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The Effect of Vibratory Stimulation and Tooth Movement on Gingival Crevicular Fluid Biomarkers

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Title:
The Effect of Vibratory Stimulation and Tooth Movement on Gingival Crevicular Fluid Biomarkers
Creator:
Mcneight, Angela Marie
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (104 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Dental Sciences
Dentistry
Committee Chair:
RODY,WELLINGTON JOSE,JR
Committee Co-Chair:
WHEELER,TIMOTHY T
Committee Members:
MCGORRAY,SUSAN P
WALLET,SHANNON MARGARET
Graduation Date:
4/30/2016

Subjects

Subjects / Keywords:
Biological markers ( jstor )
Bones ( jstor )
Gingival crevicular fluid ( jstor )
Orthodontics ( jstor )
Orthods ( jstor )
Osteoclasts ( jstor )
Rats ( jstor )
Statistical median ( jstor )
Tooth movement ( jstor )
Vibration ( jstor )
Dentistry -- Dissertations, Academic -- UF
acceledent -- crevicular -- fluid -- gcf -- gingival -- movement -- orthodontics -- tooth -- vibration
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Dental Sciences thesis, M.S.

Notes

Abstract:
In the field of orthodontics, gingival crevicular fluid (GCF) is one of the main targets for assessing biomarkers. The term GCF describes the fluid that escapes from between the tooth surface and the epithelial lining of the periodontal ligament (PDL). There are over 100 regulatory proteins that can be detected in GCF, and they can be categorized on the basis of their biological significance such as bone resorption or formation. OBJECTIVES: To investigate the effect of vibratory stimulation (AcceleDent(R) Aura) on the expression of biomarkers in GCF using a human tooth movement model with clear aligners. METHODS: Prospective, single-center, randomized controlled crossover study comparing an active vibration device to a sham device. Participants received 6 Zendura(R) plastic aligners to move a target tooth 1.98 mm over 12 weeks. Patients were crossed over to the opposite device at the 4th aligner. GCF was collected at T0 (baseline 1), T1 (baseline 2), T2 (week 1), T3 (week 7, crossover) and T4 (week 12). 11 biomarker levels quantified by multiplex bead immunoassay. A mixed model analysis was used to compare the effects of active versus sham device on GCF composition. RESULTS: 16 males and 23 females completed the protocol. No statistically significant differences were found between the biomarker levels of the Sham-Active (SA Group) and Active-Sham (AS Group). The results of each group were then combined and biomarker concentrations were compared to tooth movement. There was a negative correlation between the baseline level of IL-6 and the total amount of tooth movement. A negative correlation was seen between the baseline levels of MMP-9 and tooth movement between weeks 1-2 followed by a positive correlation between the concentration of MMP-9 at T4 and the amount of tooth movement over the course of the study. CONCLUSIONS: The results suggest that vibration does not influence the secretion of GCF biomarkers; nevertheless, tooth movement with clear aligners seems to alter the proteomic composition of GCF. This study was registered on https://clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT01962012). It was approved by the Western Institutional Review Board (WIRB #20131856). This study was funded by OrthoAccel(R) Technologies. ( en )
General Note:
In the series University of Florida Digital Collections.
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Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2016.
Local:
Adviser: RODY,WELLINGTON JOSE,JR.
Local:
Co-adviser: WHEELER,TIMOTHY T.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2017-05-31
Statement of Responsibility:
by Angela Marie Mcneight.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Angela Marie Mcneight. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
5/31/2017
Classification:
LD1780 2016 ( lcc )

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THE EFFECT OF VIBRATORY STIMULATION AND TOOTH MOVEMENT ON GINGIVAL CREVICULAR FLUID BIOMARKERS By A NGELA M. MCNEIGHT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQ UIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016

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2016 Angela M. McNeight

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To my family for their endless encouragement support and love

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4 ACKNOWLEDGMENTS Thank you to my ment ors, Dr. Wellington Rody, Dr. Timothy Wheeler, Dr. Susan P. McGorray and Dr. Shannon Wallet for your direction and guidance throughout this process Thank you to my program director and chair, Dr. Calogero Dolce, for your tireless support thr oughout these past three years. Thank you to my family and friends for your love and patience throughout dental school and residency and for always encouraging me to follow my dreams. I could not have done it without you.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 The Role of Vibration in Tooth Movement ................................ ............................... 13 GCF Biomarkers of Tooth Movement ................................ ................................ ..... 14 Osteoclastogenesis Related Factors (RANKL/OPG) ................................ ........ 15 Biomarkers of Inflammation ................................ ................................ .............. 16 Extracellular Matrix Degrad ation Factors (MMPs) ................................ ............ 17 Markers of Bone Matrix Degradation and Osteoclast Recruitment ................... 18 Specific Objectives or Hypotheses ................................ ................................ ......... 21 2 MATERIALS AND METHODS ................................ ................................ ................ 23 Trial Design ................................ ................................ ................................ ............. 23 Participants, Eligibility Criteria, and Setting ................................ ............................. 23 Interventions ................................ ................................ ................................ ........... 23 Outcomes ................................ ................................ ................................ ............... 25 Clinical Tooth Movement ................................ ................................ .................. 25 GCF Collection ................................ ................................ ................................ 26 Biomarker Analysis ................................ ................................ ........................... 27 Sample Size Calculation ................................ ................................ ......................... 28 Randomization ................................ ................................ ................................ ........ 28 Blinding ................................ ................................ ................................ ................... 29 Statistical Analysis ................................ ................................ ................................ .. 29 3 RESULTS ................................ ................................ ................................ ............... 34 Participant Flow ................................ ................................ ................................ ...... 34 Baseline Data ................................ ................................ ................................ ......... 34 Numbers Analyzed, Outcomes, and Estimation ................................ ...................... 34 Harms ................................ ................................ ................................ ..................... 38 4 DISCUSSION ................................ ................................ ................................ ......... 58

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6 Main Findings ................................ ................................ ................................ ......... 58 Limitations ................................ ................................ ................................ ............... 62 Generalizability ................................ ................................ ................................ ....... 64 5 CONCLUSIONS ................................ ................................ ................................ ..... 66 APPENDIX A BIOMARKER DATA FROM ACTIVE SHAM GROUP ................................ ............. 67 B BIOMARKER DATA FROM SHAM ACTIVE GROUP ................................ ............. 81 LIST OF REFERENCES ................................ ................................ ............................... 95 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 103

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7 LIST OF TABLES Table page 1 1 A selection of human GCF biomarkers and their clinical relevance to orthodontics (Adapted fr om Rody et al. 76 ) ................................ .......................... 22 2 1 Outline of Inclusion and Exclusion Criteria ................................ ......................... 31 3 1 Demographics of Final Population (n=39) ................................ .......................... 39 3 2 Biomarker Levels (in pg/mL) AS Group Summary Statistics .............................. 40 3 3 Biomarker Levels (in pg/mL) SA Group Summary Statistics .............................. 43 3 4 Biomarker Levels (in pg/mL) Groups Combined Summary Statistics ................. 46 3 5 Baseline Measurements of Biomarkers ................................ .............................. 53 3 6 Period Effects on Biomarkers at T2 ................................ ................................ .... 53 3 7 Sequence (AS or SA) Effects on Biomarkers ................................ ..................... 54 3 8 Treatment Effects on Biomarkers ................................ ................................ ....... 54 3 9 Significant Correlations Between Tooth Movement and GCF Levels ................. 54 A 1 IL 1 ................................ ................... 67 A 2 IL 1RA Levels (in pg/mL) for First device Active ................................ ................. 68 A 3 IL 1 1RA) Ratio in pg/mL for First Device Active ............................... 69 A 4 RANKL Levels (in pg/mL) for First Device Active ................................ ............... 70 A 5 OPG Levels (in pg/mL) for F irst Device Active ................................ ................... 71 A 6 RANKL/(RANKL+OPG) Ratio in pg/mL for First Device Active .......................... 72 A 7 Osteocalcin Levels (in pg/mL) for Firs t Device Active ................................ ......... 73 A 8 Osteopontin Levels (in pg/mL) for First Device Active ................................ ........ 74 A 9 IL 6 Levels (in pg/mL) for First Device Acti ve ................................ ..................... 75 A 10 IL 8 Levels (in pg/mL) for First Device Active ................................ ..................... 76 A 11 MMP 3 Levels (in pg/mL) for First Device Active ................................ ................ 77

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8 A 12 MMP 9 Levels (in pg/mL) for First Device Active ................................ ................ 78 A 13 M CSF Levels (in pg/mL) for First Device Active ................................ ................ 79 A 14 IFN Levels (in pg/mL) for First Device Active ................................ ................... 80 B 1 IL ................................ ................... 81 B 2 IL 1RA Levels (in pg/mL) for First Device Sham ................................ ................. 82 B 3 IL 1 1RA) Ratio in pg/mL for First Device Sham ............................... 83 B 4 RANKL Levels (in pg/mL) for First Device Sham ................................ ................ 84 B 5 OPG Levels (in pg/mL) for First Device Sham ................................ .................... 85 B 6 RANKL/(RANK L+OPG) Ratio in pg/mL for First Device Sham ........................... 86 B 7 Osteocalcin Levels (in pg/mL) for First Device Sham ................................ ......... 87 B 8 Osteopontin Levels (in pg/mL) for First Device Sham ................................ ........ 8 8 B 9 IL 6 Levels (in pg/mL) for First Device Sham ................................ ..................... 89 B 10 IL 8 Levels (in pg/mL) for First D evice Sham ................................ ..................... 90 B 11 MMP 3 Levels (in pg/mL) for First Device Sham ................................ ................ 91 B 12 MMP 9 Levels (in pg/mL) for First Device Sham ................................ ................ 92 B 13 M CSF Levels (in pg/mL) for First Device Sham ................................ ................ 93 B 14 IFN Levels (in pg/mL) for First Device Sham ................................ ................... 94

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9 LIST OF FIGURES Figure page 1 1 AcceleDent Aura Device ................................ ................................ ................... 22 2 1 Study Design Flow Diagram ................................ ................................ ............... 32 2 2 Use of Reference Planes on OrthoInsight 3D TM to determine position of maxillary left central incisor ................................ ................................ ................. 32 2 3 Calculation of Tooth Movement be tween Time Points with OrthoInsight 3D TM ... 33 2 4 Description of Actual Movement Projected onto Attempted Movement Vector ... 33 2 5 G CF Collection. A, Periopaper Absorbing Strips; B, Periopaper Insertion into Gingival Sulcus; C, Periotron 8000 ................................ ............................ 33 3 1 IL 1 Median Values ................................ ................................ ........................... 49 3 2 IL 1RA Median Values ................................ ................................ ........................ 49 3 3 RANKL/(RANKL+OPG) Median Values ................................ .............................. 50 3 4 IL 6 Median Values ................................ ................................ ............................. 50 3 5 IL 8 Median Values ................................ ................................ ............................. 51 3 6 MMP 3 Median Values ................................ ................................ ....................... 51 3 7 MMP 9 Median Values ................................ ................................ ....................... 52 3 8 Box Plots Displaying MMP 9 Concentration in GCF Samples ............................ 52 3 9 IFN Median Values ................................ ................................ .......................... 53 3 10 Correlation Between MMP 9 Levels and Tooth Movement Weeks 1 2 .............. 55 3 11 Correlation Between IL 1RA Levels and Tooth Movement Weeks 1 2 ............... 55 3 12 Correlation Between IL 6 Levels and Tooth Movement Weeks 1 12 .................. 56 3 13 Correlation Be tween MMP 9 Levels and Tooth Movement Weeks 1 12 ............ 56 3 14 Correlation Between IL 1RA Levels and Tooth Movement Weeks 1 12 ............. 57

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10 LIST OF ABBREVIATIONS AS Active th en Sham Group CBCT Cone Beam Computed Tomography ECM Extra Cellular Matrix GCF Gingival Crevicular Fluid Hz Hertz IFN Interferon Gamma IL Interleukin IL 1RA Interleukin 1 Receptor Antagonist IRB Institutional Review Board M CSF Macrophage Col ony Stimulating Factor MMP Matrix Metalloproteinase OC Osteocalcin OPG Osteoprotegerin OPN Osteopontin OTM Orthodontic Tooth Movement PDL Periodontal Ligament PV Preliminary Visit RANK Receptor Activator of Nuclear Factor Kappa B RANKL Receptor Ac tivator of Nuclear Factor Kappa B Ligand SA Sham then Active Group TNF Tumor Necrosis Factor Alpha VAS Visual Analogue Scale

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11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE EFFECT OF VIBR ATORY STIMULATION AND TOOTH MOVEMENT ON GINGIVAL CREVICULAR FLUID BIOMARKERS By Angela M. McNeight May 2016 Chair : Wellington Rody Major: Dental Sciences Orthodontics In the field of orthodontics, gingival crevicular fluid ( GCF ) is one of the main ta rgets for assessing biomarkers. The term GCF describes the fluid that escapes from between the tooth surface and the epithelial lining of the periodontal ligament (PDL). There are over 100 regulatory proteins that can be detected in GCF, and they can be ca tegorized on the basis of their biological significance such as bone resorption or formation OBJECTIVES: T o investigate the effect of vibratory stimulation ( AcceleDent Aura ) on the expression of biomarkers in GCF using a human tooth movement model with c lear aligners. METHODS: Prospective, single center, randomized controlled crossover study comparing an active vibration device to a sham device. Participants received 6 Zendura plastic aligners to move a target tooth 1.98 mm over 12 weeks. Patients were c rossed over to the opposite device at the 4 th aligner GCF was collected at T0 ( baseline 1), T1 ( baseline 2), T2 (week 1 ) T3 (week 7, crossover) and T4 (week 12 ) 11 biomarker levels quantified by multiplex bead immunoassay. A mixed model analysis was use d to compare the effects of active versus sham device on GCF composition. RESULTS: 16 males and 23 females completed the protocol No statistically significant differences were found between the biomarker levels of the

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12 Sham Active (SA Group) and Active Sha m (AS Group) The results of each group were then combined and biomarker concentrations were compared to tooth movement. There was a negative correlation between the baseline level of IL 6 and the total amount of tooth movement A negative correlation was seen between the baseline levels of MMP 9 and tooth movement between weeks 1 2 followed by a positive correlation between the concentration of MMP 9 at T4 and the amount of tooth movemen t over the course of the study. CONCLUSIONS: The results suggest that vibration does not influence the secretion of GCF biomarkers ; nevertheless, tooth movement with clear aligners seem s to alter the proteomic composition of GCF This study was registered on https://clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT01962 012). It was approved by the Western Institutional Review Board (WIRB # 20131856 ). This study was funded by OrthoAccel Technologies.

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13 CHAPTER 1 INTRODUCTION The Role of Vibration in Tooth Movement As a dynamic connective tissue, bone is constantly undergo ing turnover and remodeling to maintain stability or in response to microdamages. The key players in this highly specialized process include osteoclasts, responsible for bone resorption, and osteoblasts specialized for bone formation. 1 Adaption of the bone to orthodontic force is a complex process where mechanical force leads to signal transduction through the periodontal ligament ( PDL ) to the alveolar process and final orthodontic tooth mov ement (OTM). 2 OTM consists of three stages following the application of a force including initial displacement of the tooth, hyalinization of the PDL also known as the lag phase, and ultimately linear tooth movemen t. 3 5 Changes in the vascularity to the PDL trigger a cascade of cellular and molecular events including recruitment of inflammatory markers producing areas of resorption on the compression side and bone formation on the tension side. 3,6 Orthodontic treatment is a long process often representing obstacles to patients and practioners alike. Known disadvantages of lengthy orthodontic treatment include higher susceptibility to caries, gingival recession, and root resorption. The use of vibration to accelerate tooth movement represents a noninvasive and nonsurgical approach to the increasing demand of shortening treatment time. 7 Low molec ular, high frequency vibration (LMHF V ) has been demonstrated to be anabolic and osteogenic to skeletal bones through increasing bone mass density through bone formation 8 11 accelerating rates of angiogenesis 12 and improving fracture healing 10,13,14 The use of 60 Hertz intermittent vib ration in rats has shown significantly greater tooth movement,

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14 increased RANKL expre ssion, and increased osteoclast recruitment within the PDL without evidence of root resorption. 7 The AcceleDent Aura device (Orth oAccel Technologies, Inc Bellaire, TX USA ) (Figure 1 1) has been marketed to accelerate OTM by delivering pulsating, low magnitude forces to the dentition and has been approved by the FDA for fixed orthodontic treatment Patients are instructed to bite for 20 minutes per day on the removable mouthpiece portion of the device which vibrate s at 30 Hertz, 0.25 Newtons (25 grams ) Previous studies have demonstrated increased rates of OTM 15 17 without evidence of root resorption 18 during fixed orthodontic treatment However, other fixed appliance studies have found no evidence of increase d initial alignment rates using the device. 19 GCF Biomarkers of Tooth Movement Gingival crevicular fluid is a serum transudate that escapes from between the tooth surface and the periodontal ligament lining 20,21 and is one type of human body fluid used for proteome analysis C ollection of GCF is minimally invasive, consisting of placing a small capillary tube 20 or absorbe nt paper strip 20 at the entrance of the gingival crevice until minimum resistance is felt tactically. Apparatuses to estimate the volume of GCF collected vary, with the newest and most popular device being th e Periotron 8000 (Ora Flow, Plainview, NY, USA ) This device is very precise, determined to be able to measure volumes of GCF on the Periopaper strips (Ora Flow, Plainview, NY, USA) up to 1.2 microliters ( l ) 22 These processes lead to other advantages of GCF col lection which include minimal cost, ease of sample collection, ability for site specificity, and availability of fluid 23 However, sample handling and identification assays post collection are difficult due to the small amounts of target protein matter 24 Currently,

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15 immunoassays are the main tool that is used for biomarker quantification in GCF due to its high specificity, high sensitivity, and wide applicability 25 There are over 100 regulatory proteins that can be detected in GCF, including cytokines, chemokines, and matrix metalloproteinases. Biochemical mediators released during each sequential stage of O TM can be detected in GCF. Multiple reviews have focused on summarizing findings regarding the presence and quantities of these biomarkers and their relationships to tooth movemen t. 26 29 A summary chart of the stud ies discussed here is presented in Table 1 1 Osteoclastogenesis Related F actors (RANKL/OPG) Re ceptor activator of nuclear factor kappa B (RANK) receptor activator of nuclear factor kappa B li gand (RANKL) and the decoy receptor for RANKL, osteoprotegrin (OPG) are the three key molecules in osteoclastogenesis. Only RANKL has been proven absolutely necessary for osteoclast development from hemopoietic precursors in vivo 30 RANKL binds to RANK on the osteoclast prec ursor surface which leads to osteoclast maturation and bone resorption, while OPG binds RANKL and inhibits the binding of RANKL and RANK, thus not a llowing osteoclasts to mature. Ogasawara et al 31 studied rat peri odontal tissues during excessive orthodontic tooth movement and found RANKL in both osteoblasts and periodontal ligament cells. In addition, RANKL and OPG were both present in rat PDL in physiologic conditions, but were localize d in areas of active bone re sorp tion with osteoclasts during tooth movement 31 highlighting the transforming nature of RANKL and OPG molecules and their role in the larger picture of bone remodeling. A series of split mouth studies 32,33 at Nihon University School of Dentistry in Japan found significantly higher amounts of RANKL on the side of canine retraction

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16 than a t the control sites at all time points. However, experimental site OPG values were only lower at one time point after 24 hours. In addition, RANKL concentrations increased in the extracted premolar teeth that had stresses placed on them over time in vitro while the control teeth did not. OPG had an opposite effect and decreased more in conce ntration as compressive forces increased when compared to the control teeth. From these results, it can be inferred that both in vivo and in vitro RANKL increased and OPG decreased in response to compression forces in both time and magnitude dependent man ners, giving the conclusion that compression is most li kely leading towards bone resorp tion through these two molecules 32 Multiple other studies have found increased amounts of RANKL as well as increased ratios of RANKL to OPG at sites of compression during OTM 6,34,35 From these in vivo and in vitro studies described above, it can be concluded that the RANK/RANKL/OPG system is integral to orthodontic tooth movement. Mechan ical stresses lead to an increase in RANKL which in turn induces osteocla stogenesis, allowing bone resorp tion and tooth movement Thus, higher ratios of RANKL to OPG in GCF are indicative of areas of bone remodeling and orthodontic tooth movement. If resea rch can find non invasive ways to track this ratio, the potential for accelerating tooth movement may become a reality. Furthermore, inhibition of RANKL would be helpful in preventing unwanted tooth movement, either for anchorag e or retention afte r treatme nt Biomarkers of I nflammation Various inflammatory biomarkers have also been linked to orthodontic tooth movement. Two of the most studied cytokines found in GCF are Interleukin 1 (IL 1) and its naturally occurring receptor antagonist, Interle ukin 1 recep tor antagonist (IL 1 RA). IL

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17 1 comes in two forms, alpha and beta, with the beta form previously describe d as more potent for bone resorp tion a nd inhibition of bone formation 36 The roles that IL IL 1RA play in orthodontic tooth movement have been studied in both the rat orthodontic tooth movement model and in multiple human experiments 37 45 Iwasaki et al. 40 conducted human ex periments in which amounts of cytokine were expressed as an IL 1 Activity Index (AI) defined as t he following: AI = Experimental (IL 1RA)/ Control (IL 1 RA). The results showed that the velocity of tooth movement was related to an A I They also concluded that the following three aspects corresponded to increased tooth movement : having one copy of allele 2 at gene IL 1B (+3954), high average AI, and low IL 1RA in GCF at the experimental site during tooth movement. However, gene intera ctions and modifications such as small nucleotide polymorphisms (SNPs) are prevalent and support evidence that gene expression is affected by many factors 42 Other interleukins such as IL 6 and IL 8 are also import ant regulators of bone physiology. The primary role of IL 6 is debated since it shows both osteoc lastic and osteoblastic properties. 46 IL 8 is an inflammatory cytokine secreted by monocytes which recruits and activ ates neutrophils during the early s tages of tooth movement. 47 High levels of both IL 6 and IL 8 have been reported in chronic periodontitis pati ents 48,49 Extracellular Matrix Degradation Factors (MMPs) Matrix metalloproteinases (MMPs) derived from polymorphonuclear leukocytes and other cells are the key enzymes for degradation of the extracellular matrix. Twenty three different MMPs can be identified, and they are split into five categories: collagenases (MMP 1, 8, 13), gelatinases (MMP 2, 9), stromelysins (MMP 3, 10, 11), membrane type MMPs (MMP 14, 15, 16, 17), and miscellaneous MMPs 50 The majority

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18 of research surrounding MMPs is devoted to their use as biomarkers for periodontitis. Specifically, GCF levels of MMP 9 are elevate d in gingivitis and periodontitis, and correlate with PDL attachment loss and bleeding on probing 21 MMPs have also been linked to orthodontic tooth movement, since large amounts of re modeling of the periodontium must occur for a tooth to successfully move through bone. A pivotal study performed in rats 51 suggested that new osteoblasts are derived from PDL cells in osteogenesis, therefore showing that the PDL plays a domina nt role in remodeling of alveolar bone. Another more detailed study in rats 38 concluded that as orthodontic pressure increases, MMP 9 display s constant increases as well A s tudy performed by Bildt et al. 52 investigated the amounts of latent and active M MPs at both the resorp tion and apposition sides of teeth undergoing orthodontic forces. Results showed that more active MMP 9 was found at the resorption side Previous studi es have also confirmed that MMP 9 stimulates osteoclast migration and subsequently their activity 53,54 Markers of Bone Matrix Degradation and Osteoclast Recruitment O steopontin (OPN) osteocalcin (OC) and macroph age colony stimulating factor ( M CSF ) are three biomarkers of bone matrix degradation and osteoclast recruitment. OPN lies in the extracellular matrix of bone and is heavily glycosylated and phosphorylated 55 It is mainly secreted by osteoblast s, osteoclasts, and macrophages and is involved in the control of bone mineralization, formation, and resorption 55 Due to this involvement in bone metabolism, higher levels of OPN hav e been detected in the GCF around teeth with periodontal disease 56 Furthermore, OPN plays a role in anchoring osteoclasts to the bone surface and inhibiting bone resorption in its absence, confirmed by higher leve ls of OPN in periodontal disease progression and lower levels of OPN in health 57 When OPN is knocked out in mice, bone resorption does not occur 58

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19 and bone remodeling in r esponse to orthodontic coil mechanical pressure is suppressed 59 A recent in vitro study by Nettelhoff et al. 60 tested different strengths of compressive forc e on human PDL fibroblasts and osteoblasts and found an increase in OPN synthesis in osteob lasts under compressive forces. Osteocalcin (OC) is produced by osteoblasts and odontoblasts and is the most prominent osteoblastic specific non collagenous matrix p rotein in bone 61 Its exact role in bone metabolism is debated but known functions include perform ing as a chemoattractant for progenitor and mature osteoclasts and acting to limit bone formation without impairing bone resorption 62,63 Si gnificantly greater amounts of tooth movement were seen in the rats who re ceived injections of OC over controls. Furthermore, histological evaluations showed larger numbers of osteoclasts on the mesial (pressure) side of OC injected mice than the control rats leading to the conclusion that OC enhances the recruitment of osteocl asts thus accelerating tooth movement 64 In a human study in orthodontic patients receiving first premolar extractions 65 GCF OC levels were elevated on the side of the mou th that underwent canine retraction while the control canine on the other side of the mouth showed no elevations in OC levels. Macrophage colony stimulating factor (M CSF) has an important role in the survival, propagation, and differentiation of mononucle ar phagocytes from their progenitor cells 66 and is produced by various cells including osteoblasts and fibroblasts 67 A study in the rat model showed that heavy orthodontic tooth movement (50 g rams ) elicit ed the presence of M CSF in PDL fibroblasts and rat odontoclasts on the cementum 68 A human study by Kaku et al. 67 found higher M CSF GCF l evels on the

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20 canine retraction side t hus leading the authors to conclude that mechanical force induces osteoblasts a nd fibroblasts to release M CSF, promoting bone remodeling and ultimately orthodontic tooth movement. The role of i nterferon gamma (IFN ) is controversial in that some studies have labeled the biomarker as an inhibitor of bone resorption 69,70 while other investigations have concluded it promotes inflammation and bone resorption 71,72 In a study 73 in which an experimental group of rats was administered IFN while undergoing OTM with coil springs, histological evaluations were completed at the pressure side. Increases in trabecular bone volume and number paired with d ecreases in trabecular separation in the IFN rats led to the conclusion that the primary activity of IFN is anti osteoclastic. The authors postulated that administration of IFN could be useful for anchorage control during orthodontic treatment 73 In contrast, a rat split mouth design of OTM showed higher levels of IFN on the experimental side than on the control side 74 Human GCF studies are needed to gather more information regarding the primary role of this controversial cytokine during OTM in order to solidify any clinical applications. Few studies of the effects of vibration on GCF biomarkers are present in the litera ture. One model concluded that rat teeth undergoing resonance vibration showed increased RANKL expression in PDL fibroblasts and osteoclasts, inducing osteoclast differentiation and therefore increasing bone resorbtion 7 Leethanakul et al. 75 also evaluated the effect of vibration on human GCF biomarkers, specifically IL using vibratory stimuli from an electric toothbrush ( 125 Hz) resulting in greater tooth move ment and higher IL that vibratory stimulation increased both IL 75

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21 Specific Objectives or H ypotheses The purpose of this study was to investigate the effect of vibration and tooth movement on the expression of biomarkers in GCF. Our primary aim was to determine if there was a difference in GCF biomarker levels when orthodontic patients were using an active vibrating device ( AcceleDen t A ura) compared to when they were not Our secondary aim was to determine if any correlations existed between amount of tooth mov ement and GCF biomarker levels. Null Hypothesis # 1: There will be no changes in GCF biomarker levels when orthodontic patient s use an active vibrating device (AcceleDent A ura). Null Hypothesis # 2: There will be no correlation between tooth movement and GCF biomarker levels in patients undergoing orthodontic treat ment with clear aligners.

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22 Figure 1 1. AcceleDent Aura Devic e Table 1 1. A selection of human GCF biomarkers and their clinical relevance to orthodontics (Adapted from Rody et al. 76 ) Category of biomarker Name of biomarker Relevance to orthodontics References Osteoclastogenesis related factors RANKL OPG Monitoring of bone remodeling throughout OTM Nishijima et al., 2006 32 Kawasaki et al., 2006 33 Rody et al.., 2013 35 Grant et al., 2013 6 Barbieri et al., 2013 34 Inflammatory cytokines Proinflammatory:IL 1 Anti inflammatory: IL 1RA Influence bone remodeling Affect rate of tooth movement Iwasaki et al., 2005 41 Iwasaki et a l., 2009 42 Luppanapornlarp et al., 2010 43 Dudic et al., 2006 44 Extracellular matrix degradation factors MMPs Changes in the periodontal ligament in response to orthodontic forces Bildt et al., 2009 52 Capelli et al., 2011 54 Grant et al., 2013 6 Markers of bone matrix degradation and osteoclast recruitment OC OPN M CSF Monitoring of bone matrix degradation and recruitment of osteoclasts from hematopoetic tissues Alfeqeeh & Anil, 2011 65 Barbieri et al., 2013 34 Kaku et al., 2008 67

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23 CHAPTER 2 MATERIALS AND METHODS Trial Design The study was a prospective, single center, randomized controlled crossover clinical trial with an allocation ratio of 1:1 IRB approval was obtained from the Western Institutional Review Board ( Olympia, WA USA, WIRB# 20131856 ) and the study was listed on ClinicalTrials.gov (ClinicalTrials.gov Identifier: NCT01962012) Participants, Eligibility Crite ria and Setting Participants were recruited from the University of Florida Orthodontic clinic. Patient selection was performed using set inclusion and exclusion criteria outlined in Table 2 1. All study procedures were completed at the University of Flori da College of Dentistry Orthodontic Research Clinic (Gainesville, FL, USA) and monitored by Juno Clinical Research Services, LLP (San Antonio, TX USA). Interventions Subjects reported for biweekly study visits over a 12 week period from day 0 to day 84. On the first day of the study (Day 0), the first aligner was provided which was activated for 0.33 millimeters (mm) of anterior movement of the target tooth. Those assigned to initial active treatment received aligner treatment in conjunction with the acti ve AcceleDent Aura ( OrthoAccel Technologies, Inc, Houston, TX USA) device while those assigned to initial sham treatment received aligner treatment with a sham AcceleDent Aura device. The active device vibrates at 0.25 Newtons (25g) and 30 Hertz freque ncy while the sham had zero vibration. Subjects were asked to use the device for 20 minutes per day. Each aligner was worn for 2 weeks for a total prescribed tooth movement of 1.98mm of the target tooth over 12 weeks.

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24 The first preliminary visit was an eli gibility visit to identify potential subjects with appropriate malocclusions and the correct number of maxillary teeth and to eliminate those with exclusionary medical or dental problems. Participants reviewed and signed the informed consent forms, a medic al history was collected and reviewed, and an intra oral clinical examination was completed. Those who were deemed eligible proceeded to preliminary visit 2. At this second visit, designated the Screening Visit, participants had digital impression scans ta ken with a 3Shape Trios scanner (Copenhagen, Denmark) for fabrication of aligners and intraoral and extraoral photographs were acquired. A cone beam computed tomography scan (CBCT) (Imaging Sciences International, iCAT, Hatfield, PA, USA) was taken for di agnostic records as well as to assist in treatment planning. For all female participants, a negative urine pregnancy test was required immediately prior to the CBCT imaging procedure. Once subject eligibility was confirmed, study numbers were assigned to e ach subject and they were enrolled in the study. Selection of the target tooth was based on the tooth not being blocked out by the adjacent teeth in order to allow the anterior posterior movement of 1.98mm. The right or left maxillary central incisor was a ssigned by the principal investigator. A steolithographic (.STL) file was created from the initial digital impression and opened in 3Shape OrthoAnalyzer TM (3Shape, Copenhagen Denmark). The target tooth was digitally moved forward the prescribed amount usi ng this program, and aligners were fabricated by NorthStar Orthodontics, Inc. (Park Rapids, MN, USA). The aligners were made from Zendura (Bay Materials, LLC, Fremont, CA USA ), a polyurethane material of high performing rigid engineering resin.

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25 Participa nts were informed that the study was examining two different levels of pulsation, one of which they may not be able to detect. The sham and active devices were identical in appearance, function, and sound but one without vibration in order to blind partici pants. Study subjects were instructed to use the device for 20 minutes per day and to log start and stop times of device usage in a provided daily diary. The subjects were also asked to record the times aligners were placed and removed in order to determin e the amount of time aligners were worn daily. Over the 84 days of the study, new photos, digital impression scans, pulp testing, and periodontal probings were completed. Periodically, tooth movement was measured and GCF was collected as described below. P atients were crossed over to the opposite de vice after the delivery of the fourth aligner as shown in the study design flow diagram ( Figure 2 1 ) T hus, there were two groups of patients: those starting with the active (A) device and crossing over to the sh am (S) device (the AS group) and those starting with sham and crossing to over to th e active device (the SA group). After the study was completed, participants were given the option to continue with full orthodontic treatment at the University of Florida. Outcomes Clinical Tooth Movement Evaluation of clinical tooth movement was modeled off previous studies conducted at the University of Florida 77 79 The Intraoral scans were imported into 3Shape OrthoAnalyzer TM and then into Ortho Insigh t 3D TM (Motion View Software, LLC, Chattanooga, TN, USA) Reference planes created from posterior landmarks and superimposition on posterior teeth were used to measure the amount of anterior posterior movement of the target tooth ( Fi gure 2 2 ) The amount of tooth movement in

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26 each dimension between time points was determined by subtracting the tooth position on the latter scan from the previous scan location ( Figure 2 3 ) as used to determine how much of the projected movement was actually achieved ) ( Figure 2 4 ) The equation used to determine the percent movement actually achieved was GCF Collection GCF was collected at five time points from the buccal crev according to the following schedule: T0 (first baseline examination at time of initial scan), T1 (second baseline exami nation when the first aligner was delivered), T2 (within one week following delivery of the first aligner), T3 ( week 7, i.e., within one week following delivery of the fourth two weeks after the delivery of th e sixth aligner, which was the final study visit). GCF collection followed pr otocols previously outlined by ou r research team 35,76 Prior to GCF collection, the target tooth was air dried for ten seconds and isolated with cotton rolls. Two Periopaper absorbing strips (Oraflow, Plainview, NY USA ) (Figure 2 5 A ) were insert ed for 60 seconds into the mesiofacial and distofacial sulci of the target tooth at a depth of about 1 2mm (Figure 2 5 B ) The volume of GCF was determined by positioning the strips between the upper and lower counterparts of the pre calibrated Periotron 80 00 (Oraflow, Plainview, NY USA ) (Figure 2 5 C ) The strips were then stored in separate sealed Eppendorf tubes at 80 C for subsequent biomarker analysis

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27 Biomarker A nalysis After removal from 80C storage, 120 l of phosphate buffered saline (PBS) was a dded to the tube and the GCF collection strips were allowed to thaw at room temperature for 30 minutes. Tubes were subsequently vortexed for 15 minutes and centrifuged for 10 minutes (1500 g, 4C). Once proteins were eluted from the strip in this manner, t he strips were removed and the supernatant was separated into aliquots for use in individual kits to avoid repetitive freeze thaw cycles. The supernatant samples were analyzed using a multi analyte method by magnetic bead arrays. In this procedure, specifi along with the sample of interest.24 Four custom commercially available human magnetic bead based arrays were used to identify the following biomarkers: 1) A multiplex array for M CSF, IL 6, IL 8, IFN 3, MMP 9, and osteopontin (R&D systems, Minneapolis, MN, USA), 2) A single plex kit for RANKL (R&D systems, Minneapolis, MN, USA), 3) A single plex assay for IL 1RA (R&D systems, Minneapolis, MN, USA), and 4) a multiplex kit for OP G and osteocalcin (EMD Millipore, Chicago, IL, USA). well plates. Briefly, both samples and standards, along with the magnetic beads, were pipetted into the wells, and the plate was covered using light repelling aluminum sealers and incubated at 4C overnight while shaking at 650 rpm. The wells were subsequently washed using a hand held magnet device. The plates rested on the magnet for 60 seconds to allow for complete settling of the magnet ic beads. Contents of the wells were removed by decanting the plate while still attached to the magnet in an appropriate waste receptacle and gently tapping on absorbent pads to remove any residual liquid.

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28 Wash buffer provided by the manufacturer was added to the wells, and the plate was removed from the magnet, covered, shaken for 30 seconds, and finally allowed to settle on the magnet again for 60 seconds. This procedure was repeated three times at each wash step. A mixture of detection antibodies was th en added to each well. After a 30 minute period of incubation, streptavidin conjugated to the fluorescent protein R phycoerythrin (streptavidin RPE) was added to the beads and incubated for another 30 minutes. After a wash to remove unbound reagents, sheat h fluid (Luminex, MiraiBio, Alameda, CA, USA) was added to the wells and the beads were re suspended on a plate shaker for five minutes. Data was acquired using instrumentation (Luminex 200TM, EMD Millipore, Darmstadt, Germany) and analyzed with xPONENT software (EMD Millipore, Darmstadt, Germany) using a 5 parameter logistic or spline curve fitting method. Concentrations of chemokines/cytokines in the samples were report ed as picograms per milliliter. Sample Size Calculation The sample size was determin ed based on a primary tooth movement aim, making use of data from previous OTM studies conducted at the University of Florida College of Dentistry Department of Orthodontics. 77 79 It was powered to detect a differe nce of 15% in tooth movement Randomization Forty subjects between the ages of 18 and 40 years old seeking orthodontic treatment were enrolled in the study. The subjects were stratified by sex and randomly assigned in blocks of four to an initial device, a ctive or sham.

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29 Blinding The subjects were blinded as to which device they received since the devices were identical in appearance and sound They were told that the study was comparing two different frequencies of vibration using the AcceleDent Aura devi ce. Since the devices had no visual information regarding if they were sham or active, the clinical orthodontists were also blinded. Statistical Analysis Summary statistics (mean standard deviation, median, minimum, and maximum) and plots were used to char acterize the distributions of each biomarker at each time point, by initial device assignment (active or sham). Examination of the data led to identification of possible outliers and considerations of transforms (such as log or logit) that would better sat isfy the assumption of underlying normality. Linear mixed models were developed for each biomarker (and pre specified ratios) to test for differences due to treatment assignment. For the initial set of analyses, the outcome variable was the biomarker under consideration (possibly with outliers removed or transformed) at time points T2 and T3 (i.e. the first week s after initiation of active or sham treatment). Covariates included the baseline level of the biomarker ((biomarker at T0 + biomarker at T1)/2), se quence assigned (0 if sham then active, 1 if active then sham), period (1 if T2, 2 if T3), and treatment (0 if sham, 1 if active). The second set of analysis models focused only on T4 levels, to identify if the order of treatment had any impact on the fina l biomarker level. Covariates included baseline biomarker level (described above), and sequence (equivalent to treatment assignment order). Models were fit for original data, transformed data, and data with outliers removed, if appropriate. Results are fro m untransformed data unless otherwise

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30 indicated. For all models, a treatment p value of less than 0.05 was considered statistically significant. Correlation analysis was used to examine the relationships between biomarker level and tooth movement during a specified time interval. Biomarker related variables included baseline, T2, T3, T4, change baseline to T2, and change baseline to T4. Because the biomarkers were taken at specific time points, it is of interest to look at tooth movement during specific int ervals that match up with the specified time point. In addition, based on the modeling results which did not identify treatment differences, treatment assignment order was not considered. Tooth movement time intervals considered were the following : movemen t during weeks 1 and 2, movement during weeks 1 to 4, movement during weeks 7 to 8, movement during weeks 7 to 12, and total tooth movement (weeks 1 through 12). Pearson and Spearman correlation coefficients were estimated. A p value of less than 0.10 was considered of interest

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31 Table 2 1. Outline of Inclusion and Exclusion Criteria Inclusion Criteria 1. Males or females between and including the ages of 18 and 40 years old desiring orthodontic treatment that could be completed within two years of trea tment with either fixed appliances or aligner treatment. Subjects may have had previous orthodontic procedures. 2. Adult dentition with all upper front teeth present and any premolar and molar combination in the upper posterior of two teeth on each sid e. 3. At least one upper maxillary central incisor that is positioned to allow anterio r posterior (AP) movement (crown tipping only) of 1.98 mm. 4. Normal pulp vitality and healthy periodontal tissues as determined by intraoral exam. 5. Good heal th as determined by medical history. 6. Willingness and ability to comply with study procedures, attend study visits, and complete the study. 7. The ability to understand and sign a written informed consent form, which must be signed prior to initiat ion of study procedures. Exclusion Criteria 1. Severe malocclusions that would take longer than 2 years of treatment or require surgical intervention. 2. Significant periodontal disease (>3mm pocket depth or >1mm of recession on upper anterior teeth) 3. Active caries not under care of either a dentist or periodontist. 4. Chronic daily use of any non steroidal anti inflammatory medication, estrogen, calcitonin, or corticosteroids. 5. History of use or current use of any bisphosphonate medicati on or other medication for treatment of osteoporosis. 6. Current smoker (must not have smoked in the last 6 months). 7. Women may not be pregnant. Negative urine pregnancy tests prior to exposure to cone beam imaging is required to verify pregnancy s tatus. 8. Any condition or use of medication which in the opinion of the investigator interferes with the biology of tooth movement. 9. Any condition which in the opinion of the investigator results in increased risk to the subject

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32 Figure 2 1 Study Design Flow Diagram Figure 2 2 Use of Reference Planes on OrthoInsight 3D TM to determine position of maxillary left central incisor

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33 Figure 2 3 Calculation of Tooth Movement between Time Points with OrthoInsight 3D TM Figure 2 4 Descri ption of Actual Movement Projected onto Attempted Movement Vector Figure 2 5 GCF Collection. A, Periopaper Absorbing Strips ; B, Periopaper Insertion into Gingival Sulcus ; C, P eriotron 8000

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34 CHAPTER 3 RESULTS Participant F low Participants were recr uited from November 2013 to February 2014. 44 subjects were initially enrolled of which 3 dropped prior to assignment. 20 subjects were assigned to the active AcceleDent Aura device first (Group AS) and 19 were assigned to the sham device first (Group SA) After all data had been collected, th e trial concluded in June 2014. Baseline Data Subject demographic information is illustrated in Table 3 1. A total of 44 subjects were enrolled in the primary study and 39 patients completed the pro tocol (16 males and 23 females) The mean age of the subjects was 30.6 years ( 5.8 ) with ages rang ing from 20.2 to 40.9 years old Numbers Analyzed, Outcomes, and E stimation Two subjects (A027 and A029) withdrew from the study because they were unable to receive necessary d ental restorative treatment prior to beginning the study One subject (A041) dropped prior to day 0 reporting that she was newly pregnant. Another subject (A009) withdrew at day 14 stating that the aligners were giving her headaches. Lastly, a subject (A02 4) dropped at day 21 due to increased sensitivity and non responsive pulp testing on previously traumatized teeth #9 and #10 which were in need of root canal treatment. None of these adverse events were judged to be due to the AcceleDent Aura device. Sinc e subjects A027, A029 and A041 only had one sample of GCF collected, these samples were not included in the biomarker analysis. Statistics were calculated using the GCF samples from T0, T1 and T2 from subject

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35 A009. Since A024 dropped out of the study earl y, her T3 reading was used as T4 since she was ending t he study completely. Individual b iomarker levels ( pg/mL ) ratios, means and medians for all subjects at each time point are listed in Appendices A and B Summary statistics (median, mean, standard devi ation, st andard error, minimum, and maxi mum) of each biomarker are listed in Tables 3 2 to 3 4 Since the mean and median values were dissimilar for some of the biomarkers, median values were used to examine the distributions Transformations were used whe n mean and medians differed due to an outlier or needed log transformation to better approximate normality Figure s 3 1 to 3 8 depict graphical repre sentation s of the summary statistic medians to better visualize biomarker trends throughout the study Plea se note that d espite the trends and fluctuations seen in the graphs it is important to emphasize that there were no statistically significant differences between the SA and AS g roups. value is the average of the biomarker concentrations tak en at T0 and T1. represents overall summary statistics, ignoring treatment sequence assignment Immunoassays seem to be a sensitive, specific and selective technology to interrogate biomarkers in oral fluids. Our research w as able to reliably quantify 11 biomarkers in GCF The only analyte that was dropped from the final statistical analysis was osteocalcin (OC) because t he assay standard curve s for this biomarker were not fully fitting The results of the mixed model analys es examining treatment eff ect are presented in Tables 3 5 to 3 8 As would be expected, the baseline values were significant predictors for the T2 and T3 biomarker levels (Table 3 5) with the exception of IL 8 and M CSF Period effects were not common and are outlined in Table 3 6.

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36 There was a significant period effect for IL 1RA (p<0.0297), MMP 3 (p<0.0010), and MMP 9 (p<0.0060 ) In addition, the order of exposure (active or sham device first) was not associated with differences in GCF concentration of the biomarkers, indicating no sequence effects on biological endpoints (Table 3 7). We also did not detect statistically significant treatment effects for any of the biomarkers at T2 and T3 (Table 3 8). T hus, we failed to reject the prev iously stated null hyp othesis # 1 ; there was no significant affect seen of vibration using AcceleDent Aura on biomarker levels in GCF in this study Since there were no significant findings visualized between the sham and active treatment groups, the biomarker levels were combi ned and correlations were made to better visualize trends between tooth movement and GCF biomarker levels Tooth movement variables included tooth movement observed between weeks 1 2, 1 4, 7 8, 7 12, and 1 12. Biomarker variables included baseline biomarke r level, week 1, week 7, week 12, baseline to week 1, and baseline to week 12. Pearson correlations and Spearman rank correlations were calculated. Because of the temporal nature of the observations only some of the pairs of biomarker levels and tooth mov ement variables are considered. Since we detected positive and negative correlations between biomarker leve ls and tooth movement (Table 3 9 ), there is evidence to rejec t null hypothesis # 2. No trend differences in IL production were noticed in the AS Gr oup, SA Group, and Groups C ombined In general IL throughout orthodontic treatment as seen in Figure 3 1. IL 1RA showed different fl uctuations between the gr o u ps up until T3, after which IL 1RA decreased to below

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37 starting b aseline levels for all groups (Figure 3 2) Our data also suggests a positive correlation between baseline levels of IL 1RA and the amount tooth movement in the first two weeks, as well as a positive correlation between IL 1RA levels at the end of the stud y (T4) and tooth movement throughout the study (Table 3 9 ). Unfortunately, d ue to the much higher GCF concentration of IL 1RA compared to IL t he ratio of IL 1RA) was basically zero for all three groups generating a very abnormal distribution and making the rati o result s difficult to interpret even with log transformation As exp ected, t he RANKL /(RANKL+OPG) ratio increased for all three groups from baseline to T2 (Figure 3 3 ) This was mainly due to a decrease in OPG concentration within the first w eek after tooth movement began At T4, the RANKL/(RANKL+OPG) ratio was not as high as at T2 ; however, it remain ed above baseline measurements. No trends were observed for OPN and M CSF IL 6 concentration increased throughout the study for all three groups ( Figure 3 4 ) and a negative correlation was detected between baseline levels of th is cytokine and the amount of tooth movement throughout the study (Table 3 9 ) IL 8 responded in the opposite fashion, decreasing steadily throughout the study except for an increase at T4 for the SA group (Figure 3 5 ) The MMPs showed similar trends with strong peak s at T3 (Figure 3 6 and 3 7 ) Within the first week of treatment (T2), the actual GCF levels of MMP 3 and MMP 9 were significantly smaller than the predicted values for this time point according to our statistical model (Table 3 6 ). A significan t increase in concentration was detected from T2 to T3 for MM P 9 (ANOVA p=0.0009) (Figure 3 8 ). In addition, t here was a negative correlation between the baseline level of MMP 9 and tooth movement in the first week followed by a positive correlation betwee n the amount of MMP 9 at T4 (12 weeks) and

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38 tooth movement over the entire study (Table 3 9 ) IFN throughout the study (Figure 3 9 ) Figure 3 10 through Figure 3 15 depict graphical representations of the correlations between biomarker concentrations and tooth movement outlined in Table 3 9. Harms No significant c hanges were noted in the periodic scheduled intraoral exams, pulp testing, and periodontal probings of any subjects. No adverse effects were noted as a result of using the AcceleDent Aura device.

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39 Table 3 1 Demographics of Final Population (n=39) n Pe rcentage (%) Sex Male 16 41 Female 23 59 Race White 29 74 Black 6 15 Asian 1 3 Hispanic 3 8

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40 Table 3 2 Biomarker Levels (in pg/mL) AS Group Summary Statistics Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Ma ximum IL Baseline 21 106 129 106.8 23.3 32.95 404.6 T0 21 126.7 149.4 117.1 25.559 8.75 415.7 T1 21 63.65 108.7 121 26.405 3.98 393.6 T2 21 72.84 90.795 78.141 17.052 1.83 297.6 T3 20 57.965 94.964 115.2 25.758 5.31 416 T4 21 60.52 92.714 85. 167 18.585 6.8 293.6 IL 1RA Baseline 21 29066 33107 23171 5056.3 1989.5 92163 T0 21 39386 40476 28117 6135.6 1192 96490 T1 21 15267 25739 28230 6160.2 146.2 95976 T2 21 29202 34307 28610 6243.2 1901 90889 T3 20 36275 49376 42429 9487.4 3340 168350 T4 21 16740 26959 26798 5847.7 17.56 90992 IL (IL 1RA) Baseline 21 0.005 0.0302 0.082 0.0179 0 0.335 T0 21 0 0.0067 0.0091 0.002 0 0.03 T1 21 0 0.0538 0.162 0.0355 0 0.66 T2 21 0 0.0043 0.0093 0.002 0 0.04 T3 20 0 0.002 0.007 0.0016 0 0.03 T4 21 0 0.132 0.279 0.0609 0 0.84 RAN KL Baseline 21 23.565 24.333 6.334 1.382 14.645 44.035 T0 21 25.01 24.666 5.81 1.268 15.44 41.61 T1 21 22.09 24.001 8.047 1.756 12.08 46.46 T2 21 25.01 24.664 7.701 1.681 15.44 51.38 T3 20 25.79 24.77 7.044 1.575 15.44 46.46 T4 21 25.01 24.8 5.95 4 1.299 12.08 34.5 OPG Baseline 21 7.17 7.752 6.744 1.472 1.16 23.525 T0 21 6.06 10.852 11.638 2.54 1.59 39.64 T1 21 3.16 4.652 4.087 0.892 0.73 14.11 T2 21 3.31 14.595 35.253 7.693 0.39 160.6 T3 20 3.235 8.785 17.564 3.927 0.39 78.61

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41 T able 3 2. Continued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum OPG T4 21 3.16 9.321 11.467 2.502 0.93 38.61 R ANKL/ (RANKL+OPG) Baseline 21 0.825 0.794 0.126 0.0275 0.55 0.945 T0 21 0.78 0.742 0.19 0.0416 0.32 0.94 T1 21 0.88 0.845 0.0977 0.0213 0.67 0.97 T2 21 0.89 0.791 0.232 0.0506 0.11 0.98 T3 20 0.875 0.823 0.187 0.0418 0.23 0.98 T4 21 0.88 0.785 0.197 0.043 0.38 0.96 OC Baseline 21 22.06 46.118 44.85 9.787 0.965 154.3 T0 21 32.63 49.344 48.58 10.601 0.49 182.8 T1 21 21.55 42.891 42.493 9.273 0.49 125.8 T2 21 15.76 42.581 40.676 8.876 0.49 104.3 T3 20 15.86 37.186 37.665 8.422 0.49 95.5 T4 21 10.77 15.316 12.218 2.666 0.49 44.34 OPN Baseline 21 1922 1868.5 622.8 135.9 1143.5 3625 T0 21 2003 1891 .9 640.4 139.7 1122 3625 T1 21 1841 1845 619.1 135.1 1133 3625 T2 21 1949 1844.6 570.2 124.4 1144 3146 T3 20 1868 1826.3 623.4 139.4 1144 3200 T4 21 2135 2073.9 236.8 51.67 1503 2393 IL 6 Baseline 21 1.09 1.167 0.326 0.0712 0.82 2.285 T0 21 0.98 1.11 0.35 0.0764 0.73 2.37 T1 21 1.16 1.223 0.354 0.0773 0.74 2.2 T2 21 1.09 1.161 0.285 0.0622 0.82 2.05 T3 20 1.185 1.187 0.28 0.0627 0.59 2.05 T4 21 1.28 1.255 0.239 0.0521 0.89 1.69 IL 8 Baseline 21 131.5 176.2 199.8 43.605 6.755 882.1 T0 2 1 152.7 233 264.8 57.778 8.6 995.5 T1 21 55.22 119.4 170.1 37.121 3.65 768.8 T2 21 54.5 134.4 191.8 41.852 5.36 875.7

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42 T able 3 2. Continued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum IL 8 T3 20 43.38 98.412 1 83.4 41.014 1.8 823.2 T4 21 19.25 55.12 86.195 18.809 2.12 329.4 MMP 3 Baseline 21 2.11 2.458 1 0.218 1.405 5.02 T0 21 2.11 2.505 1.514 0.33 1.17 8.12 T1 21 1.92 2.412 1.052 0.23 0.96 5.24 T2 21 2.29 2.287 0.817 0.178 0.96 4.19 T3 20 3.27 5.955 11.587 2.591 1.38 54.77 T4 21 2.41 3.122 2.396 0.523 1.38 12.62 MMP 9 Baseline 21 3326 6062.5 6878.4 1501 152.1 22977 T0 21 3270 6414.2 8701 1898.7 132.6 34340 T1 21 2372 5710.8 8837.1 1928.4 78.71 36714 T2 21 4855 6695.2 7738.3 1688.6 113.9 30367 T3 20 6590 18911 29571 6612.4 293.5 104441 T4 21 2958 10042 14807 3231.2 96.78 62383 M CSF Baseline 21 47.89 53.337 23.737 5.18 19.085 119.8 T0 21 47.99 57.757 29.513 6.44 20.15 146.3 T1 21 46.57 48.917 25.966 5.666 13.99 108.4 T2 21 44.7 47.12 7 20.782 4.535 18.02 108.7 T3 20 46.345 48.549 20.458 4.574 20.15 83.39 T4 21 42.97 45.119 19.942 4.352 20.15 108.7 IFN Baseline 21 2.53 2.335 0.892 0.195 0.66 3.455 T0 21 2.35 2.3 1.081 0.236 0.35 3.86 T1 21 2.1 2.37 0.996 0.217 0.97 4.81 T2 21 2.18 2.334 0.978 0.213 0.35 3.86 T3 20 2.14 2.244 1.06 0.237 0.35 4.81 T4 21 1.51 1.738 0.807 0.176 0.35 2.95

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43 Table 3 3 Biomarker Levels (in pg/mL) SA Group Summary Statistics Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum IL Baseline 20 95.61 121.3 81.592 18.245 19.14 320.3 T0 20 103.4 107.8 85.84 19.194 3.61 266.1 T1 20 128.2 134.8 102.6 22.931 6.25 374.4 T2 20 75.39 84.539 62.988 14.085 3.84 218.6 T3 19 88.03 98.923 87.712 20.122 3.19 322.6 T4 19 93.44 121.4 109.9 25.216 11.46 457.2 Il 1RA Baseline 20 29601 32335 17427 3896.7 27.205 74238 T0 20 34262 375 38 29391 6572.1 40.07 99685 T1 20 26932 27132 25338 5665.7 14.34 104204 T2 20 41395 38230 26033 5821.2 70.36 82285 T3 19 37295 47854 32305 7411.2 1451 103022 T4 19 29176 39618 31073 7128.7 70.36 98420 IL (IL 1RA) Baseline 20 0.005 0.053 0.145 0.0324 0 0.505 T0 20 0 0.008 0.0221 0.005 0 0.1 T1 20 0.01 0.098 0.275 0.0614 0 0.91 T2 20 0 0.0375 0.161 0.0359 0 0.72 T3 19 0 0.0016 0.0038 0.0009 0 0.01 T4 19 0 0.0453 0.131 0.03 0 0.5 RANK L Baseline 20 24.3 22.975 4.565 1.021 15.44 28.34 T0 20 23.9 23.532 6.615 1.479 15.44 37.05 T1 20 22.295 22.417 4.543 1.016 15.44 28.34 T2 20 21.8 21.143 6.561 1.467 12.08 31.79 T3 19 21.8 21.841 4.638 1.064 15.44 30.05 T4 19 22.79 21.916 6.44 1. 478 8.98 31.79 OPG Baseline 20 5.592 8.148 8.65 1.934 0.39 36.565 T0 20 3.495 9.326 14.315 3.201 0.14 57.46 T1 20 5.155 6.971 5.6 1.252 0.39 22.02 T2 20 2.545 7.674 9.852 2.203 0.39 39.02 T3 19 4.75 10.595 17.73 4.068 0.39 79.36

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44 Table 3 3. Conti nued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum OPG T4 19 6.73 17.256 28.754 6.597 0.93 98.03 RANKL/ (RANKL+OPG) Baseline 20 0.8 0.786 0.154 0.0344 0.455 0.98 T0 20 0.87 0.793 0.206 0.0462 0.31 0.99 T1 20 0. 78 0.779 0.142 0.0316 0.52 0.98 T2 20 0.875 0.779 0.202 0.0451 0.38 0.98 T3 19 0.83 0.756 0.215 0.0493 0.19 0.98 T4 19 0.81 0.702 0.257 0.059 0.1 0.95 OC Baseline 20 18.737 22.839 25.389 5.677 0.625 93.405 T0 20 14.315 23.383 27.19 6.08 0.76 99.88 T1 20 16.52 22.296 26.031 5.821 0.49 104.3 T2 20 11.71 19.138 21.452 4.797 0.49 82.52 T3 19 11.49 18.929 27.874 6.395 0.49 97.69 T4 19 8.82 15.575 22.225 5.099 0.51 95.5 OPN Baseline 20 2140.8 2150.9 593.6 132.7 1165 4093.5 T0 20 2122 2037.7 43 5.2 97.307 1165 2758 T1 20 2122 2264.1 969.7 216.8 1165 6078 T2 20 2122 2120.9 531 118.7 1111 3676 T3 19 2135 2364.4 1431.3 328.4 1165 8143 T4 19 2068 2067.4 324.8 74.519 1165 2639 IL 6 Baseline 20 1.09 1.125 0.169 0.0378 0.86 1.485 T0 20 1.12 1 .162 0.232 0.0519 0.66 1.69 T1 20 1.065 1.088 0.162 0.0363 0.89 1.48 T2 20 1.125 1.228 0.342 0.0764 0.89 2.37 T3 19 1.18 1.264 0.377 0.0865 0.89 2.58 T4 19 1.28 1.22 0.201 0.0462 0.89 1.61 IL 8 Baseline 20 162.4 186.4 158.2 35.377 17.53 618.8 T0 20 93.46 170.2 218.8 48.917 2.62 938.4 T1 20 148.5 202.5 191.3 42.785 10.09 680.4 T2 20 104.8 123.8 105.2 23.529 0.83 396.7

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45 Table 3 3. Continued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum IL 8 T3 19 59.75 1 39.8 152.3 34.936 0.65 465.1 T4 19 94.91 119.8 101.9 23.386 3.56 353.4 MMP 3 Baseline 20 2.47 2.66 1.107 0.248 1.615 6.415 T0 20 2.36 2.472 1.041 0.233 1.38 6.18 T1 20 2.41 2.848 1.907 0.426 1.38 10.47 T2 20 2.36 2.617 1.25 0.279 1.61 6.67 T3 19 2.84 3.612 2.843 0.652 1.63 14.59 T4 19 2.84 2.878 1.369 0.314 1.85 8.13 MMP 9 Baseline 20 2366 6653.6 10945 2447.3 276.4 45891 T0 20 2265.5 3825.6 4902.4 1096.2 334.6 20307 T1 20 1906 9481.7 19282 4311.6 218.2 79402 T2 20 2821 4820.3 6438.4 1439 .7 51.18 23027 T3 19 7736 12009 13183 3024.4 33.17 43820 T4 19 5553 13593 23577 5409 511.4 105431 M CSF Baseline 20 42.073 44.696 14.349 3.208 18.055 72.61 T0 20 38.745 47.077 21.322 4.768 20.15 100.5 T1 20 42.505 42.316 16.223 3.627 15.96 85.83 T2 20 46.755 48.742 27.283 6.101 20.15 138.2 T3 19 42.97 56.962 43.023 9.87 22.23 211.4 T4 19 50.14 55.699 22.311 5.119 24.63 108.7 IFN Baseline 20 2.525 2.322 0.964 0.215 1.04 3.88 T0 20 2.1 2.159 0.917 0.205 1.04 3.86 T1 20 2.525 2.485 1.21 0.271 0.72 4.81 T2 20 2.1 2.097 1.071 0.24 0.72 3.86 T3 19 2.1 2.269 1.419 0.326 0.72 5.78 T4 19 1.51 1.981 0.948 0.218 0.72 3.86

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46 Table 3 4 Biomarker Levels (in pg/mL) Groups Combined Summary Statistics Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum IL Baseline 41 102.4 125.3 94.223 14.715 19.14 404.6 T0 41 108.1 129.1 103.9 16.231 3.61 415.7 T1 41 92.8 121.4 111.8 17.455 3.98 393.6 T2 41 72.84 87.743 70.339 10.985 1.83 297.6 T3 39 65.98 96.893 101.4 16.238 3.19 416 T4 40 68.31 106.3 97.496 15.415 6.8 457.2 IL 1RA Baseline 41 29066 32731 20319 3173.2 27.205 92163 T0 41 36322 39043 28422 4438.8 40.07 99685 T1 41 19659 26419 26531 4143.5 14.34 104204 T2 41 34335 36220 27113 4234.4 70.36 90889 T3 39 37295 48634 37350 5980.8 1451 168350 T4 40 24584 32972 29238 4623 17.56 98420 IL (IL 1RA) Baseline 41 0.005 0.0413 0.116 0.0181 0 0.505 T0 41 0 0.0073 0.0166 0.0026 0 0.1 T1 41 0 0.0754 0.222 0.0347 0 0.91 T2 41 0 0.0205 0.112 0.0175 0 0.72 T3 39 0 0.0018 0.0056 0.0009 0 0.03 T4 40 0 0.0908 0.223 0.0353 0 0.84 RANKL Baseline 41 23.565 23.67 5.516 0.862 14.645 44.035 T0 41 25.01 24.113 6.164 0.963 15.44 41.61 T1 41 22.09 23.228 6.544 1.022 12.08 46.46 T2 41 23.56 22.947 7.299 1.14 12.08 51.38 T3 39 22.79 23.343 6.099 0.977 15.44 46.46 T4 40 23.39 23.43 6.281 0.993 8.98 34.5 OPG Baseline 41 5.75 7.945 7.637 1.193 0.39 36.565 T0 41 4.16 10.108 12.871 2.01 0.14 57.46 T1 41 4.13 5.783 4.962 0.775 0.39 22.02 T2 41 3.16 11.219 26.072 4.072 0.39 160.6 T3 39 3.31 9.667 17.435 2.792 0.39 79.36

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47 Table 3 4 Continued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum OPG T4 40 3.935 13.09 21.567 3.41 0.93 98.03 R ANKL/ (RANKL+OPG) Baseline 41 0.815 0.79 0.138 0.0216 0.455 0.98 T0 41 0.83 0.767 0.198 0.0309 0.31 0.99 T1 41 0.86 0.813 0.124 0.0194 0.52 0.98 T2 41 0.88 0.785 0.215 0.0336 0.11 0.98 T3 39 0.87 0.791 0.201 0.0322 0.19 0.98 T4 40 0.85 0.746 0.228 0.0361 0.1 0.96 OC Baseline 41 21.41 34.762 38.088 5.948 0.625 154.3 T0 41 21.55 36.68 41.277 6.446 0.49 182.8 T1 41 21.55 32.844 36.515 5.703 0.49 125.8 T2 41 13.15 31.145 34.447 5.38 0.49 104.3 T3 39 11.49 28.292 34.1 5.46 0.49 97.69 T4 40 8.82 15.439 17.451 2.759 0.49 95.5 OPN Baseline 41 2101.5 2006.2 617.8 96.488 1143.5 4093.5 T0 41 2068 1963 548.1 85.601 1122 3625 T1 41 2056 2049.4 826.6 129.1 1133 6078 T2 41 2056 1979.4 562.2 87.796 1111 3676 T3 39 1949 2088.4 1113.1 178.2 1144 8143 T4 40 2135 2070.8 278.3 44.006 1165 2639 IL 6 Baseline 41 1.09 1.146 0.259 0.0405 0.82 2.285 T0 41 1.06 1.136 0.296 0.0462 0.66 2.37 T1 41 1.09 1.157 0.283 0.0442 0.74 2.2 T2 41 1.09 1.194 0.312 0.0487 0.82 2.37 T3 39 1.18 1.224 0.329 0.0527 0.59 2.58 T4 40 1.28 1.238 0.22 0.0348 0.89 1.69 IL 8 Baseline 41 134.3 181.2 178.6 27.885 6.755 882.1 T0 41 138.5 202.4 242.5 37.869 2.62 995.5 T1 41 91.27 160 183.4 28.639 3.65 768.8 T2 41 95.31 129.2 153.9 24.033 0.83 875.7

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48 Table 3 4. Continued Biomarker Time Point N Median Mean Standard Deviation Standard Error Minimum Maximum IL 8 T3 39 52.95 118.6 168.1 26.912 0.65 823.2 T4 40 35.785 85.855 98.371 15.554 2.12 353.4 MMP 3 Baseline 41 2.39 2.557 1.045 0.163 1.405 6.415 T0 41 2.36 2.489 1.289 0.201 1.17 8.12 T1 41 2.36 2.624 1.526 0.238 0.96 10.47 T2 41 2.36 2.448 1.05 0.164 0.96 6.67 T3 39 3.06 4.813 8.507 1.362 1.38 54.77 T4 40 2.41 3.007 1.955 0.309 1.38 12.62 MMP 9 Baseline 41 2797.1 6350.9 8980.2 1402.5 152.1 45891 T0 41 2674 5151.5 7140.4 1115.1 132.6 34340 T1 41 1987 7550.3 14808 2312.7 78.71 79402 T2 41 3541 5780.6 7108 .5 1110.2 51.18 30367 T3 39 6706 15549 23060 3692.6 33.17 104441 T4 40 4426.5 11729 19293 3050.5 96.78 105431 M CSF Baseline 41 44.56 49.122 19.966 3.118 18.055 119.8 T0 41 47.99 52.547 26.09 4.075 20.15 146.3 T1 41 44.7 45.697 21.755 3.398 13.99 108.4 T2 41 44.7 47.915 23.879 3.729 18.02 138.2 T3 39 44.7 52.647 33.229 5.321 20.15 211.4 T4 40 42.97 50.145 21.502 3.4 20.15 108.7 IFN Baseline 41 2.525 2.329 0.916 0.143 0.66 3.88 T0 41 2.35 2.231 0.994 0.155 0.35 3.86 T1 41 2.1 2.426 1.093 0.171 0.72 4.81 T2 41 2.1 2.219 1.019 0.159 0.35 3.86 T3 39 2.1 2.256 1.231 0.197 0.35 5.78 T4 40 1.51 1.854 0.874 0.138 0.35 3.86

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49 Figure 3 1. IL 1 Median Value s Figure 3 2. IL 1RA Median Values 0 20 40 60 80 100 120 Baseline T2 T3 T4 Concentration (pg/mL) TIme Point IL 1 AS Group SA Group Groups Combined 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Baseline T2 T3 T4 Concentration (pg/mL) Time Point IL 1RA AS Group SA Group Groups Combined

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50 Figure 3 3 RANKL/(RANKL+OPG) Median Values Figure 3 4 IL 6 Median Values 0.74 0.76 0.78 0.8 0.82 0.84 0.86 0.88 0.9 Baseline T2 T3 T4 Concentraion (pg/mL) Time Point RANKL/(RANKL+OPG) AS Group SA Group Groups Combined 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 Baseline T2 T3 T4 Concentration (pg/mL) Time Point IL 6 AS Group SA Group Groups Combined

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51 Figure 3 5 IL 8 Median Values Figure 3 6 MMP 3 Median Values 0 20 40 60 80 100 120 140 160 180 Baseline T2 T3 T4 Concentration (pg/mL) Time Point IL 8 AS Group SA Group Groups Combined 0 0.5 1 1.5 2 2.5 3 3.5 Baseline T2 T3 T4 Concentration (pg/mL) Time Point MMP 3 AS Group SA Group Groups Combined

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52 Figure 3 7 MMP 9 Median V alues Figure 3 8. Box Plots D isplaying MMP 9 Concentration in GCF Samples at Different T ime points T hroughout the Study ( 0, baseline 1; 1, baseline 2; B, average between the two baseline measurements; 2, equivalent to T2; 3, equivalent to T3 and 4; equi valent to T4 ) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 Baseline T2 T3 T4 Concentration (pg/mL) Time Point MMP 9 AS Group SA Group Groups Combined

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53 Figure 3 9 IFN Median Values Table 3 5 B aseline Measurements of Bi omarkers Biomarker Estimate S tandard Error p value IL 1 0.35 0.12 0.0048* IL 1RA 0.62 0.20 0.0036* RANKL 0.96 0.10 <.0001* OPG 0.95 0.32 0.0054* OC 0.75 0.07 <.0001* OP N 0.61 0.16 0.0004* IL 6 0.72 0.11 <.0001* IL 8 0.00 0.10 0.97 MMP 3 2.22 0.60 0.0007* MMP 9 0.49 0.22 0.0320* M CSF 0.16 0.19 0.41 IFN 0.96 0.11 <.0001* denotes significance p <0.05 Table 3 6 Period Effects on Biomarkers at T2 Biomarker Estimate Standard Error p valu e IL 1 9.37 14.05 0.51 IL 1RA 12621 5580 0.0297* RANKL 0.58 0.65 0.38 OPG 1.60 4.65 0.73 OC 2.28 2.99 0.45 OP N 103 165 0.54 IL 6 0.03 0.06 0.59 IL 8 9.53 35.96 0.79 MMP 3 0.37 0.10 0.0010* MMP 9 9714 3333 0.0060* M CSF 5.07 5.92 0.40 I FN 0.04 0.13 0.15 denotes significance p <0.05 0 0.5 1 1.5 2 2.5 3 Baseline T2 T3 T4 Concentration (pg/mL) Time Point IFN AS Group SA Group Groups Combined

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54 Table 3 7 Sequence (AS or SA) Effects on Biomarkers Biomarker Estimate Standard Error p value IL 1 2.74 21.75 0.90 IL 1RA 1414 7855 0.86 RANKL 1.84 1.07 0.10 OPG 3.04 4.89 0.54 OC 1.42 6.61 0. 80 OPN 209 190 0.28 IL 6 0.10 0.06 0.11 IL 8 15.49 37.18 0.68 MMP 3 1.49 1.25 0.24 MMP 9 4665 3979 0.25 M CSF 6.33 7.22 0.39 IFN 0.03 0.19 0.89 *denotes significance p <0.05 Table 3 8 Treatment Effects on Biomarkers Biomarker Estimate Sta ndard Error p value IL 6.31 14.05 0.66 IL 1RA 2988 5585 0.60 RANKL 0.38 0.65 0.56 OPG 4.26 4.65 0.37 OC 4.67 3.00 0.13 OPN 107 165 0.52 IL 6 0.00 0.06 0.99 IL 8 26.15 35.97 0.47 MMP 3 1.38 1.24 0.28 MMP 9 2499 3333 0.46 M CSF 3.37 5.92 0.57 IFN 0.20 0.13 0.15 *denotes significance p <0.05 Table 3 9 Significant Correlations Between Tooth Movement and GCF Levels Tooth Movement Weeks 1 2 Tooth Movement Weeks 1 12 GCF Baseline (T0) MMP 9 ( 0.35, 0.03*) IL 6 ( 0.29, 0.07*) IL 1R A (0.27, 0.09*) GCF Week 12 (T4) No significant correlations MMP 9 (0.27, 0.10*) IL 1RA (0.34, 0.03*) *denotes significance of p 0.10 All are Spearman rank correlations except IL 6 which is a Pearson correlation.

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55 Figure 3 10. Correlation Between MMP 9 Levels and Tooth Movement Weeks 1 2 Figure 3 11. Correlation Between IL 1 RA Levels and Tooth Movement Weeks 1 2

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56 Figure 3 12. Correlation B etween IL 6 L evels and Tooth Movement Weeks 1 12 Figur e 3 13. Correlation B etween MMP 9 Levels and Too th Movement Weeks 1 12

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57 Figure 3 14. Correlation Between IL 1RA Levels and Tooth Movement Weeks 1 12

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58 CHAPTER 4 DISCUSSION Main F indings W ith the advent of constantly changing technology and improved research methods, the science of orthodontics is evol ving at a rapid pace. The goal of orthodontic treatment planning is to use the most efficient methods to optimize the sp eed of tooth movement while avoiding adverse events such as root resorption or bone loss. If a practitioner could know in advance what t issue responses are most likely to occur then he or she will be able to provide individualized treatment and avo id potential problems. Through dev portable chair side assay could provide information regarding a could formulate an individualized treatment plan 76 B iomarker an alysis in GCF has been recognized as a promising non invasive tool to evaluate the complex inflammatory cascade and tissue remodeling processes elicited during orthodontic tooth movement. It is imperative that research be conducted to determine the most re levant biomarkers for diagnosis and treatment planning to include in these assays. The current study chose to evaluate protein markers based on their biological relevance as previously described In evaluating the GCF biomarker levels between the AS and SA G roups th e main time points of focus were T2 (during the first week after orthodontic movement began) and T3 ( during the first week after the crossover ) since the samples were taken within one week after aligner delivery. Therefore, these time points wou ld be more likely to show biomarker fluctuation s produced by an orthodontic force and/or a vibrating device such as Acce leDent Aura In addition, we chose to collect GCF two weeks after aligner delivery at T4 since GCF biomarker levels may

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59 behave differen t ly at a time point where a de cline in force level is taking place Our initial assumption was that if the active AcceleDent Aura device had a profound effect in periodontal tissue remodeling during tooth movement, the concentration of biomarkers in GCF w ould sense it. However, even though small fluctuations were seen (Figures 3 1 to 3 8 ) none of the biomarker levels or ratios were significantly different comparing active treatment to sham treatment In addition, no sequence (Table 3 7) or treatment (Tabl e 3 8) effects were visualized for any of the biomarker s Therefore, the results from our study suggest that biomarker quantitation in GCF is not a method sensitive enough to capture the periodontal stimulation el icited by the vibrating device. A few promi sing trends were, however, uncovered. As previously stated, baseline levels were significantly predictive of future measurements for all biomarkers except for IL 8 and M CSF (Table 3 5). This implies that baseline measurements should be incorporated in GCF studies for the information they provide about future levels Since no significant differences were found between the active and sham devices, the SA and AS Groups biomarker concentrat ions were combined at each time point to achieve our secondary aim of i nvestigating correlations between GCF biomarker levels and tooth movement. The present study confirms that the interleukin system plays a role in tooth movement since significant correlations were detected between the amount of tooth movement and the GCF l evels of IL 1RA and IL 6 The levels of the anti inflammatory cytokine IL 1RA were reported to be decreased during tooth movement by previous groups 40,42 IL 1RA acts as a receptor antagonist, blocking IL inflammatory cytokine that increases osteoclast activity and bone resorption 80 Therefore, reduced levels of IL 1RA would indicate that more IL

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60 available leading to higher levels of activated osteoclasts bone resorption, and ultimately tooth movement Surprisingly, o ur result shows an increase in IL1 RA concentration over time which was unexpected In addition, the positive correlation s between IL 1RA GCF levels and tooth movement does not substantiate the biological effect of this protein. A negative correlation would have been expected based on previous research showing that l ower GCF concentrations of I L 1RA and higher ratios of IL IL 1RA are associated with higher tooth movement velocity. 40 42,81 Since the IL 1RA) results in our study were inconclusive, this positive correlation between IL 1RA an d tooth movement cannot be supported by the ratio and the relevance of thi s finding may be insignificant. A negative c orrelation was found between the baseline level of IL 6 and the amount of tooth movement throughout the study from weeks 1 to 12 (Pearson = 0.29). Therefore, our data suggest s that if baseline levels of IL 6 are higher than normal, less OTM could be expected after 12 weeks. This is an interesting result since the exact role that IL 6 plays in bone remodeling is still very controversial 46 Previous o rthodontic studies focus ed on th is inflammatory properties, namely that IL 6 stimulates osteoclast formation through upregulating R ANKL expression by osteoblasts 37,82 Nevertheless, it is important to emphasize that other groups report the opposite implicating that IL 6 inhibits RANKL induced osteoclastogenesis by diverting progenitor cells into the macrophage lineag e. 83 These dual roles may result from the stage of differentiation the osteoblast is in. 46 M aximum levels of IL 6 have been reported in rats at day 3 followin g orthodontic force application and declining th ereafter 37 while a human stud y showed a peak in IL 6 24 hours after the start of orthodontic tooth

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61 movement and then declining until the end of the study. 84 Another stud y s how ed no increases in IL 6 after day 7 and day 21. 85 Differences in periodicity of data collection make correlations difficult, but the peak in IL 6 could have occurred undetected by our data because it was not sta tistically significant. Furthermore, the negative correlation between baseline IL 6 and OTM after 12 weeks in this study reinforces the inhibitory effect in bone remodeling. Further studies are needed to explore the diagnostic and predictive val ue of IL 6 in assessing tooth movement. Multiple studies have explored the effects of MMPs on degradati on of the extracellular matrix and tooth movement 6,53 Levels of MMP 9 were higher than those of MMP 3 confirmi ng the results of previous studies 54 One of the most interesting findings in our study was the negative correlation between baseline MMP 9 levels and tooth movement during the first week switching to a positive co rrelation between MMP 9 levels at the end of the study and total tooth movement achieved between weeks 1 12 Gelatinase MMP 9 functions to catabolize the extracellular matrix of the periodontal tissue and inhibition of this molecule in rat models decreases OTM. 86,87 The association between GCF levels of MMP 9 and orthodontic tooth movement is well documented by Capelli et al. 54 The authors found a significant increase in MMP 9 conc entration on the pressure side one hour after force appli cation which sharply decreased one day after the start of tooth movement. From this point, a steady increase in MMP 9 concentration was visualized. The highest levels of MMP 9 in the Capelli e t al. 54 study were seen at the last time point (d ay 80) after the orthodontic force was first applied to retract the upper canines I n our study MMP 9 significantly increased at approximately 40 days after tooth m ovement began (T3) and remained above baseline levels at T4 (d ay 84).

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62 This, combin ed with the positive correlation between MMP 9 levels and tooth movement at the end of our study seems to indicate that this biomarker has a delayed response, only spiking in GCF after the periodontium undergo es extensive and substantial tissue remodeling If this proves to be true, MMP 9 should be incorporated as part of a panel to yield a biomarker signature capable of assessing tissue response during orthodontic treatment The trend of increa sing RANKL / (RANKL+OPG) ratios (Figure 3 3) between baseline and T2 supports the finding of Kawasaki et al. 33 of an increased ratio 24 hours after the start of OTM. This was attributed to signific antly elevated levels of RANKL combined with significantly decre ased levels of OPG at this time point in their study The decrease in OPG during the first week of tooth movement was not statistically significant in our study, but it does support this trend J ust as the ratio decreased after T2 in our study, the RANKL/ (RANKL+ OPG ) ratio also decreased at 168 hours after force application in the Kawasaki 33 study. These findings confirm the important role of the RANKL/( RANKL+OPG) ratio in explaining the cellular dynamics of OTM. Limitations Limitations of our study include the inherent carryover effect that is most likely occurring throughout the study. Tooth movement occurred with both the sham and active devices. At th e crossover, biomarkers were still present fr om the previous period making it difficult to know if biomarker levels fluctuated due to the current intervention or due to the cumulative effect of general tooth movement. A l ongitudinal study of individual act ive and sham groups that does not involve a crossover may be more appropriate to evaluate AcceleDent Aura effects on biomarker levels. Had the study not been a crossover study, the sham group would have acted as the control group

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63 since they would have bee n devoid of any active intervention. However, the advantage of each subject serving as their own control cannot be overlooked in treatment comparison studies. In addition, the majority of biomarker studies are done in a split mouth design in order for the side without intervention to serve as a control. Due to the full coverage mouthpiece of the AcceleDent Aura device, a split mouth design was not possible. Another significant limitation of the study lies in the timing of GCF collection. For the crucial T2 and T3 time points, GCF was collected between 1 and 7 days after the new aligner and device were given. However, a recent systematic review 29 of the orthodontic effects on cytokine levels in GCF concluded that biomarker levels attain their highest values at 24 hours after a force is applied. Thus, subjects that had GCF collected on the first day after force delivery may have higher values than subjects who had GCF collected 7 days after the force was applied. T he intermittent forces generated by the aligners may evoke different patte rns of biomarker concentrations than a constant force from a coil spring for example. Biomarker levels may also be more difficult to detect in this aligner study since there is less bone remodeling occurring with incisor tipping than would be expected with bodily retraction of a canine using a coil spring There are two major prob lems that exist with all of the human studies with GCF collection, the small sample sizes and the large individual variations in biomarker measurements. With greater pools of people of different ages, races, sexes, and socioeconomic backgrounds, scientists will be able to create assays for different groups. With only a small number of patients, it is difficult to draw overarching

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64 conclusions regarding biomarker prevalence and trends Secondly, many studies were performed as short term observations upon one appliance activation. Long term studies would show the larger picture from before treatment, through the different stages of tooth movement, and past retention. Through completing longer studies with more subjects using a universal protocol for GCF collect ion time periods, and assay type, researchers around the globe would be able to collaborate and form an orthodontic clinical proteomic library from which i ndividual assays could be made. In addition to these challenges, GCF collection is inherently techni que sensitive. C ollection sites must be free of any gingival inflammation so that the factors collected are known to be associated with tooth movement, not a result of plaque induced inflammation. Furthermore, research subjects for these studies should not have a history of smoking, be taking any anti inflammatory, corticosteroid, or antibiotic medications, and have no history of periodontal disease. In addition, since the values for biomarker concentration are so minute, extreme outliers can skew the data and very small amounts can be difficult to evaluate without log transformations, both of which were done with samples in the current study Future studies should focus on calculating ratios for biomarkers and their antagonists in order to uncover the full picture of biomarker activation and inhibition occurring in a location. Generalizability To our knowledge, this is the first report of GCF collection with aligner therapy for the biomarkers studied and the first report of evaluation of GCF biomarkers with the AcceleDent Aura device. One study 88 published in 2013 examined the changes of substance P (SP) in the GCF of four young patients treated with aligner therapy (Invisalign technique). They found SP was present in aligner treated patients but not in

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65 control subjects who were not undergoing orthodontic treatment. The current study opens the door for further investigation regarding GCF biomarker levels during aligner therapy. As technology advances and additional long term longitudinal studies are completed, contemporary practitioners need to be cognizant of emerging literature in order to best treat their patients. A well formulated chair side biomarker analysis will help ort ho dontists make smarter decisions, providing the highest level of patient care while also saving time, money, and avoiding adverse orthodontic outcomes. It is up to the individual practitioner to create his or her own evidence based approach by critically reviewing biomarker studies associated with new devices and technology and subsequently treating patients accordingly.

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66 CHAPTER 5 CONCLUSIONS The objective of investigating the effect of vibratory stimulation with AcceleDent Aura on the expression of bio markers using a human tooth movement model with clear aligners was achieved. Upon combining the sham and active group data, s ome correlations were able to be ma de between specific biomarkers and tooth movement that may provide insight about their roles in each stage of orthodontic tooth movement. Future studies are needed to confirm these trends. We could not detect additional differences in biomarker expression during the use of the AcceleDent Aura device to accelerate tooth movement. Therefore, the resul ts suggest that vibration does not influence the secretion of GCF biomarkers. This study was registered on https://clinicaltrials.gov (ClinicalTrials.gov Identifier: NCT01962012 ). It was approved by the Western Institutional Review Board (WIRB # 20131856 ). This study was funded by OrthoAccel Technologies

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67 A PPENDIX A BIOMARKER DATA FROM ACTIVE SHAM GROUP Table A 1 IL 1 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days ) T3 (43 49 days) T4 (84 days) A001 59.92 10.40 37.02 9.05 26.27 A002 8.75 73.75 14.26 5.31 90.84 A003 162.52 113.41 104.85 65.98 20.64 A004 108.10 25.49 80.18 47.35 60.52 A005 326.93 182.13 31.73 107.25 134.52 A007 202.60 3.98 108.27 416.02 22.29 A 008 70.87 17.10 1.83 30.91 27.93 A010 415.69 393.55 50.27 27.21 50.46 A013 127.85 112.05 67.59 69.56 54.59 A017 126.71 92.80 150.15 159.27 63.48 A018 29.26 201.04 169.70 105.53 293.56 A020 23.62 63.65 4.61 37.06 32.41 A024 83.81 28.60 72.84 N/A* 36.7 3 A028 64.84 14.10 101.34 51.29 63.01 A030 227.35 321.64 126.20 100.62 224.67 A032 173.37 308.23 297.56 168.75 203.63 A033 36.12 29.78 61.37 22.68 45.39 A034 200.78 11.16 4.16 14.84 169.11 A037 61.74 15.03 50.00 7.92 67.39 A042 373.50 259.03 254.37 388.04 252.76 A044 252.14 5.46 118.40 64.64 6.80 Mean 149.36 108.68 90.80 94.96 92.71 Median 126.71 63.65 72.84 57.97 60.52 *N/A: not assessed

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68 Table A 2. IL 1RA Levels (in pg/mL) for First device Active 1st device: Active 2nd Device: Sham Time p oints Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 39386.00 18746.00 34335.00 58430.00 52939.00 A002 6444.00 21659.00 56774.00 8076.00 79956.00 A003 8019.00 28817.00 19323.00 31750.00 17.56 A004 38221.00 35826.00 74449 .00 115188.00 14653.00 A005 50074.00 15267.00 7883.00 33160.00 20663.00 A007 31738.0 79277.0 2527.0 14935.0 38443.00 A008 2386.00 1593.00 3509.00 27478.00 2742.00 A010 62908.00 30232.00 29202.00 41375.00 11343.00 A013 88350.00 95976.00 90889.00 168350 .00 45777.00 A017 11503.00 146.16 47449.00 53114.00 4500.00 A018 56407.00 35526.00 30794.00 91058.00 79.47 A020 1192.0 11794.0 1901.0 13497.0 39131.00 A024 66999.00 86638.00 12949.00 N/A* 90992.00 A028 72487.00 8327.00 6655.00 27857.00 31627.00 A030 96490.00 12849.00 90016.00 44919.00 12616.00 A032 29979.00 158.64 23902.00 14074.00 37.40 A033 14761.0 8888.0 41952.0 39390.0 28526.00 A034 52201.0 13334.0 12716.0 3340.0 136.66 A037 49380.0 4244.0 12280.0 18007.0 13478.00 A042 17653.00 29153.00 53418 .00 86093.00 61740.00 A044 53412.00 2073.00 67518.00 97421.00 16740.00 Mean 40475.71 25739.23 34306.71 49375.60 26958.91 Median 39386.00 15267.00 29202.00 36275.00 16740.00 *N/A: not assessed

PAGE 69

69 Table A 3. IL 1 1RA) Ratio in pg/mL for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 0.00 0.00 0.00 0.00 0.00 A002 0.00 0.00 0.00 0.00 0.00 A003 0.02 0.00 0.01 0.00 0.54 A004 0.00 0.00 0.00 0. 00 0.00 A005 0.01 0.01 0.00 0.00 0.01 A007 0.01 0.00 0.04 0.03 0.00 A008 0.03 0.01 0.00 0.00 0.01 A010 0.01 0.01 0.00 0.00 0.00 A013 0.00 0.00 0.00 0.00 0.00 A017 0.01 0.39 0.00 0.00 0.01 A018 0.00 0.01 0.01 0.00 0.79 A020 0.02 0.01 0.00 0.00 0.00 A024 0.00 0.00 0.01 N/A* 0.00 A028 0.00 0.00 0.01 0.00 0.00 A030 0.00 0.02 0.00 0.00 0.02 A032 0.01 0.66 0.01 0.01 0.84 A033 0.00 0.00 0.00 0.00 0.00 A034 0.00 0.00 0.00 0.00 0.55 A037 0.00 0.00 0.00 0.00 0.00 A042 0.02 0.01 0.00 0.00 0.00 A044 0. 00 0.00 0.00 0.00 0.00 Mean 0.01 0.05 0.01 0.00 0.13 Median 0.00 0.00 0.00 0.00 0.00 *N/A: not assessed

PAGE 70

70 Table A 4 RANKL Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 22.09 22.09 28.05 28.05 25.01 A002 28.05 26.54 26.54 28.05 30.05 A003 23.56 22.09 28.05 22.09 28.34 A004 26.54 29.58 22.09 25.04 25.01 A005 28.05 28.05 22.09 26.54 23.39 A007 25.04 22.09 25.04 26.54 21.80 A008 28.05 20.64 25.04 28.05 28.34 A010 25.01 28.34 28.34 28.34 18.72 A013 25.01 21.80 25.01 30.05 28.34 A017 25.01 28.34 28.34 23.39 33.55 A018 26.66 35.33 25.01 28.34 31.79 A020 21.80 31.79 31.79 28.34 31.79 A024 30.05 25.01 25.01 N/A* 28.34 A028 17.21 15.44 22.79 19.02 19.02 A030 15.44 15.44 19.02 15.44 20.88 A032 22.79 19.02 19.02 22.79 22.79 A033 30.76 12.08 19.02 17.21 19.02 A034 19.02 22.79 15.44 15.44 19.02 A037 19.02 19.02 15.44 17.21 12.08 A042 17.21 12.08 15.44 19.02 19.02 A044 41.6 1 46.46 51.38 46.46 34.50 Mean 24.67 24.00 24.66 24.77 24.80 Median 25.01 22.09 25.01 25.79 25.01 *N/A: not assessed

PAGE 71

71 Table A 5 OPG Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (d ay 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 35.89 5.48 7.32 5.48 27.27 A002 1.76 2.71 1.42 1.76 2.96 A003 3.11 2.90 3.31 3.31 1.69 A004 1.94 3.31 4.59 3.31 4.13 A005 4.16 3.73 1.76 2.13 3.24 A007 14.87 1.94 6.86 26.47 23.63 A008 2.32 1.42 1.42 1.76 2.96 A010 12.58 3.53 1.92 1.69 1.05 A013 16.85 9.58 9.58 7.43 9.58 A017 32.94 14.11 43.86 78.61 38.61 A018 1.92 13.34 34.16 4.13 3.53 A020 6.06 12.19 1.92 5.39 1.47 A024 11.44 2.96 3.83 N/A* 16.45 A028 1.59 0.73 0.39 0.39 2.61 A030 3.16 2.61 0.73 1.83 2.61 A032 3.16 2.09 2.09 2.09 1.14 A033 13.14 3.16 160.62 1.14 15.51 A034 8.78 0.73 0.55 1.59 3.16 A037 39.64 4.33 12.12 3.16 0.93 A042 8.78 5.56 3.16 16.87 31.06 A044 3.81 1.29 4.88 7.16 2.15 Mean 10.85 4.65 14.59 8.79 9.32 Median 6 .06 3.16 3.31 3.24 3.16 *N/A: not assessed

PAGE 72

72 Table A 6 RANKL/(RANKL+OPG) Ratio in pg/mL for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 0.38 0.80 0.79 0.84 0.48 A002 0.94 0.91 0.95 0.94 0.91 A003 0.88 0.88 0.89 0.87 0.94 A004 0.93 0.90 0.83 0.88 0.86 A005 0.87 0.88 0.93 0.93 0.88 A007 0.63 0.92 0.78 0.50 0.48 A008 0.92 0.94 0.95 0.94 0.91 A010 0.67 0.89 0.94 0.94 0.95 A013 0.60 0.69 0.72 0. 80 0.75 A017 0.43 0.67 0.39 0.23 0.46 A018 0.93 0.73 0.42 0.87 0.90 A020 0.78 0.72 0.94 0.84 0.96 A024 0.72 0.89 0.87 N/A* 0.63 A028 0.92 0.95 0.98 0.98 0.88 A030 0.83 0.86 0.96 0.89 0.89 A032 0.88 0.90 0.90 0.92 0.95 A033 0.70 0.79 0.11 0.94 0.55 A034 0.68 0.97 0.97 0.91 0.86 A037 0.32 0.81 0.56 0.84 0.93 A042 0.66 0.68 0.83 0.53 0.38 A044 0.92 0.97 0.91 0.87 0.94 Mean 0.74 0.85 0.79 0.82 0.78 Median 0.78 0.88 0.89 0.88 0.88 N/A: not assessed

PAGE 73

73 Table A 7 Osteocalcin Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 88.98 93.32 95.50 95.50 21.55 A002 91.14 86.82 95.50 91.14 21.55 A003 95.50 78.25 97.69 91.14 26.99 A 004 88.98 91.14 80.38 86.82 8.82 A005 117.65 95.50 95.50 84.66 44.34 A007 82.52 97.69 86.82 91.14 38.42 A008 76.13 82.52 104.29 57.45 11.49 A010 11.49 21.55 11.49 11.49 8.82 A013 182.78 125.84 68.84 32.63 8.82 A017 32.63 11.49 21.55 8.82 16.35 A018 21.55 32.63 56.46 21.55 32.63 A020 32.63 21.55 8.82 8.82 8.82 A024 8.82 8.82 8.82 N/A* 8.82 A028 15.76 10.77 10.77 13.15 10.77 A030 10.77 15.76 13.15 18.57 8.61 A032 15.76 8.61 15.76 8.61 13.15 A033 13.15 9.66 8.61 3.56 4.99 A034 7.61 3.56 3.56 7.61 1.88 A037 2.37 0.49 1.07 1.44 0.51 A042 0.49 1.44 0.49 0.49 0.49 A044 39.52 3.30 9.13 9.13 23.82 Mean 49.34 42.89 42.58 37.19 15.32 Median 32.63 21.55 15.76 15.86 10.77 *N/A: not assessed

PAGE 74

74 Table A 8 Osteopontin Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 1122.00 1185.00 1165.00 1224.00 2135.00 A002 1165.00 1175.00 1185.00 1165.00 2201.00 A003 1205.00 1133.00 1185.00 1 185.00 2101.00 A004 1185.00 1185.00 1144.00 1144.00 2265.00 A005 1185.00 1260.00 1165.00 1165.00 2265.00 A007 1175.00 1165.00 1205.00 1242.00 2168.00 A008 1122.00 1165.00 1185.00 1144.00 2393.00 A010 3625.00 3625.00 3146.00 3200.00 2265.00 A013 2639. 00 2201.00 2265.00 1931.00 2068.00 A017 2233.00 2330.00 2135.00 2265.00 2265.00 A018 2168.00 2135.00 2669.00 2330.00 2135.00 A020 2135.00 2517.00 2330.00 2361.00 2068.00 A024 2330.00 2265.00 2393.00 N/A* 2135.00 A028 2003.00 1841.00 1949.00 1949.00 22 64.00 A030 1949.00 1730.00 1949.00 1674.00 2161.00 A032 2161.00 2213.00 1949.00 1895.00 2161.00 A033 1730.00 1786.00 2161.00 1841.00 1503.00 A034 2264.00 2264.00 1730.00 1949.00 1730.00 A037 2366.00 1841.00 1949.00 2900.00 1618.00 A042 1949.00 1841.0 0 2056.00 1445.00 1895.00 A044 2019.00 1888.00 1822.00 2516.00 1756.00 Mean 1891.90 1845.00 1844.62 1826.25 2073.90 Median 2003.00 1841.00 1949.00 1868.00 2135.00 *N/A: not assessed

PAGE 75

75 Table A 9 IL 6 Levels (in pg/mL) for First Device Active 1st devi ce: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 0.73 1.16 1.16 1.35 1.48 A002 1.16 0.98 0.82 0.98 1.28 A003 0.82 0.82 1.16 0.98 1.48 A004 0.82 1.16 0.82 0.98 1.38 A005 0.98 1.35 0.98 0.98 1.69 A007 0.90 1.35 1.16 1.07 1.38 A008 0.98 1.16 0.98 1.16 1.69 A010 2.37 2.20 2.05 2.05 1.28 A013 1.28 1.79 1.09 1.28 1.28 A017 0.90 1.48 1.28 1.28 1.09 A018 1.48 1.69 1.48 1.38 1.28 A020 1.09 1.09 1.09 1.48 1.09 A024 1.28 1.48 1.69 N/ A* 1.48 A028 0.89 0.89 1.06 0.59 1.23 A030 1.23 1.15 1.06 1.23 0.97 A032 0.97 0.74 1.23 1.23 1.23 A033 0.97 1.06 1.06 1.06 0.89 A034 1.42 1.06 1.06 1.15 1.06 A037 1.06 1.06 0.89 1.23 0.89 A042 0.97 0.81 1.06 1.06 0.89 A044 1.01 1.21 1.21 1.21 1.31 Mean 1.11 1.22 1.16 1.19 1.25 Median 0.98 1.16 1.09 1.19 1.28 *N/A: not assessed

PAGE 76

76 Table A 10 IL 8 Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 d ays) T4 (84 days) A001 152.69 11.72 54.23 28.83 5.79 A002 57.60 205.45 11.89 3.44 19.25 A003 71.83 42.53 180.71 147.91 14.68 A004 13.79 32.93 122.41 52.95 10.37 A005 710.98 260.88 51.28 59.50 206.99 A007 437.76 3.65 135.27 823.23 23.39 A008 213.41 5 5.22 5.36 70.50 6.73 A010 995.50 768.79 27.95 1.80 39.83 A013 166.54 111.64 280.43 39.25 16.26 A017 196.23 163.51 158.44 203.84 20.56 A018 10.92 24.63 875.66 28.90 15.24 A020 138.51 91.27 10.19 51.26 2.12 A024 128.85 188.23 304.80 N/A* 95.11 A028 20 5.55 42.58 220.57 54.88 31.74 A030 186.76 199.28 41.12 34.97 55.25 A032 134.56 58.77 134.97 47.51 44.22 A033 8.60 4.91 60.08 16.13 8.76 A034 713.94 21.36 6.01 10.93 329.44 A037 43.03 12.11 44.04 2.01 2.69 A042 255.50 204.34 42.03 258.29 205.00 A044 51.43 4.54 54.50 32.10 4.09 Mean 233.05 119.44 134.38 98.41 55.12 Median 152.69 55.22 54.50 43.38 19.25 *N/A: not assessed

PAGE 77

77 Table A 11 MMP 3 Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 2.67 2.67 1.58 4.79 2.41 A002 1.75 4.34 1.58 1.58 2.41 A003 1.58 1.92 1.92 3.69 2.41 A004 1.58 1.92 2.29 2.67 3.27 A005 2.11 2.67 1.92 8.12 2.01 A007 8.12 1.92 3.07 54.77 12.62 A008 1.5 8 1.92 0.96 4.34 4.43 A010 4.54 5.24 3.86 4.20 2.01 A013 3.27 3.27 3.05 3.27 3.27 A017 2.84 3.73 2.84 5.16 2.41 A018 1.63 2.84 4.19 2.41 2.84 A020 2.41 3.27 2.41 3.27 2.41 A024 2.21 2.01 1.63 N/A* 5.65 A028 1.38 1.85 1.38 1.38 1.38 A030 1.85 1.61 2 .36 2.90 2.36 A032 1.17 1.85 1.85 2.36 1.85 A033 3.46 1.38 2.90 3.46 1.85 A034 2.36 1.85 1.38 2.36 3.17 A037 1.85 0.96 2.10 1.85 1.38 A042 1.85 1.85 2.36 3.46 3.46 A044 2.39 1.58 2.39 3.06 1.97 Mean 2.50 2.41 2.29 5.96 3.12 Median 2.11 1.92 2.29 3. 27 2.41 *N/A: not assessed

PAGE 78

78 Table A 12 MMP 9 Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 12170.00 4918.00 7707.00 6863.00 2669.00 A002 3270.00 36714.00 6012.00 475.37 4288.00 A003 34340.00 11614.00 16079.00 63141.00 657.32 A004 983.33 1802.00 16818.00 19790.00 16722.00 A005 8019.00 21075.00 1826.00 14563.00 1674.00 A007 9715.00 408.39 18002.00 104441.00 23467.00 A008 80 8.12 1620.00 186.32 15817.00 4741.00 A010 26259.00 9510.00 1880.00 653.45 1002.00 A013 3522.00 3130.00 5592.00 25310.00 19911.00 A017 3539.00 11572.00 5668.00 83626.00 29679.00 A018 278.76 1663.00 30367.00 6474.00 1097.00 A020 4050.00 3761.00 147.42 5 765.00 936.00 A024 9338.00 2669.00 6453.00 N/A* 62383.00 A028 2293.00 351.23 1188.00 327.01 2958.00 A030 2674.00 4952.00 10175.00 6706.00 9003.00 A032 2400.00 895.44 2483.00 3535.00 1355.00 A033 132.57 171.57 1570.00 293.53 327.01 A034 5635.00 529.75 113.86 1398.00 16120.00 A037 1045.00 120.09 1163.00 311.82 96.78 A042 3031.00 2372.00 2313.00 5691.00 9645.00 A044 1196.00 78.71 4855.00 13043.00 2152.00 Mean 6414.23 5710.82 6695.17 18911.21 10042.05 Median 3270.00 2372.00 4855.00 6590.00 2958.00 N/A: not assessed

PAGE 79

79 Table A 13. M CSF Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 46.57 50.89 46.57 78.54 42.97 A002 38.23 1 08.36 42.35 38.23 42.97 A003 38.23 50.89 50.89 50.89 31.02 A004 57.55 38.23 42.35 71.39 69.10 A005 83.39 88.28 69.04 83.39 47.99 A007 46.57 42.35 50.89 83.39 74.59 A008 36.21 46.57 34.23 50.89 38.08 A010 146.28 93.38 46.00 38.96 42.97 A013 69.10 58. 36 69.10 47.99 42.97 A017 53.12 61.02 58.36 66.39 42.97 A018 47.99 53.12 108.71 58.36 38.08 A020 47.99 69.10 24.35 35.68 24.35 A024 126.33 66.39 47.99 N/A* 108.71 A028 39.41 24.63 24.63 20.15 44.70 A030 50.14 34.28 44.70 36.83 42.04 A032 58.54 44.70 73.09 61.40 44.70 A033 20.15 18.02 44.70 29.35 29.35 A034 67.20 13.99 18.02 20.15 34.28 A037 55.71 15.96 39.41 20.15 20.15 A042 44.70 24.63 20.15 44.70 61.40 A044 39.49 24.11 34.14 34.14 24.11 Mean 57.76 48.92 47.13 48.55 45.12 Median 47.99 46.57 4 4.70 46.35 42.97 *N/A: not assessed

PAGE 80

80 Table A 14 IFN Levels (in pg/mL) for First Device Active 1st device: Active 2nd Device: Sham Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A001 3.05 3.23 2.18 2.35 1.04 A002 2.35 2.35 3.42 2.87 1.04 A003 2.35 2.01 3.05 2.1 8 1.51 A004 2.35 2.18 3.05 2.01 1.51 A005 2.01 3.05 3.42 3.79 1.51 A007 3.05 2.69 3.05 3.05 1.04 A008 2.01 3.42 2.35 2.35 1.04 A010 3.23 3.23 1.66 1.66 1.51 A013 0.72 1.04 2.02 0.72 2.02 A017 0.72 1.51 1.51 1.51 0.72 A018 1.04 1.51 1.27 1.04 1.51 A020 1.51 1.51 1.51 1.04 1.51 A024 1.04 1.04 1.04 N/A* 2.02 A028 2.95 2.10 2.95 2.10 2.10 A030 3.86 2.10 1.32 2.95 2.95 A032 2.95 3.86 3.86 2.10 2.95 A033 2.95 2.95 2.95 2.95 1.32 A034 3.86 2.10 2.10 2.95 2.95 A037 3.86 2.10 2.10 2.10 2.95 A042 2.1 0 4.81 3.86 4.81 2.95 A044 0.35 0.97 0.35 0.35 0.35 Mean 2.30 2.37 2.33 2.24 1.74 Median 2.35 2.10 2.18 2.14 1.51 *N/A: not assessed

PAGE 81

81 APPENDIX B BIOMARKER DATA FROM SHAM ACTIVE GROUP T able B 1. IL 1st devic e: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 266.10 374.41 137.86 166.19 53.37 A009 140.80 120.62 138.89 N/A* N/A* A011 102.60 73.17 45.18 46.17 106.51 A012 193.85 6.25 122.48 88.23 27.27 A014 173.61 41.87 154.71 69.07 345.27 A015 199.07 191.23 5.20 3.19 69.23 A016 3.61 135.83 7.64 19.98 31.09 A019 195.48 145.27 92.15 3.33 155.04 A021 28.43 9.85 179.48 19.31 11.46 A022 35.07 34.90 103.67 225.93 77.76 A023 4.61 151.13 22.2 9 148.20 152.11 A025 7.39 173.02 21.37 109.94 181.20 A026 18.57 163.77 58.63 22.30 90.20 A031 79.91 76.11 35.37 157.19 54.60 A035 186.18 343.48 218.61 322.55 124.59 A036 231.64 263.34 118.17 202.21 457.18 A038 104.21 100.62 48.89 65.02 60.09 A039 37 .23 30.15 121.02 4.00 93.44 A040 21.93 75.38 55.33 88.03 106.18 A043 125.49 186.00 3.84 118.70 109.22 Mean 107.79 134.82 84.54 98.92 121.36 Median 103.41 128.23 75.39 88.03 93.44 *N/A: not assessed

PAGE 82

82 T able B 2 IL 1RA Levels (in pg/mL) for First Devi ce Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 36431.00 19659.00 52702.00 24288.00 3553.00 A009 13100.00 3663.00 67946.00 N/A* N/A* A011 77210.00 49795.00 40547. 00 91095.00 21395.00 A012 61320.00 992.03 71451.00 61681.00 60431.00 A014 19782.00 29982.00 60505.00 28412.00 29176.00 A015 36322.0 43981.0 42242.0 37295.0 34178.00 A016 4171.00 104204.00 16746.00 31406.00 70.36 A019 18309.00 51804.00 56985.00 1451.00 16221.00 A021 9152.00 23881.00 70.36 6612.00 12564.00 A022 15047.00 35627.00 11708.00 79144.00 22038.00 A023 40.07 14.34 11925.00 17240.00 151.05 A025 5780.0 31747.0 11858.0 28553.0 50689.00 A026 33648.00 19515.00 9474.00 20348.00 62860.00 A031 9968 5.00 48791.00 67259.00 92976.00 75334.00 A035 69052.00 42.81 53414.00 40302.00 27129.00 A036 66693.00 6849.00 8738.00 93436.00 21342.00 A038 40898.00 30730.00 82285.00 72387.00 96020.00 A039 84842.0 1464.0 36199.0 23021.0 98420.00 A040 34875.0 8398.0 53526.0 56560.0 76689.00 A043 24409.0 31508.0 9011.0 103022.0 44483.00 Mean 37538.30 27132.36 38229.57 47854.16 39618.07 Median 34261.50 26931.50 41394.50 37295.00 29176.00 *N/A: not assessed

PAGE 83

83 Table B 3 IL 1 1RA) Ratio in pg/mL for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 0.01 0.02 0.00 0.01 0.01 A009 0.01 0.03 0.00 N/A* N/A* A011 0.00 0.00 0.00 0.00 0.00 A012 0.00 0.01 0.00 0.00 0.00 A014 0.01 0.00 0.00 0.00 0.01 A015 0.01 0.00 0.00 0.00 0.00 A016 0.00 0.00 0.00 0.00 0.31 A019 0.01 0.00 0.00 0.00 0.01 A021 0.00 0.00 0.72 0.00 0.00 A022 0.00 0.00 0.01 0.00 0.00 A023 0.10 0.91 0.00 0.01 0.50 A025 0.00 0.01 0.00 0.00 0.00 A 026 0.00 0.01 0.01 0.00 0.00 A031 0.00 0.00 0.00 0.00 0.00 A035 0.00 0.89 0.00 0.01 0.00 A036 0.00 0.04 0.01 0.00 0.02 A038 0.00 0.00 0.00 0.00 0.00 A039 0.00 0.02 0.00 0.00 0.00 A040 0.00 0.01 0.00 0.00 0.00 A043 0.01 0.01 0.00 0.00 0.00 Mean 0.01 0.10 0.04 0.00 0.05 Median 0.00 0.01 0.00 0.00 0.00 *N/A: not assessed

PAGE 84

84 Table B 4 R ANKL Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 da ys) A006 28.05 28.05 23.56 22.09 25.04 A009 25.04 28.05 28.05 N/A* N/A* A011 28.34 25.01 21.80 28.34 28.34 A012 28.34 28.34 28.34 23.39 23.39 A014 28.34 21.80 28.34 28.34 23.39 A015 28.34 18.72 25.01 21.80 31.79 A016 31.79 23.39 26.66 26.66 30.05 A 019 31.79 21.80 26.66 25.01 26.66 A021 18.72 28.34 21.80 25.01 21.80 A022 21.80 28.34 28.34 30.05 28.34 A023 25.01 26.66 31.79 21.80 28.34 A025 22.79 19.02 13.73 19.02 19.02 A026 37.05 19.02 15.44 22.79 19.02 A031 15.44 15.44 15.44 19.02 10.49 A035 15.44 19.02 17.21 19.02 22.79 A036 15.44 15.44 12.08 15.44 15.44 A038 19.02 20.88 12.08 15.44 8.98 A039 15.44 22.79 12.08 20.88 17.21 A040 15.44 15.44 15.44 15.44 15.44 A043 19.02 22.79 19.02 15.44 20.88 Mean 23.53 22.42 21.14 21.84 21.92 Median 23. 90 22.30 21.80 21.80 22.79 *N/A: not assessed

PAGE 85

85 Table B 5 OPG Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 6.86 3.73 4.37 2.90 1.42 A009 1.59 4.16 2.13 N/A* N/A* A011 11.06 12.58 12.58 13.34 20.02 A012 2.96 0.70 3.53 4.75 4.75 A014 57.46 15.67 39.02 6.73 94.67 A015 14.11 8.85 1.25 1.47 12.19 A016 1.05 22.02 1.92 1.92 6.73 A019 25.25 4.75 1.47 0.70 14.89 A021 1.92 4.13 18. 03 5.72 7.43 A022 1.92 1.47 2.96 3.53 2.96 A023 1.05 4.44 1.05 1.25 1.47 A025 0.14 2.09 2.09 19.59 9.44 A026 2.09 8.78 8.78 9.77 27.04 A031 33.72 9.44 20.27 79.36 98.03 A035 5.56 13.47 8.78 15.51 4.33 A036 8.78 5.56 2.09 17.55 3.74 A038 4.03 7.47 1 9.59 12.12 14.15 A039 0.39 0.39 1.59 0.39 0.93 A040 0.39 1.59 1.59 2.61 2.09 A043 6.19 8.12 0.39 2.09 1.59 Mean 9.33 6.97 7.67 10.59 17.26 Median 3.50 5.16 2.55 4.75 6.73 *N/A: not assessed

PAGE 86

86 Table B 6 RANKL/(RANKL+OPG) Ratio in pg/mL for First De vice Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 0.80 0.88 0.84 0.88 0.95 A009 0.94 0.87 0.93 N/A* N/A* A011 0.72 0.67 0.63 0.68 0.59 A012 0.91 0.98 0.89 0.83 0 .83 A014 0.33 0.58 0.42 0.81 0.20 A015 0.67 0.68 0.95 0.94 0.72 A016 0.97 0.52 0.93 0.93 0.82 A019 0.56 0.82 0.95 0.97 0.64 A021 0.91 0.87 0.55 0.81 0.75 A022 0.92 0.95 0.91 0.89 0.91 A023 0.96 0.86 0.97 0.95 0.95 A025 0.99 0.90 0.87 0.49 0.67 A02 6 0.95 0.68 0.64 0.70 0.41 A031 0.31 0.62 0.43 0.19 0.10 A035 0.74 0.59 0.66 0.55 0.84 A036 0.64 0.74 0.85 0.47 0.81 A038 0.83 0.74 0.38 0.56 0.39 A039 0.98 0.98 0.88 0.98 0.95 A040 0.98 0.91 0.91 0.86 0.88 A043 0.75 0.74 0.98 0.88 0.93 Mean 0.79 0 .78 0.78 0.76 0.70 Median 0.87 0.78 0.88 0.83 0.81 N/A: not assessed

PAGE 87

87 Table B 7. Osteocalcin Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (8 4 days) A006 99.88 78.25 82.52 97.69 95.50 A009 82.52 104.29 65.65 N/A* N/A* A011 32.63 11.49 21.55 21.55 44.34 A012 8.82 11.49 8.82 94.04 11.49 A014 56.46 8.82 11.49 11.49 26.99 A015 32.63 21.55 16.35 11.49 11.49 A016 11.49 32.63 26.99 8.82 8.82 A 019 21.55 32.63 8.82 11.49 8.82 A021 21.55 11.49 32.63 8.82 8.82 A022 8.82 21.55 21.55 8.82 8.82 A023 8.82 21.55 8.82 21.55 11.49 A025 24.77 21.58 28.14 13.15 17.14 A026 24.77 24.77 24.77 17.14 24.77 A031 17.14 24.77 11.93 17.14 6.68 A035 4.99 4.99 4.99 7.61 3.56 A036 4.99 9.66 4.25 4.99 2.37 A038 3.56 2.93 1.07 1.44 1.44 A039 0.76 0.49 1.44 1.44 0.51 A040 0.76 0.49 0.49 0.49 2.37 A043 0.76 0.49 0.49 0.49 0.51 Mean 23.38 22.30 19.14 18.93 15.58 Median 14.32 16.52 11.71 11.49 8.82 *N/A: not as sessed

PAGE 88

88 Table B 8 Osteopontin Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 1185.00 1205.00 1111.00 1165.00 1165.00 A009 1165. 00 1165.00 1242.00 N/A* N/A* A011 2068.00 2233.00 2424.00 2135.00 2639.00 A012 2265.00 2034.00 2068.00 1931.00 2265.00 A014 2455.00 2393.00 2135.00 2265.00 2265.00 A015 2135.00 2068.00 2265.00 2135.00 2455.00 A016 2265.00 2393.00 2201.00 2201.00 2330. 00 A019 2758.00 2135.00 2517.00 2330.00 2068.00 A021 2517.00 2265.00 2330.00 2135.00 2330.00 A022 2393.00 2517.00 3676.00 2135.00 2068.00 A023 2330.00 2330.00 2265.00 2265.00 2068.00 A025 1730.00 2056.00 2109.00 8143 .00 1841.00 A026 2109.00 6078 .00 2 565.00 2516.00 2056.00 A031 1674.00 2056.00 2056.00 2315.00 2056.00 A035 1841.00 2315.00 2315.00 2056.00 1841.00 A036 2315.00 1949.00 1674.00 2109.00 2264.00 A038 2264.00 2466.00 1841.00 1841.00 1949.00 A039 1387.00 1674.00 1949.00 1730.00 1730.00 A0 40 1841.00 2109.00 2056.00 1730.00 2161.00 A043 2056.00 1841.00 1618.00 1786.00 1730.00 Mean 2037.65 2264.10 2120.85 2364.37 2067.42 Median 2122.00 2122.00 2122.00 2135.00 2068.00 *N/A: not assessed

PAGE 89

89 Table B 9 IL 6 Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 1.16 0.98 1.55 1.35 1.07 A009 0.98 1.07 1.16 N/A* N/A* A011 1.69 1.28 1.48 0.99 1.09 A012 1.28 1.28 1.09 2.58 1.48 A014 1.58 1.28 1.48 1.18 1.28 A015 1.28 1.18 1.48 1.09 1.28 A016 1.38 1.09 1.28 1.28 1.28 A019 1.09 1.09 1.28 1.28 1.28 A021 1.28 0.90 1.09 1.28 1.28 A022 1.28 1.48 2.37 1.18 1.38 A023 1.28 1.09 1.28 1.69 1.48 A025 0.66 1.06 0.89 0.89 1.06 A026 0. 97 1.23 1.06 1.15 1.61 A031 0.89 1.06 1.06 1.33 1.06 A035 1.06 0.89 1.23 1.15 1.42 A036 1.06 0.89 1.06 0.89 1.06 A038 1.15 1.06 1.06 1.42 0.89 A039 1.06 1.06 0.89 1.06 0.89 A040 1.06 0.89 0.89 0.89 1.23 A043 1.06 0.89 0.89 1.33 1.06 Mean 1.16 1.09 1.23 1.26 1.22 Median 1.12 1.07 1.13 1.18 1.28 *N/A: not assessed

PAGE 90

90 Table B 10 IL 8 Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 938.44 299.11 150.70 150.24 94.91 A009 474.52 477.84 396.71 N/A* N/A* A011 276.34 113.39 171.86 57.89 126.55 A012 89.86 24.81 47.95 28.14 6.65 A014 282.24 11.46 202.50 13.29 231.27 A015 43.11 44.27 0.83 0.65 27.19 A016 2.71 353.33 23.02 4.12 9.6 5 A019 231.85 241.93 95.31 0.65 65.16 A021 66.54 23.02 285.99 18.87 3.56 A022 51.26 52.03 98.75 422.90 81.93 A023 20.00 40.21 15.24 59.75 18.97 A025 2.62 680.39 64.56 238.45 295.19 A026 44.58 93.95 116.70 54.97 159.17 A031 97.06 268.83 70.77 465.12 228.00 A035 152.07 364.40 264.95 389.91 93.17 A036 213.20 25.68 19.00 111.33 171.00 A038 233.43 309.91 110.93 213.20 136.19 A039 24.97 10.09 194.03 0.81 23.91 A040 17.08 183.63 137.93 246.47 353.39 A043 141.32 432.65 7.45 179.97 150.82 Mean 170.16 2 02.55 123.76 139.83 119.83 Median 93.46 148.51 104.84 59.75 94.91 *N/A: not assessed

PAGE 91

91 Table B 11 MMP 3 Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 d ays) T4 (84 days) A006 6.18 3.07 2.29 3.07 2.29 A009 1.41 1.92 2.11 N/A* N/A* A011 2.41 2.62 3.27 3.27 3.27 A012 2.84 2.41 6.67 3.50 2.84 A014 2.84 2.01 2.41 2.41 3.27 A015 3.27 2.84 2.41 2.84 2.41 A016 1.63 3.73 2.41 2.41 2.84 A019 2.84 2.84 3.05 2.41 2.84 A021 2.41 2.41 2.41 2.84 2.21 A022 2.21 2.41 5.24 3.27 3.05 A023 2.84 2.41 1.82 1.63 2.01 A025 1.61 2.36 1.61 5.87 1.85 A026 2.36 10.47 2.36 14.59 8.13 A031 2.36 1.38 1.85 1.85 2.90 A035 1.85 4.04 2.90 4.04 1.85 A036 2.90 2.36 1.85 2.90 3 .46 A038 2.36 1.61 1.85 2.62 2.90 A039 1.38 1.85 1.85 1.85 1.85 A040 1.38 1.85 2.36 4.63 2.36 A043 2.36 2.36 1.61 2.62 2.36 Mean 2.47 2.85 2.62 3.61 2.88 Median 2.36 2.41 2.36 2.84 2.84 *N/A: not assessed

PAGE 92

92 Table B 12 MMP 9 Levels (in pg/mL) for F irst Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 20307.00 18361.00 7315.00 25718.00 1502.00 A009 2284.00 13133.00 5296.00 N/A* N/A* A011 7818.00 2958.00 3 541.00 12501.00 26428.00 A012 887.70 235.46 20510.00 11516.00 5553.00 A014 6319.00 932.39 1812.00 1568.00 3720.00 A015 2501.00 4241.00 51.18 83.66 3577.00 A016 443.42 3290.00 902.21 13718.00 8205.00 A019 1653.00 1303.00 242.26 33.17 3752.00 A021 5165 .00 429.19 5596.00 1977.00 511.41 A022 2719.00 1213.00 3830.00 11961.00 13830.00 A023 975.71 2379.00 622.42 2076.00 2780.00 A025 741.42 43262.00 2101.00 36846.00 8127.00 A026 12380.00 79402.00 10475.00 43820.00 105431.00 A031 444.10 1362.00 983.18 365 3.00 13478.00 A035 3118.00 10812.00 4331.00 4154.00 7732.00 A036 3707.00 1825.00 204.24 7736.00 3903.00 A038 1104.00 1097.00 4724.00 6266.00 4565.00 A039 334.59 218.19 731.38 49.74 3530.00 A040 1363.00 1987.00 23027.00 31698.00 29155.00 A043 2247.00 1193.00 110.75 12803.00 12495.00 Mean 3825.60 9481.66 4820.28 12009.35 13593.39 Median 2265.50 1906.00 2821.00 7736.00 5553.00 *N/A: not assessed

PAGE 93

93 Table B 13 M CSF Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time p oints Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 90.75 26.62 73.76 50.89 42.35 A009 55.31 85.83 53.09 N/A* N/A* A011 53.12 53.12 50.54 58.36 74.59 A012 35.68 33.33 138.23 69.10 38.08 A014 50.54 33.33 53.12 42.97 108 .71 A015 63.69 38.08 24.35 22.23 80.14 A016 38.08 53.12 42.97 42.97 47.99 A019 69.10 47.99 53.12 26.52 63.69 A021 50.54 28.75 74.59 33.33 38.08 A022 38.08 28.75 68.75 74.59 42.97 A023 28.75 42.97 31.02 38.08 38.08 A025 24.63 44.70 29.35 211.36 50.14 A026 39.41 67.20 31.79 29.35 61.40 A031 24.63 24.63 24.63 67.20 88.16 A035 100.52 44.70 55.71 85.11 50.14 A036 34.28 29.35 20.15 50.14 36.83 A038 55.71 55.71 34.28 29.35 52.91 A039 20.15 15.96 29.35 24.63 34.28 A040 34.28 50.14 61.40 94.31 85.11 A 043 34.28 42.04 24.63 31.79 24.63 Mean 47.08 42.32 48.74 56.96 55.70 Median 38.75 42.51 46.76 42.97 50.14 *N/A: not assessed

PAGE 94

94 Table B 14 IFN Levels (in pg/mL) for First Device Sham 1st device: Sham 2nd Device: Active Time points Subject T0 (PV 2) T1 (day 0) T2 (1 7 days) T3 (43 49 days) T4 (84 days) A006 2.35 3.05 2.35 2.69 3.05 A009 3.79 3.23 3.79 N/A* N/A* A011 1.51 3.14 1.04 1.04 1.51 A012 1.04 1.51 0.82 1.27 1.51 A014 1.04 1.51 1.04 1.04 1.51 A015 1.51 1.51 1.04 1.51 1.27 A016 1.04 1.04 1.04 1.04 0.82 A019 1.51 1.04 0.72 0.72 1.04 A021 1.51 1.51 1.51 0.72 1.04 A022 1.51 0.72 1.66 0.72 0.72 A023 1.04 1.51 0.72 1.51 1.51 A0 25 2.95 4.81 2.95 5.78 1.25 A026 2.95 4.81 2.95 2.95 2.95 A031 2.10 2.95 3.40 3.86 2.95 A035 2.95 2.95 2.10 3.86 2.10 A036 3.86 2.10 3.86 3.40 2.10 A038 2.95 2.10 2.10 2.10 2.95 A039 2.95 3.40 2.95 3.86 3.86 A040 2.10 2.95 2.95 2.10 3.40 A043 2.52 3.86 2.95 2.95 2.10 Mean 2.16 2.49 2.10 2.27 1.98 Median 2.10 2.53 2.10 2.10 1.51 *N/A: not assessed

PAGE 95

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100 64. Hashimoto F, Kobayashi Y, Mataki S, Kobayashi K, Kato Y, Sakai H. Administration of osteocalcin accelerates orthodontic tooth movement induced by a closed coil spring in rats. Eur J Orthod 2001;23:5 35 545. 65. Alfaqeeh SA, Anil S. Osteocalcin and N telopeptides of type I collagen marker levels in gingival crevicular fluid during different stages of orthodontic tooth movement. Am J Orthod Dentofacial Orthop 2011;139:e553 559. 66. Hume DA, Pavli P, D onahue RE, Fidler IJ. The effect of human recombinant macrophage colony stimulating factor (CSF 1) on the murine mononuclear phagocyte system in vivo. J Immunol 1988;141:3405 3409. 67. Kaku M, Motokawa M, Tohma Y, Tsuka N, Koseki H, Sunagawa H et al. VEGF and M CSF levels in periodontal tissue during tooth movement. Biomed Res 2008;29:181 187. 68. Nakano Y, Yamaguchi M, Fujita S, Asano M, Saito K, Kasai K. Expressions of RANKL/RANK and M CSF/c fms in root resorption lacunae in rat molar by heavy orthodont ic force. Eur J Orthod 2011;33:335 343. 69. Bemiller LS, Roberts DH, Starko KM, Curnutte JT. Safety and Effectiveness of Long Term Interferon Gamma Therapy in Patients with Chronic Granulomatous Disease. Blood Cells, Molecules, and Diseases 1995;21:239 24 7. 70. Steinmller C, Franke Ullmann G, Lohmann Matthes M L, Emmendrffer A. Local Activation of Nonspecific Defense against a Respiratory Model Infection by Application o f Interferon 2000;22:481 490. 71. Key LL, Rodriguiz RM, Willi SM, Wright NM, Hatcher HC, Eyre DR et al. Long Term Treatment of Osteopetrosis with Recombinant Human Interferon Gamma. New Englan d Journal of Medicine 1995;332:1594 1599. 72. Cornish J, Gillespie MT, Callon KE, Horwood NJ, Moseley JM, Reid IR. Interleukin 18 Is a Novel Mitogen of Osteogenic and Chondrogenic Cells. Endocrinology 2003;144:1194 1201. 73. Mermut S, Bengi AO, Akin E, K Gamma on Bone Remodeling during Experimental Tooth Movement. The Angle Orthodontist 2007;77:135 141. 74. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic movement induces high numbers of cells expressing IFN gamma at mRNA and protein levels. J Interferon Cytokine Res 2000;20:7 12.

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101 75. Leethanakul C, Suamphan S, Jitpukdeebodintra S, Thongudomporn U, Charoemratrote C. Vibratory stimulation increases interleukin 1 beta secretion during orthodontic tooth mov ement. Angle Orthod 2015. 76. Rody W, Iwasaki L, Krokhin O. Oral Fluid based Diagnostics and Applications in Orthodontics. In: McNamara J, editor. Taking Advantage of Emerging Technologies in Clinical Practice. Ann Arbor MI: University of Michigan; 2012: p. 223 261. 77. Drake CT, McGorray SP, Dolce C, Nair M, Wheeler TT. Orthodontic tooth movement with clear aligners. ISRN Dent 2012;2012:657973. 78. McGorray SP, Dolce C, Kramer S, Stewart D, Wheeler TT. A randomized, placebo controlled clinical trial on the effects of recombinant human relaxin on tooth movement and short term stability. Am J Orthod Dentofacial Orthop 2012;141:196 203. 79. Chisari JR, McGorray SP, Nair M, Wheeler TT. Variables affecting orthodontic tooth movement with clear aligners. Am J Orthod Dentofacial Orthop 2014;145:S82 91. 80. Dinarello CA. Interleukin 1, interleukin 1 receptors and interleukin 1 receptor antagonist. Int Rev Immunol 1998;16:457 499. 81. Iwasaki LR, Gibson CS, Crouch LD, Marx DB, Pandey JP, Nickel JC. Speed of too th movement is related to stress and IL 1 gene polymorphisms. Am J Orthod Dentofacial Orthop 2006;130:698.e691 699. 82. Ohsaki Y, Takahashi S, Scarcez T, Demulder A, Nishihara T, Williams R et al. Evidence for an autocrine/paracrine role for interleukin 6 in bone resorption by giant cells from giant cell tumors of bone. Endocrinology 1992;131:2229 2234. 83. Duplomb L, Baud'huin M, Charrier C, Berreur M, Trichet V, Blanchard F et al. Interleukin 6 inhibits receptor activator of nuclear factor kappaB ligand induced osteoclastogenesis by diverting cells into the macrophage lineage: key role of Serine727 phosphorylation of signal transducer and activator of transcription 3. Endocrinology 2008;149:3688 3697. 84. Uematsu S, Mogi M, Deguchi T. Interleukin (IL) 1 beta, IL 6, tumor necrosis factor alpha, epidermal growth factor, and beta 2 microglobulin levels are elevated in gingival crevicular fluid during human orthodontic tooth movement. J Dent Res 1996;75:562 567. 85. Basaran G, Ozer T, Kaya FA, Hamamci O. In terleukins 2, 6, and 8 levels in human gingival sulcus during orthodontic treatment. Am J Orthod Dentofacial Orthop 2006;130:7.e1 6.

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103 BIOGRAPHICAL SKETCH Angela M. McNeight was born and raised in Indian Harbour Be ach, Florida by her parents Richard and Teresa McNeight. In 2004, she moved to Atlanta, Georgia to attend Emory University where she received her Bachelor of Science degree, double majoring in Neuroscience and Behavioral Biology and general Biology and als o acquiring a minor in Spanish. During her undergraduate career, Angela served as collegiate chapter president of her sorority, Kappa Kappa Gamma, and was selected as one of the prestigious Emory 100 Senior Honorary members. After graduation from Emory, An gela worked as a National Leadership Consultant for her sorority, traveling to over 30 universities across North America and Canada. She then returned to her home state of Florida to attend the University of Florida College of Dentistry where she graduated cum laude in 2013. Her dental honors include induction into Omicron Kappa Upsilon (Xi Omicron Chapter), Hinman Dental Society Scholar, A. Lewis Leo Outstanding Student Award, Clinical and Professional Excellence Award, and the Orthodont ic Clinical Achievement Award through her Advanced Education Honors Certificate Program in Orthodontics. She served as both on the UFCD Admissions Committee and class Vice President for all four years of dental school. After completing her DMD degree, she was accepted into the University of Florida Graduate Orthodontic Program to continue her training. She has presented her research at the Florida Association of Orthodontists and Southern Association of Orthodontists meetings, and her research recently won Honorable Mention in the Dentsply GAC GCare University Research Award competition. She was selected as one of the AAO Emerging Leaders and is a finalist for the 2016 Eugene L. Gottleib JCO Student of the Year. Upon

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104 completion of orthodontics residency and Master of Dental Sciences requirements in May 2016, Angela plans to practice in Florida in a private practice setting.