it today

(Weir, 2017). More recently, in 1999, the Invisalign system was introduced as the first commercial brand of FDA-approved clear aligners (“Align Technology,” n.d.).

Since the introduction of Invisalign, there have been many different clear aligner systems on the market, including at-home systems, increasing the appeal of these types of treatments. All existing aligner’s systems work as a succession of clear aligners that function similarly to traditional fixed braces, moving the teeth incrementally as aligners are progressively changed

(“Braces vs. Clear Aligners,” 2019). One of the biggest attractions to this system is that they are clear and removable and, therefore, much more appealing to individuals that want to have the effects of braces without the aesthetics drawbacks of traditional braces.

The overall goal of orthodontic treatment is to correctly align the teeth and jaw resulting in a physiologic occlusion with an aesthetically pleasing smile (“Why Orthodontics?,” n.d.). As mentioned previously, the majority of the individuals undergoing orthodontic treatment are children between the ages of 9 and 18. This is a critical period for craniofacial development

(Sadowsky, 1998). During this crucial developmental point, many young people experience abnormalities that can result in malocclusion. Malocclusion occurs as a consequence of interferences when the opposing teeth come together or during excursive movements of the mandible (“Malocclusion of teeth,” n.d.).

Additionally, orthodontic treatment attempts to correct crowding, excessively spaced teeth, crossbite, underbite, missing teeth, and thumb sucking that may adversely affect the jaw and teeth

(Prabhakar et al., 2014). If these conditions are present, it is essential to treat them as soon as possible to allow for healthy craniofacial development. The American Association of Orthodontics recommends that young people be referred to an orthodontist as soon as the issue arise and no later than age 7; this gives the patients the best chance for a healthy smile (“Frequently Asked

Questions,” n.d.).

Even though orthodontic treatment has been deemed relatively safe, there are some potential risks associated with the treatment. The most common risk factor is pain caused by orthodontic treatment. Studies have shown that 70-95% of patients undergoing orthodontic treatment have experience pain associated with the appliance (Talic, 2011). The pain includes discomfort, tension, pressure, and tenderness caused by the orthodontic appliance. In severe cases, patients prematurely ended the treatment due to the pain (Talic, 2011).

The oral cavity, commonly referred to as the mouth, is surrounded by the lips and is composed of two different regions- first the vestibule, which is the area between the lips and cheeks

and the teeth. Secondly, the oral cavity proper, which consists of the tongue, which is surrounded by teeth toward the anterior and sides. Posteriorly is the oropharynx, superiorly is the hard palate, which consists of the palatine process of the maxilla and the horizontal plate of the palatine bone, all covered by mucosa (Kamrani and Sadiq, 2020). Teeth come in different shapes and sizes; however, all teeth consist of the same general structures: a hard crown that consists of a layer of enamel, dentin, and pulp that sits above the gumline and is attached to the root via the neck of the tooth (Arola et al., 2017). Unlike the crown, the root of the tooth is not covered with enamel, but instead with cementum. Cementum is less mineralized as enamel and therefore does not provide the same amount of protection. It is also the site of fibers that attaches the tooth to the bone, called the periodontal ligament. These fibers anchor the tooth to the jaw. In healthy individuals, gum tissue sits below the crown and protects the root of the tooth from food and debris that might accumulate in the oral cavity (Yamamoto et al., 2016).

A common risk of orthodontic treatment is root resorption, described as a process resulting in the loss of cementum and dentine surrounding teeth (Dindaroglu and Dogan, 2017). It is a complex and not very well understood process that includes a genetic predisposition and environmental factors. The length of treatment also plays a role in root resorption, and the longer the treatment time, the higher the likelihood of root resorption (Talic, 2011).

Decalcification of the enamel is another common side effect of orthodontic treatment. It is characterized as the loss of mineralization in the outermost layer of the tooth and is often associated with the appearance of white spots on teeth. It is also considered to be a precursor to the formation of caries in teeth. Previous studies demonstrated that decalcification occurs on 50% of patients that undergo orthodontic treatment (Talic, 2011).

Another widespread drawback of braces is the risk of developing periodontal disease. Over half of Americans older than 30 have been diagnosed with periodontal disease (“Periodontal

Disease | Oral Health Conditions | Division of Oral Health | CDC,” 2018). It includes gingivitis, periodontitis, and loss of attached gingival support. Gingivitis is defined as “an inflammatory lesion resulting from interactions between the dental plaque biofilm and the host’s immune- inflammatory response, which remains within the gingiva” (Chapple et al., 2018). It is one of the more prevalent diseases and considered a mild form of periodontal disease. Symptoms include inflammation of gingival tissue, redness, and bleeding (“Periodontal Disease | Oral Health

Conditions | Division of Oral Health | CDC,” 2018). While gingivitis is reversable, if left untreated, it usually progresses into a more severe condition called periodontitis, however, there are forms of periodontitis that present with minor or absent gingival inflammation. Periodontitis is defined as

“a chronic multifactorial inflammatory disease associated with dysbiotic plaque biofilms and characterized by progressive destruction of the tooth-supporting apparatus. It’s primary features include the loss of periodontal tissue support, manifested through clinical attachment loss (CAL) and radiographically assessed alveolar bone loss, presence of periodontal pocketing and gingival bleeding” (Papapanou et al., 2018). A biofilm is a community of microorganisms found on the tooth surface (Marsh, 2006).

As described in a study done by H. Löe (1965), the accumulation of soft and hard debris can result in gingival inflammation over 12-15 days if left untreated. The average time of orthodontic appointments is between 3-4 weeks (Moresca, 2018), which gives plenty of time for debris to accumulate potentially resulting in gingivitis if proper oral hygiene is not performed by the patient. Brushing alone is not enough to totally remove debris from teeth. Multibracketed teeth require both brushing and interdental hygiene to remove debris entirely (Bock et al., 2010).

With the potential risked posed by orthodontic treatments, the goal of this study was to compare the periodontal status of individuals who underwent traditional orthodontic appliances and clear aligners. In addition, the effects that both these systems had on the microbiome of the oral cavity was investigated.

Chapter 2: Literature Review

Orthodontic treatment is becoming more and more appealing since the introduction of

Invisalign in 1999, especially in the adult population (“Align Technology,” n.d.). It is estimated that 27% of new patients seeking orthodontic treatment are adults (“Clear Aligners For Adults|The

How, Who, Where & Why,” 2018). Naturally we want to compare current techniques or therapies to their advances as a way investigate further. A few studies have been done comparing clear aligners and fixed orthodontics, covering the topics of their effectiveness, the time frame of each system, and their effects on the periodontium, which is of interest to us. In general, it has been shown that clear aligner treatment resulted in improved periodontal health indexes that include plaque index, bleeding on probing, and probing depth when compared to fixed orthodontics. The clinical parameters described, are all related to the diagnosis of plaque-induced periodontal diseases.

The primary objective of this review is to review papers on the periodontal status of individuals who underwent fixed orthodontic treatment as compared it to studies of individuals who underwent orthodontic treatment using clear aligners.

Several papers that explored the differences between clear aligners and fixed orthodontics and their effects on periodontal health. The first two papers we reviewed followed a similar methodology to this thesis and, hence, are thoroughly described as it follows.

The study “Periodontal status of adult patients treated with fixed buccal appliances and removable aligners over one year of active orthodontic therapy” (Karkhanechi et al., 2013) was performed in the United States. The sample size included 42 participants, 20 were assigned to receive clear aligner treatment, and the remaining 22 were treated with fixed buccal orthodontics.

One week before treatment, both groups received professional oral hygiene treatment. Before the

appliances were delivered, a clinical exam was performed that included plaque index, gingival index, bleeding on probing, and probing depth using a North Carolina 15 periodontal probe.

Follow-up examinations were done at six weeks, six months, and 12 months. During the yearlong study, 4 participants in the fixed buccal group and 3 in the clear aligner group failed to be reevaluated. The plaque index was recorded for the fixed buccal appliance group and the clear aligner group; at the 6-week examination, no difference was noted between the groups. However, at the 6-month and 12-month evaluations a there was a significant increase in PI for the fixed buccal appliance group. A similar result was observed for the gingival index. At 6-weeks bleeding on probing was similar between the two groups, but an increase in BOP was observed at the 6- month and 12-month mark for the fixed buccal appliance group. Unlike the previous results, an increase in probing depth was observed at 6-weeks and throughout the study for the fixed buccal appliance group. They concluded that treatment with clear aligners resulted in improved periodontal health compared to fixed buccal appliances.

The second study, we reviewed the paper “Periodontal health status in patients treated with the Invisalign system and fixed orthodontic appliances: A 3 months clinical and microbiological evaluation” (Levrini et al., 2015) was done in Italy. Seventy-seven participants were selected, ranging in age from 16 to 30, and randomly assigned them to an Invisalign or fixed orthodontics groups with ten being assigned to a control group. One month before treatment started, all the participants received professional oral hygiene treatment and follow-up personal oral hygiene instructions, which included a schedule of brushing for 2 minutes and flossing, performed three times a day. The clinical examinations were performed according to the modified PI of Loe and

Silness, PD was measured with a Goldman-Fox periodontal probe to the nearest millimeter, and

BOP was recorded 20 seconds after probing. Clinical evaluation and microbiological samples of

oral biofilm was collected at the beginning, one month, and three-month intervals. Clinical evaluations and biofilm sample collection was done on the upper right first molar and upper left central incisor. The investigators looked for the presence or absence of Prevotella intermedia,

Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia.

Real-time PCR was performed using a LightCycler instrument and a LightCycler DNA Master

SYBR Green I kit according to the manufacture’s protocol. The statistical analysis was performed using the Mann-Whitney test and the statistical package SPSS.

The results showed a statistically significant difference between the Invisalign and the fixed orthodontics group, with all periodontal parameters performing better for the participants in the Invisalign group. They found a significant increase in PI and BOP in participants treated with fixed orthodontic; however, there was no significant change in probing depths. Additionally, they found subjects treated with fixed orthodontics had a bacterial count of 8,187 and subjects treated with clear aligners had 2,739. The investigators did not find any periodontal pathogens in the group treated with Invisalign and found Aggregatibacter actinomycetemcomitans, at one month and three months into the study, in one subject treated with fixed orthodontics. The study concluded that the participants treated with fixed orthodontics had increased PI, BOP, and PD indices, which could make them more susceptible to gingival inflammation that could lead to periodontal disease.

A secondary aim of this paper is to compare the microbiome status between subjects with fixed orthodontics and clear aligners. We focused on one publication that had this similar goal as ours.

The first study we reviewed at was “Profiling of subgingival plaque biofilm microbiota in female adult patients with clear aligners: a three-month prospective study” (Guo et al., 2018) was performed in China. The sample size included ten subjects who underwent clear aligner treatment.

A periodontal exam and subgingival samples were taken at three different times, before treatment, one month after treatment, and three months after treatment. All the subgingival samples underwent DNA extraction, PCR amplification, and 16s rRNA gene sequencing using the Illumina

MiSeq Platform. The sequenced data was processed through the QIIME pipeline. Using UCLUST, the data was separated and referenced using the Human Oral Microbiome Database. They found that plaque index and bleeding on probing increased from before treatment started to three months later, however, it was not a significant increase. They found the average number of operational taxonomic units was 245 ±30 per subject. Alpha diversity analysis was performed that included

Chao 1 and ACE, which showed no significant difference. Additionally, microbial community diversity estimators were performed, that included Simpson and Shannon indices, that showed more diversity before treatment than at one and three months. Suggesting that microbial richness and evenness decreased over time with no statistical significance. Beta diversity estimators, including Weighted Unifrac and Principal Coordinates Analysis, were performed and indicated bacterial communities tended before treatment began cluster separately when compared to samples collected at one and three months. Suggesting a more diverse bacterial community at one and three months than before treatment started. The top 3 genera before treatment were Streptococcus

(11.95%), Neisseria (7.29%), and Actinomyces (7.27%) and Rothia (11.39%), Actinomyces

(10.53%), and Fusobacterium (8.36%) after three months. They found that majority of the genera did not change significantly form before treatment to three months. However, a lower proportion of phylum Firmicutes and genus Mycoplasma were found during the first three months of clear aligner treatment. Both phylum Firmicutes and genus Mycoplasma were recognized as periodontal pathogens. Additionally, five other periodontal pathogens (including Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Fusobacterium nucleatum

and Treponema denticula) were detected, however, no significant changes were observed during the three-month treatment time. They also found that in the first three months, clear aligners did not induce gingival inflammation. This was considered to be a stable periodontal status and one that could not cause periodontal inflammation. They concluded that clear retainers could significantly change the oral microbiome by changing the microbial diversity. However, these changes did not affect the periodontal pathogens and core microorganisms as they were considered stable.

In addition, some literature reviews on the topic, worth describing lacked a significant sample size and were meta-analyzed together for greater statistical power. “Clear aligner therapy might provide a better oral health environment for orthodontic treatment among patients at increased periodontal risk” (Flores-Mir, 2019), consists in a meta-analysis of a sample size of 464 participants, from which 207 underwent clear aligners treatment, and 257 underwent fixed orthodontic treatment, the average duration was 21 months. Their results showed patients who underwent clear aligner treatment had a decreased plaque index and probing depth when compared to fixed orthodontics.

Three additional papers that investigated the oral microbiome, we also reviewed. The first of these being “Alterations of the oral microbiome in patients treated with the Invisalign system or with fixed appliances” (Wang et al., 2019), a study performed in China. The sample size included 26 subjects, seven received Invisalign, 12 received fixed orthodontics, and seven did not undergo any form of orthodontic treatment. Unstimulated saliva from each subject that was used for microbiome analysis. They found the average number of operational taxonomic units (OTUs) were 255.4, 224.8, and 300.8 for subjects treated with Invisalign, fixed orthodontics, and controls

respectively. The top 10 genera for the samples were Veillonella, Streptococcus, Prevotella,

Haemophilus, Neisseria, Porphyromonas, Prevotella, Selenomonas, Rothia, and Fusobacterium.

Overall, they found few significant differences between the two groups treated with orthodontics.

However, three genera were found to be different, Neisseria, Rothia, and TM7, which was not in the top 10 bacteria proportions for either group. Neisseria and TM7 showed higher proportions in subjects treated with Invisalign, while Rothia showed higher proportion in subjects with fixed orthodontics. These findings were significant since Neisseria has been shown to be associated with improved oral health, in some studies. While TM7 has been linked to gingivitis and periodontal disease. They concluded that both Invisalign and fixed orthodontics caused microbial dysbiosis and that there was no significant difference between the two groups. Additionally, they did not find an improved oral microbiome in patients that underwent Invisalign treatment.

Another study reviewed was “Which orthodontic appliance is best for oral hygiene? A randomized clinical trial” (Chhibber et al., 2018) performed in the United States. The sample size consists of 71 participants, 27 were placed in the clear aligner group, 22 were placed in the self- ligating brackets group, and 22 were placed in the conventional brackets group. Plaque index, gingival index, and bleeding on probing was recorded for each participant before treatment, nine months after treatment, and 18 months after treatment. Their results showed no significant difference between all three groups after 18 months. However, after nine months of treatment the participants in the clear aligner group exhibited better gingival index and bleeding on probing scores, when compared to the other groups.

The last study included was “Salivary levels of cariogenic bacterial species during orthodontic treatment with thermoplastic aligners or fixed appliances: a prospective cohort study”

(Sifakakis et al., 2018), performed in Switzerland. The sample size included 30 patients, 15

received fixed orthodontic treatment, and the remaining 15 received clear aligner treatment. They found no difference for S. mutans and L. acidophilus between the two groups after one month of treatment. However, lower salivary levels of S. sanguinis were found in patients treated with clear aligners.

Chapter 3: Objectives

With the increase in the type of orthodontic treatments available, each with its own set of benefits and potential risks to the periodontal status, a comparison of the periodontium under the effect of different orthodontic methods is needed. With the stated studies in chapter 2, our alternative hypothesis is that periodontal health is better sustained with clear aligner treatment.

This study investigated this topic with the following objectives.

The primary objective of this study is to evaluate the periodontal status in individuals undergoing fixed orthodontic treatment and compare it to subjects who underwent orthodontic interventions utilizing removable clear retainers. The comparison of the periodontal status between subjects involved two individuals that never received any orthodontic treatment before as control subjects.

The secondary objective is to compare the oral microbiome of the four subjects described above in order to examine the differences in the oral microbiome that could be influenced by the orthodontic appliances leading to dysbiosis or to pathogenic traits.

Chapter 4: Methodology

1. Subject Selection

In order to achieve the goals outlined by the objectives, four subjects volunteered to assist

with this study. All of the participants were part of Creighton School of Dentistry at the

time of the study. One was currently under traditional orthodontic treatment, one was

currently under a clear aligner treatment, and two participants had not undergone any

form of orthodontic treatment in their life that served as control (IRB # 2000866).

2. Clinical Evaluation of Periodontium

Clinical evaluation of the periodontium was performed according to the guidelines

established by the American Dental Association (Smiley et al., 2015); three subjects were

asked to perform personal oral hygiene and one was asked to refrain from performing

personal oral hygiene. Each participant underwent a clinical exam used to determine the

status of their periodontium. The clinical parameters included bleedings on probing,

probing depths, clinical attachment levels, and the amount supragingival plaque present

around each present tooth. Each measurement was recorded at six sites (distal buccal,

buccal, mesial buccal, distal lingual, lingual, and mesial lingual) around each tooth

according to Axium (Exan software Company, Mellville, NY) periodontal charting

guidelines.

2.1 Supragingival Plaque

Visibly identifiable plaque on any tooth surface in the oral cavity was recorded.

2.2 Probing Depth

Probing depth was determined through the use of a University of North Carolina UNC 15

periodontal probe (Hu-Friedy, Chicago, IL) and the walking stroke technique (Boyd et al.,

2019). A measurement from the gingival margin to the most coronal level of the junctional

epithelium was recorded in millimeters (mm).

2.3 Bleeding on Probing

Any visible bleeding in the surrounding gingival margin as a result of probing was

recorded.

2.4 Clinical Attachment Level (CAL)

The distance between the cementum-enamel junction (CEJ) to the most coronal point of

the junctional epithelium was recorded, in mm, as CAL.

2.5. Gingival recession.

Wherever the gingival margin presented an apical location in relation to the CEJ, the

distance was recorded in mm.

3. Oral Hygiene Treatment

A professional oral hygiene treatment was performed on each subject one week before the

sample collection occurred, to establish an equal baseline for subgingival plaque collection.

The hygiene treatment was performed by a member of the Department of Periodontology

at Creighton School of Dentistry withthe use of ultrasonic devices (Cavitron, Dentsply-

Sirona, York, PA) and hand instruments (Hu-Friedy, Chicago, IL).

4. Sample Collection

At the time of the clinical periodontal examination, the following samples were collected.

- A pool of the subgingival plaque from all gingival crevices from each subject, with the

use of sterile periodontal curettes (Hu-Friedy, Chicago, IL). The plaque pooled per

subject was placed in a sterile vial containing RNase free water.

- Unstimulated saliva samples were collected from each subject, during a two-minute

interval. The saliva collected was directly placed is a sterile vial.

- Swabs from the brackets, wires and bands of the subject with fixed orthodontic

appliances. Facial and lingual aspects from the upper and lower appliances were

swabbed with a sterile swab, resulting in a total of two swabs placed into a sterile vial

and freeze-dried in liquid nitrogen.

- Swabs from the clear aligners of the subject under this treatment modality. Internal and

external surfaces of the upper and lower aligners were swabbed with a sterile swab,

resulting in a total of two swabs placed into a sterile vial and freeze-dried in liquid

nitrogen.

All samples were placed in a -80°C freezer for storage until DNA extraction.

5. Laboratory Analysis

DNA extraction of the plaque, saliva, and swabs sample was achieved through the use of

DNeasy Power Soil Kit (MO BIO Laboratories Inc., Carlsbad, CA) according to protocol

provided by the manufacturer.

5.1 DNA Extraction

All products used are from the kit mentioned in section 5. Approximately 0.25g of plaque and saliva samples were placed into the PowerBead tubes, respectively, and vortexed gently until the solution was homogeneous. Next, 60µl of C1 solution was added to the

PowerBead tube and inverted several times. After the tubes were inverted, they were vortexed horizontally at maximum speed (12,000-13,000 rpm) for 10 minutes. The samples were then centrifuged at 10,000 x g for 30 seconds; the supernatant was collected without disturbing the precipitant and transferred to a clean 2ml collecting tube. Once in the collecting tube, 250µl of solution C2 was added and vortexed for 5 seconds and placed in a 4°C refrigerator. The samples were allowed to rest for 5 minutes at 4 °C and then centrifuged again, at 10,000 x g for 1 minute. Up to 600µl of the supernatant was collected, without disturbing the pellet, and transferred to a clean 2ml collection tube. 200µl of C3 solution was added to the sample and again briefly vortexed and allowed to rest in a 4°C refrigerator for 5 minutes. The sample was then centrifuged at 10,000 x g for 1 minute after being removed from the refrigerator. 750µl of the supernatant was transferred to a clean

2ml collection tube without disturbing the pellet. Next, 1200µl of solution C4 was added to the collecting tube and vortexed for 5 seconds. After being vortexed, 675µl was loaded onto an MB Spin Column and centrifuged at 10,000 x g for 1 minute. The flow-through was discarded and the process repeated once more. Then 500µl of solution C5 was added to the spin column and centrifuged at 10,000 x g for 30 seconds. Again, the flow-through was discarded, and the MB Spin Column was carefully placed in a clean 2ml collecting tube, and 100µl of solution C6 was added to the center of the filter membrane. It was then centrifuged at 10,000 x g for 30 seconds.

The flow-through was collected and stored in a -80°C freezer for future PCR amplification

of the hypervariant 16s sequence of the rRNA and its analysis.

DNA was extracted at two separate times to guarantee satisfactory levels for sequencing.

For this purpose, the subject treated with fixed orthodontics provided a second saliva

sample at a different timepoint, as the first collection was insufficient for two DNA

extractions.

6. 16S Sequencing

All the samples were sent to University of Nebraska Medical Center Sequencing core for

PCR amplification and sequencing through the use of the MiSeq System (Illumina, San

Diego, CA).

6.1 PCR Amplification

A reaction containing the following was set up: 2.5µl of sample microbial DNA (5ng/µl),

5µl of Amplicon PCR Forward Primer 1µM

(TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG),

5µl of Amplicon Reverse Primer

(GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTA

ATCC), 12.5µl of 2x KAPA HiFi HotStart Ready Mix for a total of 25µl. Next, the plate

was sealed, and PCR was performed in a thermal cycle using the following program: 95°C

for 3 minutes, then 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30

seconds. Then, 72°C for 5 minutes and finally held at 4°C.

6.2 PCR Clean-up 1

The AMPure XP (Agencount AMPureXP, Brea, CA) beads were brought up to room

temperature, then, the Amplicon PCR plate was centrifuged at 1,000 x g at 20°C for one

minute, and the seal was removed. Next, the AMPure XP beads were vortexed for 30

seconds. 20µl of AMPure XP beads were pipetted to each well of the PCR plate. The plate

was shaken for 1800 rpm for 2 minutes using a MIDI plate. Next, the plate was incubated

at room temperature for 5 minutes, then placed on a magnetic stand for 2 minutes until the

supernatant cleared. The supernatant was removed and discarded. The beads were washed

with freshly prepared 80% ethanol by adding 200µl of ethanol to each well and left to

incubate for 30 seconds. The supernatant was then discarded. This process was repeated

one more time, and the excess ethanol removed using a fine-tipped pipette. Next, 52.5µl of

10 mM Tris pH 8.5 was added to each well. Again, the plate was shaken for 1800 rpm for

2 minutes until the beads were fully resuspended and left to incubate at room temperature

for 2 minutes. The plate was then placed on the magnetic stand for 2 minutes until the

supernatant cleared. 50µl of supernatant was transferred to a clean PCR plate.

6.3 PCR Index

Indexing was performed using the Nextera XT Set A kit (Illumina, San Diego, CA). 5µl

from each well was transferred to a new plate, and the remaining 45µl was stored for other

use. Index 1 primers were arranged horizontally, aligned with columns 1 through 12, while

Index primer 2 tubes were arranged vertically, aligned with rows A through H, and the

plate was placed into the TruSeq Index Plate Fixture. The following reaction was set up in

the PCR plate: 5µl Nextera XT Index Primer 1 (N707, N710-N715), 5µl Nextera XT Index

Primer 2 (S502-S511), 25µl 2x KAPA HiFi Hotstart Ready Mix, and 10µl of PCR Grade

water. The solution was gently mixed ten times by pipetting up and down. The plate with

Mircoseal ‘A’ was covered and centrifuged at 1000 x g at 20°C for one minute. Next, PCR

was performed on a thermal cycle using the following program: 95°C for 3 minutes, then

25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds. Then, 72°C

for 5 minutes and finally held at 4°C.

6.4 PCR Clean-up 2

The PCR plate was centrifuged at 280 x g at 20°C for one minute. The AMPure XP beads

were vortexed for 30 seconds to make sure they are homogeneous. Next, 56µl of AMPure

XP beads were pipetted into each well and gently mixed by pipetting up and down ten

times. The plate was left to incubate for 5 minutes at room temperature then the plate was

placed on a magnetic stand for two minutes until the supernatant cleared. The supernatant

was removed and discarded. The beads were washed with freshly prepared 80% ethanol by

adding 200µl of ethanol to each well and left to incubate for 30 seconds, and the supernatant

was then discarded. This process was repeated one more time, and the excess ethanol

removed using a fine-tipped pipette. Next, 27.5µl of 10 mM Tris pH 8.5 was added to each

well. Again, the plate was shaken for 1800 rpm for 2 minutes until the beads were fully

resuspended and left to incubate at room temperature for 2 minutes. The plate was then

placed on the magnetic stand for 2 minutes until the supernatant cleared. 25µl of

supernatant was transferred to a clean PCR plate.

6.5 Library Denaturing and MiSeq Sample Loading

A heat block with 1.7ml microcentrifuge tubes was set to 96°C. The MiSeq reagent

cartridge was removed from -15°C freezer and allowed to thaw at room temperature. An

ice bath was also set up. The final DNA was diluted with 10µl of 0.1 N NaOH in a

microcentrifuge tube, the sample solution was vortexed briefly and centrifuged at 280 x g

at 20°C for one minute. The solution was left to incubate at room temperature to allow the

DNA to denature into single strands. 5µl of 4nM pooled sample and 5µl of 0.1 N NaOH

were combined and allowed to rest at room temperature for five minutes. It was combined

with 990µl of pre-chilled HT1 (Hybridization buffer) to make 20pM of denatured library.

Next, 2µl of 10nM PhiX library and 3µl of 10nM Tris pH 8.5 to dilute the PhiX library to

4nM. The diluted sample was combined with 5µl of 0.2 N NaOH in a microcentrifuge tube

and vortexed. The solution was left to incubate at room temperature for 5 minutes to

denature the PhiX library into single strands. 390µl of pre-chilled HT1 and 210µl of

denatured PhiX library were combined to give a final concentration of 7 pM of PhiX

library. The solution was inverted several times and vortexed to mix the solution and placed

on ice. A 20% PhiX control spike-in was added along with 30µl of the denatured and

diluted PhiX control, and 570µl of the denatured and diluted amplicon library were

combined and placed on ice. The tubes were inverted twice and placed in an ice bath for

five minutes. The samples were loaded onto the MiSeq using a 600 cycle MiSeq v3 reagent

cartridges and sequenced. Upon completion the data from sequencing was hosted in the

cloud for download to be processed. All sequences were organized by sample, with their

barcodes removed and demultiplexed.

7. Microbiome Analysis

Once downloaded, the data from sequencing was analyzed using the Qiime 2 environment

(Qiime 2), with additional analysis done in the phyloseq package for R (phyloseq) and

Microsoft Excel (Microsoft Corporation, Redmond, WA). The DNA strains were paired

by their ends, denoised, and aligned by their metadata. The metadata was then compared

to the Greengene version 13.8 database (Greengene) for clustering and identification into

Operational Taxonomic Units (OTU) for phylogenetic identification. The OTUs were

quantified in a sample as alpha diversity and summarized into distinct species. Faith’s

phylogenetic distance was used to present equivalences or differences on the OTUs found

per sample. Beta diversity was also performed in the same way, with underweight and

weighted UniFrac tests plotted in distinct Principal Coordinate Analysis (PCoA) plots.

Shannon’s diversity was also used to identify the OTUs richness or evenness per sample.

Taxonomic proportions were performed to sort OTUs by their quantities per sample.

Chapter 5: Results

1. Demographics

Four subjects volunteered for this project. Demographic data collected included age,

gender, ethnicity, and periodontal remarks (Table 1). Subject ages ranged from 24-29. The

majority (75%) of the subjects were male and Caucasian and 1 out of 4 (25%) identified as

an Asian female. Three out of four subjects exhibited clinical periodontal conditions such

as altered passive eruption, thin, and very thin gingival biotype.

Demographic data Clear Aligners Conventional Control 1 Control 2 Orthodontics Age 24 23 25 29 Gender Male Female Male Male Ethnicity Caucasian Asian Caucasian Caucasian Altered Passive Very Periodontal Eruption Thin Gingival Thin None Remarks Biotype Gingival Biotype Table 1: Demographic information on study subjects. .

2. Clinical Examination

2.1 Supragingival Plaque

The number of surfaces with supragingival plaque for each subject is presented in Figure

1a. Each tooth was visually examined, and visible plaque was recorded for all 6 surfaces

and recorded. All participants performed personal oral hygiene, except Control 1 subject.

The total number of surfaces with supragingival plaque was comparable between clear

aligners, conventional orthodontics, and control 2. The data was also converted to show

the percentages of tooth surfaces that exhibited supragingival plaque (Figure 1b).

Number of Surfaces with Supragingival Plaque

77*

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure.1a: Absolute number of surfaces with supragingival plaque. *subject with no oral hygiene before the clinical exam

Percentage of Surfaces with Supragingival Plaque

53.47%*

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 1b: Percentage of surfaces with supragingival plaque. *subject with no oral hygiene before the clinical exam

2.2 Probing Depth

The average probing depth was taken from 6 surfaces of the tooth for each subject (in mm),

shown in Figure 2. All measurements are within a 0.37mm interval, ranging from 1.58-

1.95mm, of each other and are comparable with each other. Probing depth greater than

3mm was also recorded and shown in Figure 3a. The data was also converted to show the

percentages of periodontal pockets greater than 3mm (Figure 3b). The subject with clear

aligners exhibited no sites greater than 3mm while both the subject with conventional

orthodontics and control one exhibited 3 sites of periodontal pockets greater than 3mm.

Average Probing Depths (mm)

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 2: Average probing depths in mm

Number of Probing Depths > 3 mm

3 3

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 3a: Absolute number of probing depth greater than 3mm.

Percentage of Probing Depths > 3mm

2.08% 2.08%

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 3b: Percentage of probing depths greater than 3mm.

2.3 Bleeding on Probing

The number of bleeding sites after probing was recorded for each subject and presented in

Figure 4a. Bleeding sites ranged from 27 to 37 sites, with control 2 exhibiting the highest

number of bleeding sites. Control 2 exhibited a very thin gingival biotype. The data was

converted to show the percentages of sites that exhibited bleeding upon probing shown in

Figure 4b.

Number of Bleedings on Probing

37*

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 4a: Absolute number of bleedings on probing. * indicates participant with very thin gingival biotype

Percentage of Bleedings on probing

25.69%*

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 4b: Percentage of Bleedings on probing, * indicates participant with very thin gingival biotype

2.4 Clinical Attachment Level (CAL)

The average clinical attachment level for each subject is presented in Figure 5. Both subjects

treated with orthodontic appliances exhibited less attachment loss than the control subjects.

The total number of sites with gingival recession for each subject is presented in Figure 6a.

Both the control subjects exhibited an increased number of sites with gingival recession when

compared to the subjects who underwent orthodontic treatment. The data was also converted

to show the percentages of sites with gingival recession (Figure 6b).

Average Clinical Attachment Levels

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 5: Average clinical attachment level

Number of Sites with Gingival Recession 46

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 6a: Number of Sites with Gingival Recession

Percentage of Sites with Gingival Recession 31.94%

Clear Aligner Conventional Control 1 Control 2 Orthodontics Figure 6b: Percentage of sites with gingival recession

3. Microbiome Analysis

3.1 Quantitative Analysis

Table 2 presents the total number of Operational Taxonomic Units (OTUs) and total

number of species found in the samples. The highest number of OTUs and species are

found in the fixed orthodontics saliva 1 and swab samples while the lowest numbers are

found on the swab from the clear aligner. Figure 7a presents the total number of Operational

Taxonomic Units (OTUs) and total number of species found in the samples. The number

of pooled subgingival plaque bacteria and species is presented in Figure 7b. The clear

aligner shows the highest number of bacteria and species for the pooled subgingival

samples. The number of bacteria and species of bacteria found on the swabs of each

appliance is presented in Figure 7c. The swabs for the orthotic appliances show a higher

number of both bacteria and species for fixed orthodontics. The number of bacteria and

species of bacteria found in the saliva of each subject is presented in Figure 7d. The first saliva sample from the fixed orthodontic subject shows the highest number of bacteria and species at 185 and 75 respectively.

sample-source Total_OTUs Total_species clear-aligner-subgingival plaque 146 68 clear-aligner-saliva 127 60 clear-aligner-swab 70 29 clear-aligner-saliva2 121 53 fixed-orthodontic- subgingival plaque 98 46 fixed- orthodontic -saliva1 185 75 fixed- orthodontic swab 179 68 fixed- orthodontic saliva2* 119 53 control-nohygiene- subgingival plaque 128 60 control-nohygiene-saliva 103 51 control-nohygiene-saliva2 97 49 control-subgingival plaque 92 52 control-saliva1 163 71 control-saliva2 137 60

Table 2: Total number of OTUs and total number of species found in each sample. * indicates sample was taken at a different time.

Bacteria Count and Species Count per Sample 200 180 160 140 120 100 80 60 40 20 0

Figure 7a: Bacteria and species count for all samples. * indicates sample was collected at a different time.

Bacteria Count and Species per Sample for Pooled Plaque Samples

160

140

120

100

s -

Figure 7b: Pooled bacteria and species number in subgingival plaque samples.

Bacteria Count and Species Count per Swab 200 180 160 140 120 100 80 60 40 20 0 Clear aligner-swab Fixed orthodontics-swab Figure 7c: Bacteria and species number between Clear Aligners and Fixed Orthodontics with swabs.

Bacteria Count and Species Count per Sample 200 180 160 140 120 100 80 60 40 20 0

1 2 v a v v v a a

Figure 7d: Bacteria and Species number between Clear Aligners and Fixed Orthodontics in Saliva. *saliva was collected a different time.

3.2 Qualitative Analysis

Figure 8a demonstrates Faith’s phylogenetic distance, showing majority of the plots

between the range of 10-14. Three outliers are observed in the swab for the clear aligner

sample, the first saliva sample form fixed orthodontics, and the subgingival plaque sample

for the Control 2 subject, at 7, 16, and 8 respectively.

Figure 8a: Faith's phylogenetic distance between all samples.

Figure 8b shows the Unweighted Unifrac for all the samples collected. Most samples are

shown to be clustering per subject, with four plots not following this trend. The swab from

the clear aligner, the subgingival plaque from Control 1, the subgingival plaque from

Control 2, and the one saliva sample from fixed orthodontics so not cluster with the rest of

the samples.

Figure 8b: Unweighted Unifrac PCoA of all samples.

Weighted Unifrac is shown in Figure 8c for all the samples collected. Majority of the

samples are clustering. All subgingival plaque samples do not follow this trend and are

independent form the rest of the plots.

Figure 8c: Weighted Unifrac PCoA of all samples.

The Shannon Index shows majority of the plots clustering between 5.5 and 7, in Figure 8d.

Two plots do not follow this trend in the swab form the clear aligner and the subgingival

plaque from Control 2, at 4.5 and 5 respectively.

Figure 8d: Shannon Index of all samples.

The proportion of most prevalent bacteria genera and species is presented in Figure 9a. In

the majority of samples, the most prevalent genus is Streptococcus, except for the

subgingival plaque sample of the subject with fixed orthodontics and the control 2 subject,

which has the genus Capnocytophaga and the species Rothia dentocariosa as the most

prevalent bacteria, respectively.

Top 10 Species Taxonomic proportions 100% f Micrococcaceae;g Rothi a;s mucilaginosa 90% f Porphyromonadaceae;g 80% Porphyromonas;s 70% f Veillonellaceae;g Veillon ella;s dispar 60% f Pasteurellaceae;g Haem 50% ophilus;s parainfluenzae 40% f Flavobacteriaceae;g Cap nocytophaga;s 30% f Actinomycetaceae;g Act 20% inomyces;s 10% f Veillonellaceae;g Veillon ella;s parvula 0%

1 2 1 2 2

1 1 2

f Neisseriaceae;g Neisseri Swab Swab Swab Swab a; Plaque Plaque Plaque Plaque Saliva Saliva Saliva Saliva

Saliva Saliva Saliva Saliva f Micrococcaceae;g Rothi a;s dentocariosa f Streptococcaceae;g Stre Subgingival Subgingival Subgingival Subgingival ptococcus;

Figure 9a: Top 10 Taxonomic Species between all samples.

The top 10 bacteria proportions for the subject with clear aligners is shown in Figure 9b,

the genus Streptococcus makes up the greatest proportion in all samples except for the

subgingival plaque sample. The subgingival plaque also shows a greater proportion of the

genus Neisseria, the species Rothia dentocariosa, the genus Actinomyces, and the genus

Capnocytophaga. Both saliva samples show elevated proportions of Actinobacillus

parahaemolyticus and the genus Haemophilus. The swab sample show elevated

proportions of the genus Capnocytophaga and Porphyromonas.

Top 10 Bacteria prevalent on samples from subject with a Clear aligner 100%

f__Gemellaceae; ; 90%

f__Porphyromonadaceae;g Porphyro 80% monas;s f__Pasteurellaceae;g Haemophilus;s_ 70% _ f__Flavobacteriaceae;g Capnocytoph 60% aga;s f__Pasteurellaceae;g Haemophilus;s_ 50% _parainfluenzae

f__Actinomycetaceae;g Actinomyces; 40% s

f__Micrococcaceae;g Rothia;s dent 30% ocariosa f__Pasteurellaceae;g Actinobacillus;s 20% parahaemolyticus f__Neisseriaceae;g Neisseria;s 10%

f__Streptococcaceae;g Streptococcus 0% ; Subgingival Saliva 1 Swab Saliva 2 Plaque Figure 9b: Top 10 Taxonomic Species between the samples from the subject treated with a Clear Aligner.

The top 10 bacteria proportions for the subject with fixed orthodontics is shown in Figure

9c, the genus Streptococcus makes up the greatest proportion in all samples except for the

subgingival plaque sample. The subgingival plaque shows a greater proportion of the genus

Capnocytophaga, the species Veillonella dispar, the genus Lautropia, the genus Neisseria,

the genus Actinomyces, and the genus Fusobacterium. The saliva samples show elevated

proprtions of Veillonella parvula.

Top 10 Bacteria prevalent on samples from subject with Fixed Orthodontics

100%

f__Fusobacteriaceae;g Fusobacteriu 90% m;s

f__Porphyromonadaceae;g Porphyro 80% monas;s

f__Actinomycetaceae;g Actinomyces; 70% s f__Neisseriaceae;g Neisseria; 60% f__Pasteurellaceae;g Haemophilus;s_ 50% _parainfluenzae f__Veillonellaceae;g Veillonella;s p 40% arvula

f__Burkholderiaceae;g Lautropia;s 30%

f__Veillonellaceae;g Veillonella;s di 20% spar

f__Flavobacteriaceae;g Capnocytoph 10% aga;s

f__Streptococcaceae;g Streptococcus 0% ; Subgingival Saliva 1 Swab Saliva 2 Plaque Figure 9c: Top 10 Taxonomic Species between the samples from the subject treated with Fixed Orthodontics.

Figure 9d, shows a comparison between the top 10 bacteria proportions for orthodontic appliances. The genus Streptococcus is the most prominent between the two subjects. The subject with clear aligners exhibits higher proportions of the genus Neisseria, the genus

Prophyromonas, and the genus Capnocytophaga. While the subject with fixed orthodontics exhibits higher proportions of the species Veillonella dispar and the genus Actinomyces.

Top 10 Bacteria prevalent on Orthodontic Devices 100% f__Fusobacteriaceae;g Fusobacteriu 90% m;s f__Aerococcaceae;g Abiotrophia;s 80% f__Actinomycetaceae;g Actinomyces; 70% s f__Flavobacteriaceae;g Capnocytoph 60% aga;s f__Gemellaceae; ; 50% f__Veillonellaceae;g Veillonella;s di 40% spar f__Porphyromonadaceae;g Porphyro 30% monas;s f__Pasteurellaceae;g Haemophilus;s_ 20% _parainfluenzae f__Neisseriaceae;g Neisseria;s 10% f__Streptococcaceae;g Streptococcus ; 0% Clear aligner Fixed Figure 9d: Top 10 Taxonomic Species found colonizing Orthodontic Devices.

Figure 9e, shows a comparison between the top 10 bacteria proportions for subgingival plaque samples. Both controls exhibit higher proportions of the species Rothia dentocariosa. The subject with clear aligners exhibits higher proportion of the genus Streptococcus, the genus Neisseria, the genus Actinomyces, and the genus Neisseria. The subject with fixed orthodontics exhibits higher proportions of the genus Capnocytophaga, the species Veillonella dispar, the genus Lautropia, the genus Aggregatibacter, and the genus Fusobacterium. The control 2 subject also exhibits higher proportion of the genus Aggregatibacter and the genus Fusobacterium.

Top 10 Bacteria prevalent in the subgingival Plaque

100% f__Fusobacteriaceae;g Fusobacteriu 90% m;s f__Pasteurellaceae;g Haemophilus;s_ 80% _parainfluenzae f__Pasteurellaceae;g Aggregatibacter 70% ;s f__Burkholderiaceae;g Lautropia;s 60% f__Veillonellaceae;g Veillonella;s di 50% spar f__Neisseriaceae;g Neisseria;s 40% f__Flavobacteriaceae;g Capnocytoph 30% aga;s f__Actinomycetaceae;g Actinomyces; 20% s f__Streptococcaceae;g Streptococcus 10% ; f__Micrococcaceae;g Rothia;s dent 0% ocariosa Clear aligner Fixed Control no Oral Control Hygiene Figure 9e: Top 10 Taxonomic Species between Subgingival Plaque Samples.

Chapter 6: Discussion

The results of this study were based on the clinical data collection and microbiologic samples from four healthy volunteers. These four individuals were chosen to test our hypothesis could be tested within the limited scope of a small sample size.

The subjects involved in this study were within the same age range. All subjects were male and of Caucasian descent except for the subject with conventional orthodontics, as shown in Table

1. Due to the differences in the subject with conventional orthodontics from the other subjects, there may be factors that influence her clinical and microbiological results. All the subjects received professional oral hygiene treatments after the examination of the periodontium one week before the sample collection, which aimed to establish an equal time frame for the samples collected for microbiome analysis.

All volunteers presented good clinical periodontal conditions, with their status ranging from a healthy periodontium to a periodontium with few areas of loss of attachment, and probing depths and bleedings on probing that may relate to some very mild degree of inflammation of the periodontium.

The subject who underwent conventional orthodontic treatment exhibited a normal periodontium, while the subject who underwent clear aligner treatment exhibited altered passive eruption.

Altered passive eruption (APE) is defined as a clinical periodontium where “the gingival margin in the adult is located incisal to the cervical convexity of the crown and removed from the cementoenamel junction of the tooth” (Alpiste-Illueca, 2011). AEP leads to an excessive gingival overlap at the crown, increasing probing depth but not increasing the clinical attachment level, creating pseudo-pockets. APE can also adversely affect periodontal health, especially in patients

that have a higher predisposition to periodontitis due to its hampering of oral hygiene (Alpiste-

Illueca, 2011).

The volunteers were not instructed to perform their personal oral hygiene before examination and sample collection. However, both subjects under orthodontic treatment did perform their oral hygiene before the clinical examination. Control 1 exhibited a thin gingival biotype and did not perform any personal oral hygiene before the clinical examination. Control 2 exhibited a very thin gingival biotype and performed personal oral hygiene before the clinical examination.

Gingival biotype is the thickness of the gingiva in the faciopalatal region (Rana, 2015), with many existing classifications. These classification range from very thick, thick, thin, and very thin biotypes (Maynard and Wilson, 1980). Previous studies suggest that some biotypes are more vulnerable to disease; for instance, as thin and very thin gingival biotypes have less soft tissue, they, therefore, have a higher susceptibility to periodontitis (Claffey and Shanley, 1986). As a consequence of their gingival biotype, both controls presented clinical signs of controlled periodontitis, due to their loss of attachment, both placed at stage I and II for this disease (Figures

5, 6a and 6b) (Papapanou et al., 2018).

The amount of detectable supragingival plaque for all the subjects showed a percentage of the surfaces with detectable dental plaque ranging from 21 to 30%, a satisfactory result. Control 1 was an exception with over 50% of surfaces with plaque (Sreenivasan and Prasad, 2017). This individual did not perform personal oral hygiene prior to sampling. (Figure 1a and 1b). It is also important to note that all subjects are part of a population that is educated on oral hygiene techniques.

Of the three subjects who performed personal oral hygiene, the subject with conventional orthodontics had the highest amount of supragingival plaque, with13 more sites with plaque or a

9.03% increase when compared to the subject with clear aligners. Comparing control two and the volunteer with clear aligners, showed a 1.39% increase of plaque in the subject with clear aligners, which is the equivalent of two additional surfaces with detectable plaque. These results suggest that conventional orthodontics may have a direct impact on dental plaque accumulation, possibly due to the obstacle that the fixed orthodontic appliances represent for personal oral hygiene.

Similar results were noticed on the microbiological data, (Figure 7c), indicating that the swabs from the fixed orthodontics’ appliances had a higher OTU (bacteria) count than the clear aligner. Our results are consistent with previous studies by both Levrini et al. and Karkhanechi et al., who found an increased plaque index scores on participants treated with conventional orthodontics (Levrini et al., 2015) (Karkhanechi et al., 2013).

The average probing depth of all participants ranged from 1.58mm to 1.95mm, with all subjects have a relatively similar probing depth (Figure 2). Probing depths greater than 3mm

(Figure 3a) were also evaluated. A pocket depth greater than 3 mm is often associated with active periodontitis, which is linked to clinical attachment loss and bone loss (Tonetti et al., 2018). Along with the adverse effects previously described, a pocket deeper than 3 mm has also been shown to create a more favorable environment for anaerobe proliferation (Greenstein, 1984).

It should be noted that the subject with clear aligner exhibited altered passive eruption at the time of the clinical exam, however, this subject presented no sites greater than 3mm. The subject with conventional orthodontics had three sites with a probing depth greater than 3mm, suggesting that conventional orthodontics could lead to local vulnerabilities or become a local risk factor for periodontal breakdown. The findings of Levrini et al. (2015) and Karkhanechi et al.

(2013) again reflect our results. Karkhanechi et al. noticed a significant increase in probing depth, starting at the six-week reexamination, for the 22 participants with conventional orthodontics

(Karkhanechi et al., 2013).

Similar results for bleeding on probing were shown when comparing subjects, except for control 2, which is considerably higher and exhibited a very thin biotype at the time of examination

(Figure 4a and 4b). It should be noted that generally thin and very thin gingival biotypes are more vulnerable to tissue loss and damage (Shah et al., 2015). While the second control subject had the highest number of bleeding sites, he also had the second lowest subgingival OTU count, shown in figure 7b. It should be noted that control 2 exhibited a very thin gingival biotype. Suggesting that a thin gingival biotype can present signs of gingival inflammation even when exposed to a lower number of bacteria, if pathogenic.

For the three other subjects, bleeding sites ranged between 18.75 to 20.14%, which is equivalent to two more bleeding sites across the subjects. When comparing the subject with clear aligners to the subject with conventional orthodontics, two fewer bleeding sites were identified in the subject with clear aligners. Bleeding on probing has long been used to determine periodontal health and the possible progression of disease (Checchi et al., 2009). Both Levrini et al., 2015 and

Karkhanechi et al., 2013 also found significant increases in bleeding on probing for participants with conventional orthodontics.

Both control subjects demonstrated a higher average clinical attachment level (CAL) when compared with the subjects who underwent orthodontic treatment. Two volunteers presented CAL values between 2.77 and 2.98mm, which is considered within the stage 2 of periodontitis

(Papapanou et al., 2018) (Figure 5).

The subject with clear aligner, who exhibits altered passive eruption, did not display any site with attachment loss. Both controls exhibited the greater CAL (Figure 5) and many sites with gingival recession (Figures 6a and 6b), which can be partially explained by the fact that both controls exhibited thin and very thin gingival biotypes at the time of examination. The subject with clear aligners showed no sites with gingival recession, which is expected with altered passive eruption (Pulgaonkar and Chitra, 2015).

Gingival recession is typical in adults, and it is prevalent in 20%, according to a previous study (Handelman et al., 2018). Apical migration of the gingival tissue and the gradual exposure of the root structure to the external environment has been linked to orthodontic treatment (Jati et al., 2016). It should be noted that the subjects who underwent orthodontic treatment had the fewest sites with gingival recession, with clear aligners having zero sites.

The total number of Operational Taxonomic Units (OTUs) and the total number of bacterial species found in each sample after the microbiome are shown in Table 2. OTUs are a genetic sequence that can be identified as bacterial species based on their phylogenetic and taxonomic relationships when they are compared to a database of known bacterial DNA sequences, establishing the alpha diversity of a microbiome (Willis, 2019).

After all DNA sequences that can result in the hyper variant 16s rRNAs are identified at the sequencing, the redundancies are filtered from the OTU count, resulting in the number of species present in each sample. The same relationships between the number of OTUs and the number of species as a bar graph are shown in Figure 7a. The saliva and swab samples for the participant with fixed orthodontics have the highest number of bacteria; this could be due to a higher amount of bacterial accumulation around the brackets and wires.

This subject was the only volunteer to sample saliva at two different times. The quantity variation between these two saliva samples collections for this subject with conventional orthodontics could be due to factors not directly related to the oral cavity or the use of a fixed orthodontic device, which are harder to account, since the samples were collected at different times.

The lowest number of bacteria was seen with the clear aligners (Figure 7c). This could be partially explained by how much easier it is to perform hygiene of the clear aligner, as well as the patient’s oral hygiene during use of aligners. Since clear aligners can be removed more easily and the frequency the aligners were replaced during orthodontic treatment. However, the number of bacteria in the subgingival plaque was the highest when compared to the other participants; this could partially be explained by the ease with which bacteria can colonize an individual who experiences altered passive eruption (APE), due to the fact that APE is usually associated with deeper subgingival areas, called pseudopockets (Alpiste-Illueca, 2011).

Another interesting comparison can be made when looking at the number of bacteria seen in between the control that performed oral hygiene before and the control who did not perform oral hygiene before sample collection. The participant who did not perform oral hygiene showed a lower number of bacteria, suggesting that personal oral hygiene does not alter the oral microbiome in the very short turn.

When comparing the number of bacteria and the number of species in the subgingival plaque (Figure 7b) it is notable that the subject with clear aligners has the highest amount than the one with fixed orthodontics. This result does not align with the data presented by Guo et al., who showed higher amounts of OTUs for subgingival plaque samples (Guo et al., 2018). However, when comparing the clinical measurement, such as clinical attachment loss and probing depth, the

subject with clear aligners exhibits similar results for clinical attachment level, however, better probing depths compared to both controls, as this volunteer is the only subject without a probing depth higher than 3 mm (Figures 3a and 3b). This suggests that the bacteria present are not pathogenically affecting the tissue. The volunteer with clear aligners also exhibited healthier probing depth measurements compared to the subject with fixed orthodontics (Figures 3a and 3b).

These findings suggest that the anatomy presented by the controls, thin and very thin biotypes, is associated with more sites presenting attachment loss.

The swabs from clear aligners and fixed orthodontics shows a drastic difference between the two samples (figure 7c). The clear aligner had 60% fewer OTUs and 57% fewer bacterial species present when compared to the fixed orthodontics’ appliances.

The amount of OTUs and species in saliva for each participant are shown in Figure 7d; two saliva samples were requested for the core facility to guarantee robust levels of DNA being present in these samples. The amount of OTUs found in the samples from the subject with clear aligners was lower than the amount found in the subject with fixed orthodontics. The saliva samples for the participant with fixed orthodontics showed the greatest difference between the two samples; this could partially be explained by the samples being collected at a different time due to the insufficient volume of the first sample.

Faith’s phylogenetic diversity, which measures species’ richness within a sample is shown in Figure 8a. Each plot represents the average distances within the branches of the phylogenetic tree of all bacterial species in a particular microbiome; the higher is the average of plots within the matrix, the more diverse are the species in the microbiome (Faith and Baker, 2007). Most of the plots are in the range of 10 to 14; however, there are some outliers.

The first outlier is the swab from the clear aligner is around 7, suggesting a less diverse bacteria population. This may suggest that potential periodontal pathogens can easily colonize clear aligners however this needs to be explored with a longitudinal study.

Another outlier shown in Faith’s (Figure 8a) is the first saliva sample from the subject with fixed orthodontics, which exhibits a distinct bacterial population, even from the second sample.

This observation should be further investigated.

The difference between each microbiome was compared by two beta-diversity tests: weighted and unweighted unifrac. Weighted unifrac shows the diversity between samples based on the relative abundance of species. In contrast, unweighted unifrac shows the diversity based on the absolute number and presence or absence of species shared between samples (Lozupone et al.,

2011).

In the unweighted unifrac data for all samples; the closer the plots are to each other, the less diverse the microbiome represented by the plots, as shown in Figure 8b. The majority of samples cluster per volunteer, indicating similarities between the microbiomes of the same individual. This does not hold true for the saliva sample from the subject with fixed orthodontics.

This could partially be explained by the fact that the saliva was collected at different times. It may also suggest a temporal shift in the sample’s microbiome between the two collections.

When comparing the data from the weighted unifrac graph, presented in Figure 8c, most of the plots forms a large cluster, suggesting less diversity when the relative abundance of the bacteria species is accounted. However, the subgingival plaque samples presented more diversity from the other plots, which can be explained by environmental differences from the subgingival region, leading to microbial diversity. Additionally, these subgingival plaque samples are the only samples demonstrating less or no predominance of the genus Streptococcus when compared to

clear aligners (Figure 9e). The genus Streptococcus is a typically a commensal genus of bacteria but has some species like Streptococcus mutans that are associated with dental caries (Forssten et al., 2010).

The weighted unifrac of the swab from the clear aligner, clustered with the majority of the other samples. This was in contrast to what was seen previously, at Faith’s Phylogenetic distances and unweighted unifrac. This could be due to the predominance of the genus Streptococcus that mitigates the impact of the other bacteria in this particular microbiome, as shown by Figure 9c.

Streptococcus mutans are associated with dental caries (Forssten et al., 2010). Our results reflect the findings of Guo et al., who showed little distance between plots in weighted unifrac (Guo et al., 2018).

Lastly, it is possible to compare the predominance of bacteria between samples through the use of the Shannon Index (Figure 8d). This analysis measures the predominance of bacterial species within the microbiome in terms of richness or evenness (Morris et al., 2014). The results show that the overall richness of all samples was very high (Figure8d). Similar to Faith’s phylogenetic diversity, the Shannon Index presents similar findings with most of the plots ranging from 5.5 to 6.5, suggesting that many bacterial species share dominancy, however, there were a few outliers.

The first outlier observed is the swab of the clear aligner, around 4.5, suggesting less dominant bacterial species. This is further demonstrated in Figure 9a which shows a lower proportion of the genus Streptococcus and higher proportions of the bacteria and the genera

Neisseria and Porphyromona. Streptococcus mutans are associated with dental caries (Forssten et al., 2010). While Neisseria is generally associated with health and exhibits low pathogenicity, it does include a periodontal pathogens species, Neisseria mucosa (Awdisho and Bermudez, 2016;

C. Chen et al., 2018). The genus Porphyromona has a well-known periodontal pathogen in

Porphyromona gingivalis (Rafiei et al., 2017)This could be partially due to fewer bacteria being found colonizing the clear aligner swab, as shown in figure 7c, and as a result of the shorter colonization time for new bacteria as the aligners are replaced more frequently.

The second outlier is the subgingival sample from Control 2 (higher levels of clinical attachment loss) which has a value of around 5. Also, suggesting a less dominant bacteria population in this sample. Figure 9a shows the highest proportion as the bacteria Rothia dentocariosa along whit greater proportions of the genus Actinomyces. Rothia dentocariosa is linked to tooth decay and Actinomyces odontolyticus, Actinomyces meyeri, Actinomyces iseaelii are associated with periodontal disease (Fridman et al., 2016; Vielkind et al., 2015). Both samples are more closely related to each other than the other samples regarding phylogenetic diversity, as seen in figure 8a.

The top 10 species of bacteria found in each sample is shown in Figure 9a. The genus

Streptococcus is the most common form of bacteria present except in the subgingival plaque sample from the conventional orthodontics and both control subjects; confirming the lack of clustering in the weighted unifrac for these samples, as shown in Figure 8c.

One interesting result showed that the swab for the subject with clear aligners has a higher proportion of genus Porphyromona; one well-known periodontal pathogen is Porphyromona gingivalis (Rafiei et al., 2017). This genus is also more prevalent in the saliva of Control 2, who performed oral hygiene before sample collection and who also exhibited increased attachment loss.

The subject with fixed orthodontics showed the highest level of genus Capnocytophaga in the subgingival sample. Capnocytophaga is usually a commensal bacteria but has some species associated with gingivitis, Capnocytophaga gingivalis (Pudakalkatti et al., 2016). Also, higher

proportions of Veillonella dispar were found in the saliva samples from the subject with fixed orthodontics (Figure 9a), which has been linked to oral infections (Mashima et al., 2015). Both the control subjects exhibited a higher proportion of Rothia dentocariosa, which is linked to tooth decay and periodontal disease (Fridman et al., 2016).

The proportions of bacteria found in the saliva samples from each subject, was also investigated (Figure 9a). The genus Streptococcus had the highest proportions, similar to all the samples except the subgingival plaque samples. Streptococcus mutans is associated with dental caries (Forssten et al., 2010).

The control subject who did not perform personal oral hygiene (Control 1) exhibited higher proportions of genus Neisseria and the species Veillonella parvula. Veillonella parvula has been linked to periodontitis (Mashima et al., 2015). While Neisseria is generally associated with health and exhibit low pathogenicity, it does include a periodontal pathogens species, Neisseria mucosa

(Awdisho and Bermudez, 2016; C. Chen et al., 2018).

Potential genera with periodontal pathogens were found at low proportions, including

Porphyromona and Actinomyces, in both subjects treated with orthodontic appliances.

Porphyromona gingivalis and several species of Actinomyces including; Actinomyces odontolyticus, Actinomyces meyeri, Actinomyces iseaelii have been linked to periodontal disease

(Rafiei et al., 2017; Vielkind et al., 2015).

Higher proportions of Neisseria were found in the subject treated with clear aligners, supporting the results of Wang. However, unlike Wang et al., the results did not show the higher numbers of potential periodontal pathogens (Porphyromona and Actinomyces) in the subject treated with clear aligners when both saliva samples were averaged. Additionally, Wang et al.

found periodontal pathogens on both groups treated with orthodontic appliances (Wang et al.,

2019).

The ten most abundant bacteria from the subject with the clear aligner are shown in Figure

9b. The subgingival plaque sample showed higher proportions in the genera Neisseria,

Capnocytophaga, Actinomyces, as well as the bacteria Rothia dentocariosa. Both genus Neisseria and Capnocytophaga are generally considered commensal, but have species that have been linked to periodontal disease, Neisseria mucosa and Capnocytophaga gingivalis (Awdisho and

Bermudez, 2016; Pudakalkatti et al., 2016).The genus Actinomyces also has species associated with periodontal disease (Actinomyces odontolyticus, Actinomyces meyeri, Actinomyces iseaelii), as previously mentioned (Vielkind et al., 2015). Additionally, Rothia dentocariosa has a pathogenic role as previously described (Fridman et al., 2016). The saliva and swab samples also contained Actinobacillus parahemolyticus and the genus Porphyromona, which has a well-known periodontal pathogen, Porphyromona gingivalis (Rafiei et al., 2017). Actinobacillus parahemolyticus has been shown to be elevated in saliva of subjects with Rheumatoid arthritis (B.

Chen et al., 2018; Drago et al., 2019). In this sample, the genus Porphyromona was the third most abundant.

The study also looked at the prevalence of bacteria found only in the samples from the subject using fixed orthodontics (Figure 9c). The subgingival plaque sample showed a higher prevalence of genus Capnocytophaga and Lautropia, which both have species that have been linked to periodontal disease, Capnocytophaga gingivalis and Lautropia mirabilis (Pudakalkatti et al., 2016; Rossmann et al., 1998). An elevated count of Veillonella dispar was observed in the subgingival sample and Veillonella parvula in the saliva sample. Veillonella parvula and

Veillonella dispar have been linked to periodontal disease and to other oral infections, respectively

(Mashima et al., 2015). Additionally, the genus Fusobacterium, which has a well know periodontal pathogen in Fusobacterium nucleatum, had a higher prevalence in the subgingival plaque sample of the subject with conventional orthodontics, when compared to the swab and saliva (Han, 2015).

When comparing the bacteria prevalent in the swabs from the two orthodontic devices,

(Figure 9d), the clear aligners has a higher prevalence of bacteria from the genera Neisseria,

Porphyromonas, and Capnocytophaga. Although some species in the genera Neisseria and

Capnocytophaga are associated with periodontal disease (Neisseria mucosa, Capnocytophaga gingivalis) they are generally associated with health and exhibit low pathogenicity (Awdisho and

Bermudez, 2016; Pudakalkatti et al., 2016). The genus Porphyromona also possesses a well know periodontal pathogen in Porphyromona gingivalis (Rafiei et al., 2017).The fixed orthodontic appliances had a higher prevalence of bacteria from the genera Actinomyces and Abiotrophia and species Haemophilus parainfluenzae and Veillonella dispar. The genus Abiotrophia contains a

specie, Abiotrophia adiancens that has been linked to periodontal disease and can lead to periodontal disease (Mikkelsen et al., 2000). Haemophilus parainfluenzae has been linked to oral biofilm formation and can cause opportunistic infections such as periodontal infections (Pang and

Swords, 2017).

Lastly, a comparison was made between the ten most abundant bacteria found exclusively in the subgingival plaque from each subject (Figure 9e). All subjects show varying prevalence of genus Fusobacterium, which has a well know periodontal pathogen in Fusobacterium nucleatum

(Han, 2015) with the highest amount in the subject with conventional orthodontics. Both control subjects showed higher proportions of Rothia dentocariosa, which is associated with periodontal disease. This could partially be explained by the fact that both control subjects exhibited higher amounts of attachment loss. The subjects who underwent orthodontic treatment also presented an

elevated prevalence of the genus Neisseria when compared to the control subjects. Interestingly, both the subject with fixed orthodontics and Control 2, who exhibited the highest level of attachment loss, showed the highest prevalence of the genus Aggregatibacter. One of the species of Aggregatibacter, Aggregatibacter actinomycetemcomitans, has been linked to aggressive periodontitis (Fine et al., 2019). These results support the findings of Leverini et al., (2015) who found one subject with fixed orthodontics had Aggregatibacter actinomycetemcomitan (Levrini et al., 2015). When comparing our data from the subgingival plaque samples to the data collected from the third month of treatment for subjects undergoing clear aligner treatment from Guo et al.,

(2018) there are some similarities. Both data sets exhibit higher amounts of the genera Rothia,

Actinomyces, Fusobacterium, and Neisseria (Guo et al., 2018).

Chapter 7: Conclusion

Overall, all the subjects presented good periodontal conditions with their status ranging from a healthy periodontium to a periodontium with few areas of attachment loss, it is important to address that all volunteers were individuals with a higher awareness on oral health and methods for effective oral hygiene.

Our results indicated improved clinical parameters for the subject that underwent clear aligner treatment. The amount of supragingival plaque increased 9.03% in the subject with conventional orthodontics. Suggesting that conventional orthodontics had a direct impact on dental plaque accumulation.

Furthermore, both subjects have equivalent number of sites with bleeding on probing, however, the subject with clear aligners had zero sites that measured greater than 3mm or with attachment loss verified by gingival recession, while that subject with conventional orthodontics had three sites with probing depths greater than 3 mm and three sites with gingival recession.

Suggesting that clear aligners provided a healthier periodontal environment when compared to fixed orthodontics, which may be related with site vulnerability and a greater chance for the onset of periodontitis.

When comparing the oral microbiome between the two appliances, our study found fewer bacteria on the swab from the clear aligners. The subject with fixed orthodontics had the highest bacteria count in the swab from the appliances and one of the saliva samples provided.

Overall, all samples presented high richness. Interestingly, the swab for clear aligners exhibited higher proportions of genus Porphyromona. The subgingival plaque for the subject with clear aligners exhibited higher proportions of the genera Neisseria, Capnocytophaga,

Actinomyces, as well as the bacteria Rothia dentocariosa. The subgingival plaque sample for the

subject with conventional orthodontics presented higher proportions of genera Capnocytophaga,

Lautropia, Fusobacterium and species Veillonella parvula and Veillonella dispar. All of which has been linked to tooth decay or periodontal disease. Furthermore, the subject with conventional orthodontics and control two, who exhibited the highest level of attachment loss, present the highest proportion of the genus Aggregatibacter, which has been associated with aggressive periodontitis. These results indicate that both clear aligners and fixed orthodontics influence the oral microbiome and can lead to microenvinromental changes that favor colonization with periodontal pathogens.

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