PROTEOMIC CHARACTERIZATION OF EXOSOMES RELEASED BY RESORBING OSTEOCLASTS AND ODONTOCLASTS IN CELL CULTURE

By

ALYSSA KATHLEEN EMORY

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2017

© 2017 Alyssa Kathleen Emory

To my husband and family for their endless encouragement, support, and love

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ACKNOWLEDGMENTS

I want to thank my mentor, Dr. Wellington Rody, for his guidance on this project,

Dr. Shannon Holliday, Dr. Kevin McHugh, and Dr. Shannon Wallet for sharing their

expertise, Casey Chamberlain for teaching me the cell culture protocol, and Manitoba

Center of Proteomics for the mass spectrometry analysis. I also thank the American

Association of Orthodontists Foundation and the Southern Association of Orthodontists

for funding this project. In addition, I thank the full-time and part-time residency

instructors for their support the past three years. Lastly, thank you to my family for your

continual encouragement throughout my college education, and to my husband, who

has sacrificed much for me to fulfill my dreams.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 11

CHAPTER

1 INTRODUCTION, BACKGROUND, AND SIGNIFICANCE ..................................... 12

External Root Resorption ........................................................................................ 12 Cellular Mechanisms of Mineralized Tissue Resorption.......................................... 15 Biomarkers of Root Resorption ............................................................................... 18 Exosomes ............................................................................................................... 19 Purpose and Hypotheses ........................................................................................ 20

Purpose ............................................................................................................ 20 Null Hypotheses ............................................................................................... 21

2 MATERIALS AND METHODS ................................................................................ 22

Cell Culture Protocol ............................................................................................... 22 Exosome Isolation .................................................................................................. 23 Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry (2D-

LC-MS/MS) .......................................................................................................... 23 Protein Identification and Quantitation .................................................................... 24 Data Analysis .......................................................................................................... 25

3 RESULTS ............................................................................................................... 27

Cell Culture and Characterization of Clastic Cells .................................................. 27 Proteomic Analysis of Exosomes Extracted from Osteoclasts, Odontoclasts,

and Non-resorbing Clastic Cells .......................................................................... 27 Pathway Analysis of Significantly Changed Proteins .............................................. 29 Functional Enrichment Analysis .............................................................................. 31 Screening of Exosomal Proteins as Potential Biomarkers of Dentin Resorption ..... 36

4 DISCUSSION ......................................................................................................... 39

Oxidative Phosphorylation Pathway ....................................................................... 39 Stabilin-1 ................................................................................................................. 41 Plexin B1 ................................................................................................................. 44

6

Serpin A11 .............................................................................................................. 46 Limitations and Final Remarks ................................................................................ 47

5 CONCLUSIONS ..................................................................................................... 48

APPENDIX

A ISOLATION OF PRIMARY BONE MARROW STEM CELLS AND OSTEOCLAST/ODONTOCLAST CELL CULTURING FOR EXOSOME ANALYSIS .............................................................................................................. 49

B OSTEOCLAST/ODONTOCLAST TRAP STAINING TECHNIQUE ......................... 52

C ACTIN RING STAINING TECHNIQUE ................................................................... 53

D EXOSOME ISOLATION FROM OSTEOCLAST/ODONTOCLAST CELL CULTURE USING EXOQUICK-TC* ....................................................................... 54

E PROTEINS PRESENT DAY 7 BY MATRIX TYPE .................................................. 55

LIST OF REFERENCES ............................................................................................... 90

BIOGRAPHICAL SKETCH .......................................................................................... 102

7

LIST OF TABLES

Table page 3-1 Molecular pathways and associated proteins that were significantly enriched

(Z > 1.65) in dentin and plastic at day 7 of cell culture........................................ 29

3-2 Molecular pathways and associated proteins that were significantly enriched (Z > 1.65) in bone and plastic at day 7 of cell culture ......................................... 30

3-3 Molecular pathways and associated proteins that were significantly enriched (Z > 1.65) in bone and dentin at day 7 of cell culture .......................................... 30

3-4 Significantly upregulated proteins in odontoclasts exosomes when compared to osteoclasts (Z >1.65) with at least 4 peptides in odontoclast samples ........... 37

E-1 Proteins Present in Dentin Day 7, but not Bone Day 7 or Plastic Day 7. ............ 55

E-2 Proteins Present in Bone Day 7, but not Dentin Day 7 or Plastic Day 7. ............ 58

E-3 Proteins Present in Dentin Day 7 and Bone Day 7, but not Plastic Day 7 .......... 71

E-4 Proteins Present in Dentin Day 7, Bone Day 7, and Plastic Day 7 ..................... 83

E-5 Proteins Present in Plastic Day 7, but not Dentin Day 7 or Bone Day 7. ............ 87

8

LIST OF FIGURES

Figure page 2-1 Experimental design. .......................................................................................... 26

3-1 Mouse marrow-derived cells cultured on plastic, bone, and dentin slices. ......... 27

3-2 Three-way Venn diagram showing the overlap and differences between exosomal proteins originated from osteoclasts, odontoclasts and non-resorbing clastic cells at day 7 of the cell culture. ............................................... 28

3-3 Enrichment analysis of exosomes by FunRich. Comparison of biological processes, cellular components, and molecular function of proteins that were exclusively found in one media but not the other (dentin vs bone). .................... 33

3-4 Enrichment analysis of exosomes by FunRich. Comparison of biological processes, cellular components, and molecular function of proteins found in media from resorbing clastic cells (dentin, bone, and bone + dentin) versus those exclusive to inactive clastic cells (plastic). ................................................ 35

9

LIST OF ABBREVIATIONS

2D Two Dimensional

2D-LC-MS/MS Two Dimensional liquid chromatography-tandem mass spectrometry

3D Three Dimensional

ALARA

BCA

As Low As Reasonably Achievable

Bicinchoninic Acid

BMSC Bone Marrow Stem Cells

CBCT Cone Beam Computed Tomography

CCC

CT

Coagulation Cascades

Computer Tomography

DAVID Database for Annotation, Visualization, and Integrated Discovery

DPP Dentine Phosphoprotein

DSP Dentine Sialoprotein

ERR

ESI

External Root Resorption

Electrospray Ionization

EV

FASP

Extracellular Vesicles

Filter-aided Sample Preparation

FBS Fetal Bovine Serum

GCF Gingival Crevicular Fluid

IACUC Institutional Animal Care and Use Committee

KEGG Kyoto Encyclopedia of Genes and Genomes

M-CSF Macrophage Colony Stimulating Factor

MEM Minimum Essential Media

MGF Mascot Generic File

MS Mass Spectrometry

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MVB

OCR

OPG

OxPhos

PANTHER

Multivesicular Bodies

Oxygen Consumption Rate

Osteoprotegerin

Oxidative Phosphorylation

Protein Analysis Through Evolutionary Relationships

PBS

PDL

RA

RANK

Phosphate Buffered Saline

Periodontal Ligament

Rheumatoid Arthritis

Receptor Activator of Nuclear Factor Kappa Beta

RANKL Receptor Activator of Nuclear Factor Kappa Beta Ligand

RNA Ribonucleic Acid

Sema 4D

SPARC

TIC

TFA

Semaphorin 4D

Secreted Protein Acidic and Rich in Cysteine

Total Ion Count

Trifluoroacetic Acid

TRAP Tartrate Resistance Acid Phosphatase

VEGF Vascular Endothelial Growth Factor

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

PROTEOMIC CHARACTERIZATION OF EXOSOMES RELEASED BY RESORBING

OSTEOCLASTS AND ODONTOCLASTS IN CELL CULTURE

By

Alyssa Kathleen Emory

May 2017

Chair: Wellington J. Rody Jr. Major: Dental Sciences - Orthodontics

Osteoclasts and odontoclasts secrete vesicles called exosomes, which are an

appealing source of biomarkers. The purpose was to identify exosomal proteins that

can distinguish between bone and dentin resorption. Mouse ‘clasts were cultured on

either plastic, dentin or bone. Exosomes were isolated from the media and analyzed for

proteomic differences using two-dimensional mass spectrometry. For each protein, the

difference between the total ion count (TIC) values were mapped to an expression ratio

histogram (Z-score) in order to detect proteins differentially expressed. 2,183 proteins

were identified. Plexin-B1 (Z=12.4) and Serpin A11 (Z=12.3) were uniquely present in

odontoclast exosomes. Stabilin-1 and oxidative phosphorylation pathway proteins were

enriched in exosomes from osteoclasts and odontoclasts, but not in exosomes from

non-resorbing cells on plastic. Exosomal proteins are potential biomarkers of root

resorption and/or bone remodeling.

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CHAPTER 1 INTRODUCTION, BACKGROUND, AND SIGNIFICANCE

External Root Resorption

Root resorption refers to the breakdown of mineralized dental tissues, such as

cementum and dentin. Although considered a normal physiological response in

deciduous teeth, the process of dentin resorption in the permanent dentition is

pathological. Pre-cementum and cementum line the outer most root surface and serve

a protective function against root resorption. 1,2 External root resorption (ERR) occurs

when there is damage to these tissue layers, which may be followed by damage to the

dentin. 1,3-7 Trauma, systemic disease, individual susceptibility, genetics, and

orthodontic treatment are some factors that may cause root resorption. 8-11

It was recognized as early as 1927 that root resorption is related to orthodontic

treatment. 12,13 Subsequent and recent studies indicate that this unwanted sequel and

adverse event will occur at some level, ranging from insignificant to severe, in most

orthodontic patients. 12,14-17 There are many factors associated with the extent of

orthodontically induced external root resorption. 10,15,18-20 Treatment-related factors

include duration of treatment, direction of tooth movement, type of appliance, and

magnitude and type of force (continuous or intermittent). 19-23 Patient risk factors

include nutrition, ethnicity, hereditary disposition, systemic factors including asthma or

allergies, impacted teeth, premolar extractions, age, anatomical differences in root

morphology, and genetics. 10,15,22,24-26 These multi-faceted and complex causes of root

resorption make it difficult to predict its incidence and severity. 10,15,18,25

Studies show that 70% to 80% of orthodontic cases will involve mild root

resorption, 20% to 25% will involve moderate root resorption, and up to 5% will involve

13

severe resorption. 14,27-31 Mild root resorption involves only the surface layer in which

cementum is resorbed, but will subsequently remodel. In moderate cases, the outer

dentin layer is also affected and permanent damage to the root varies, depending on

the depth of resorption. Severe root resorption involves root shortening of 5mm or

greater than one third of the root length 1,10,28,31 and the tissues are irreversibly lost. 32

This root shortening causes tooth instability and possibly early tooth loss. 6,15,25 When

the cause of root resorption is known, the etiological factor can be removed and

resorption will generally stop. 9,33 It has been reported that orthodontically induced

external root resorption ceases when the pressure within the periodontal ligament drops

below that needed for optimal tooth movement. 34,35 When root resorption is diagnosed

early during the initial stage of occurrence, the prognosis is fair, but if left undiagnosed

the prognosis begins to decline. 9 Therefore, early and accurate detection and

diagnosis is essential in order to prevent or cease adverse effects and allow for

treatment success. 2,36

Early signs and symptoms of external root resorption do not present themselves

clinically, therefore detection and diagnosis in orthodontic practice is commonly based

on routine imaging procedures, such as panoramic, periapical, occlusal, or lateral

cephalometric radiographs. 9,22,25,37,38 Reasons for using two-dimensional (2D) imaging

techniques include ease of use, low radiation dose, relatively low cost, and availability of

equipment in most dental offices. 38 However, 2D radiographic imaging has its

limitations. Compression of a three-dimensional (3D) object into a 2D image causes

anatomic structures to overlap, compromising the diagnostic accuracy. 2,9,11,25,39 One of

the biggest challenges using 2D imaging is encountered when lesions are on the buccal

14

or lingual tooth surfaces, 32,40 and studies show that determining the root surface

affected and the location of the defect, as well as the size and depth of the lesion is

difficult and inconsistent. 4,9,25 Magnification, distortion, and projection orientation and

angulation represent other obstacles that contribute to the inability to reliably repeat and

reproduce 2D images. In addition, conventional 2D radiography cannot detect a change

in root structure until there is 40% to 70% loss of mineralized tissue and therefore some

root shortening is required for detection. 4,22 For these reasons, some authors

recommend 3D scanning technology for earlier and more accurate detection of external

root resorption. 9,32,41,42

Conventional three-dimensional computer tomography (CT) technology provides

an image with high resolution and increased accuracy to detect and diagnose external

root resorption, 6,41-43 however, there are many drawbacks that make this undesirable

for use by orthodontists. CT machines are too expensive to be practical in most

orthodontic offices, 9,22,44,45 and many practitioners are not trained in CT image reading

and must consult a radiologist to interpret the scan. 38 In addition, exposure to radiation

is much higher with CT imaging than with 2D radiography. 22,38,45

Another three-dimensional imaging technique is cone beam computed

tomography (CBCT). CBCT uses a scanner that radiates a beam from the x-ray source

in a cone shape, covering a larger volume with significantly less radiation than

conventional CT scanners. 22,32,38,46 CBCT is more commonly used in the dental

profession and has benefits of ease of use, short scan time, and high resolution and

accuracy, all with a lower cost and radiation dose than CT. 2,22,47 When compared to

2D imaging techniques, CBCT eliminates superimpositions and magnification errors, but

15

increases radiation dose and cost. 22,46,48 Recently, a few studies have reported that

CBCT outperforms 2D radiography in diagnosing external root resorption 5,22,45 and that

more accurate treatment choices were made when diagnosing from CBCT. 2 While

CBCT has these advantages over conventional CT as well as 2D radiography in

detecting external root resorption, it should be used with caution. The amount of

radiation exposure is still significantly greater than 2D radiography. Though the dose of

radiation is dependent on many variables, studies show effective dose of a 2D

panoramic at 4-23 μSv and of a CBCT at 30-90 μSv. 22,46-49 In general, radiography

methods are unable to determine whether resorption is currently active or inactive 1 and

serial x-rays are needed to determine this, thus increasing radiation exposure. It is

recommended by the United States Nuclear Regulatory Commission that radiation

exposure for patients be as low as reasonably achievable (ALARA). 50 With these

limitations, there is a need for improvement in prognostic and diagnostic methods to

detect early root resorption that are sensitive, economical, and void of side-effects.

Cellular Mechanisms of Mineralized Tissue Resorption

Clastic cells are multinuclear giant cells responsible for the physiological and

pathological breakdown of mineralized tissues. Their full names are dependent on the

matrix that they resorb. Thus, osteoclasts are the primary bone-resorbing cells, while

odontoclasts are the primary dentin-resorbing cells. It is believed that osteoclasts and

odontoclasts have identical origins, but little is known about the timing and mechanism

of their differentiation. 28,51 In spite of the similarities, odontoclasts generally have fewer

nuclei than osteoclasts, are smaller in size, and form smaller resorption lacunae. 51 In

addition, key differences in odontoclastic and osteoclastic activity have been reported in

the literature. For instance, it appears that odontoclasts are not regulated by

16

parathyroid hormone (PTH) as osteoclasts are. 51 It has been reported that

odontoclasts show higher activity in forming resorption lacunae, and interleukin-1 (IL-1)

has been found to stimulate the formation of resorption lacunae on dentin to a higher

extent than bone. 52 Under specific circumstances, osteoclasts and odontoclasts may

behave and function differently.

Osteoclast formation involves division of bone marrow stem cells into

hematopoietic monocytes. These mononuclear osteoclast progenitor cells migrate from

hematopoietic tissues to sites of bone resorption, where they become committed pre-

osteoclastic cells, and in the presence of appropriate signals fuse to form multinuclear

osteoclasts. 28,51,53 This process of osteoclastogenesis occurs in the presence of many

cytokines including receptor activator of nuclear factor kappa-β ligand (RANKL) and

macrophage colony-stimulating factor (M-CSF). M-CSF is necessary for the survival,

proliferation, and differentiation of precursors, and RANKL identifies and binds its

receptor, receptor activator of nuclear factor kβ (RANK), which is found on the surface

of clastic precursor cells and activates a signaling pathway to form mature osteoclasts.

28,54-56 Upon activation, the osteoclast or odontoclast contacts and adheres to the bone

or root, and forms a ruffled border and its cytoskeletal structure changes to form an

actin ring. The ruffled border is the cell’s resorptive organelle and the actin ring acts a

sealant and isolates the resorptive microenvironment from the extracellular space. 28,54

Due to the presence of proteolytic enzymes and pumping of protons across the

membrane, the extracellular area under the ruffled border becomes acidified and

resorption begins. 51,57

17

Immediately after the application of orthodontic force, an orchestrated process

involving both osteoclasts and odontoclasts begins. Stimulation of clastic cells is a

consequence of compression of the periodontal ligament, which results in increased

capillary pressure and tissue necrosis/hyalinization. 3,21 Although the removal of this

damaged tissue by clastic cells is necessary for orthodontic tooth movement, 34,53,58-60

the necrotic tissue elimination process also plays a role in orthodontically induced

external root resorption. 61 Initially, mono-nuclear phagocytic cells appear between the

hyalinized tissue and vital periodontal tissue. These peripheral cells are negative for

tartrate resistance acid phosphatase (TRAP), which is an enzyme known to be

characteristic of clastic cells. 61,62 In addition, TRAP-positive multi-nucleated giant cells

are present, which are similar to osteoclasts and odontoclasts, but do not have a ruffled

border. Both of these cell types are not only responsible for elimination of the necrotic

tissue, but also appear to penetrate into the protective layers of the root surface. 61,63,64

During the lag phase of orthodontic tooth movement, undermining alveolar bone is

resorbed by osteoclasts until the periodontal ligament is restored. Odontoclasts, on the

other hand, appear several days after the repair process of hyalinized tissue began.

These mature clastic cells have a ruffled border and are found in the resorption lacunae

of cementum and dentin. 1 Resorption lacunae are predominately seen on the

compression side and rarely on the tension side of the periodontium. 64 The resorption

process continues until no hyaline tissue is present and/or the force level decreases.

61,64-67 Continual increased clastic activity may result in extensive external root damage

and compromise the benefit of orthodontic treatment. 2,6,15,33

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Biomarkers of Root Resorption

Gingival crevicular fluid (GCF) is a serum exudate and/or an inflammatory

transudate that can be isolated from the gingival sulcus. 1,68 GCF contents contain

biochemical molecules and other cellular products, the quality and quantity of which is

dependent upon the health of the periodontium. It has been shown that these

biomolecules are indicative of activity and metabolic change of the periodontal tissues.

68,69 Periodontists were the first to utilize GCF biomarkers as a diagnostic tool for

periodontal health and disease. 69,70

Orthodontic forces induce sterile inflammation, and thus host cell-derived

breakdown byproducts, such as extracellular matrix components, tissue degrading

enzymes and acids, and inflammatory mediators from the surrounding bone and dentin

are released into the gingival fluid. 69,70 Sample collection of GCF is of minimal risk or

harm to patients and has the benefit of being site-specific and readily accessible.

Therefore, GCF analysis has potential value in diagnostic testing for biomarkers of

orthodontic tooth movement and external root resorption. 1,68-70 In order for biochemical

assay of gingival fluid to be diagnostic for root resorption, the follow criteria must be

met: proteins and cytokines associated with root resorption must be released into the

gingival fluid and identified differences in these markers must exist between treatment

and control groups and between treatment groups ranging in severity. 70

Currently, orthodontic literature pertaining to the analysis of GCF for root

resorption biomarkers are relatively few. 1,69 Three studies were found relating to the

potential for presence of dentine sialoprotein (DSP) and dentine phosphoprotein (DPP)

in the GCF to indicate root resorption. 68,69,71 Other studies investigating the levels of

cytokines, such as osteoprotegerin (OPG) and RANKL in GCF, detected an increase in

19

RANKL and a decrease in OPG in areas of root resorption. 14,29,70,72,73 The findings

were not conclusive, as they highlighted the potential for these proteins to originate from

non-dentinal tissues, such as cementum and bone. 68 Even though these studies

introduce novel approaches, the required testing and separation required to identify

these possible biomarkers is difficult and is clinically impractical at this time. 1 However,

the results of these studies suggest analyzing GCF has potential to open new avenues

for analyzing the biology of tooth movement and root resorption. 69

An innovative method to identify biomarkers is mass spectrometry (MS), a highly-

specialized technique which enables qualitative and quantitative analysis of protein

contents by sorting and separating proteins according to their mass-to-charge ratio

(m/z). Ionization is a critical step in MS techniques since only ionized molecules can be

further separated in mass analyzers. Electrospray ionization (ESI) results in a plume

containing protein/peptide molecules carrying multiple positive charges. Analysis of the

ionized molecules allows for exploration of differential protein expression levels between

media taken from the osteoclasts and odontoclasts at higher protein/peptide loads, and

results in detection of up- and down- regulated proteins in dentin and bone resorption

supernatants. Readers are referred to a current review for further explanation. 74

Exosomes

Exosomes are cell-derived vesicles that carry proteins, lipids, and ribonucleic

acids (RNAs) and are present in biological fluids, for example, blood, urine, saliva,

mucus, breast milk, and bile. 1,75-78 Until recent years, it was thought that the main

function of exosomes was to allow cells to discharge waste proteins. 1,79 It is now

known exosomes reflect characteristics of their parent cells 80-83 and play a key role in

intercellular communication and signaling. 75,76,79,84 With this knowledge, it may be

20

possible to read these lipid, protein, and RNA containing exosomal messages, which

would give insight to the condition of the originating cells. The potential for exosomes to

be used for detection of biomarkers of specific pathological conditions, and even

vehicles for drug delivery, is being studied in many areas of the medical profession.

75,76,79,85-88

Recent research has shown that regulatory exosomes are released by

osteoclasts. 89-93 As clastic cells resorb, vesicles containing degradation products are

transported across the cell and released into the extracellular space. 57 Another recent

study using a highly-specialized mass spectrometry technique to analyze GCF contents

found that approximately 50% of proteins in gingival fluid have been observed in

exosomes and some of these proteins may have significant potential to detect

odontoclast activity. 1,53 Given this scope of information, the use of gingival fluid to

collect and analyze exosomes potentially offers numerous benefits, including non-

invasiveness, ease of use, earlier determination of resorptive activity and severity, and

even administration of therapeutics, as well as assessment of treatment responses. 69

Purpose and Hypotheses

Purpose

This proposal is based on the innovative concept that exosomes from ‘clastic’

cells may contain proteins that can distinguish between osteoclast and odontoclast

activity. Thus, the purpose of this study is to identify proteomic differences between

exosomes derived from resorbing osteoclasts and odontoclasts in vitro. Consequently,

molecules identified here may in the future serve as 1) novel biomarkers to distinguish

between dentin and bone resorption, or 2) targets for therapies of external root

resorption.

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Null Hypotheses

Null Hypothesis #1: No proteomic differences exist between exosomes

secreted from bone-resorbing cells (‘osteoclasts’) and dentin-resorbing cells

(‘odontoclasts’).

Null Hypothesis #2: No proteomic differences exist between exosomes

secreted by active and inactive clastic cells.

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CHAPTER 2 MATERIALS AND METHODS

Cell Culture Protocol

The purpose of cell culturing was to extract bone marrow stem cells and expose

them to the signals needed to differentiate into clastic cells. 94 The detailed step-wise

protocol of the cell culture method employed in this research is found in Appendix A.

Adhering to Institutional Animal Care and Use Committee (IACUC) protocols, primary

bone marrow cells were extracted from bone marrow of femurs of 4-6 week old female

C57BL/6 mice. Bone marrow stem cells (BMSC) were then isolated and cultured in

triplicates for 11 days on either bone slices, dentin discs, or plastic wells (control).

Experiments were performed in triplicates for each condition to reduce random error.

The cells were grown in media that contained alpha minimum essential media (α-MEM),

which provides the bare essentials for cells to survive, and exosome-depleted fetal

bovine serum (Exo-FBS) (System Biosciences, Mountain View, CA), which supplies

additional nutrients allowing cells to grow. They were also grown in the presence of

RANKL and M-CSF, which are the signals needed to stimulate bone marrow stem cells

to differentiate into clastic cells. Visual inspection of clastic cell formation was carried

out under a light microscope on days 3, 7, and 11. On day 7, the conditioned media

was collected and replaced with fresh media (α-MEM + Exo-FBS) containing RANKL

and M-CSF. On Day 11, the conditioned media was also collected, but not replaced,

and the wells containing the bone and dentin slices were subjected to TRAP staining

and actin ring staining as described in detail in Appendices B and C.

23

Exosome Isolation

Exosomes were isolated from media using ExoQuick TC (System Biosciences,

Mountain View, CA) as described in Appendix D. In summary, on the day in which the

media was collected, differential ultracentrifugation was used to rid the media of large

unwanted tissues, which formed a pellet. The remaining media, excluding the pellet,

was combined with ExoQuick TC solution (proprietary composition) and left overnight,

allowing for a matrix formation that traps exosomes. The following day, the sample was

put through centrifuge and airfuge and a pellet of isolated exosomes was formed.

Pellets from dentin, bone, and plastic were then pooled across the three rounds of

experiment, thereby providing one pooled sample from odontoclasts, one pooled

sample from osteoclasts, and one pooled sample from inactive clastic cells grown in

plastic at each time point (days 7 and 11). Pellets were frozen until ready to be sent for

proteomic analysis via mass spectrometry technique. These pooled samples were

analyzed by two-dimensional liquid chromatography-tandem mass spectrometry (2D-

LC-MS/MS) as described below in an attempt to provide extended proteome coverage

and confirm findings from 1D analyses that are published elsewhere.93

Conditioned media collected from day 7 was used for exosome characterization

because this time point showed a higher number of multinuclear cells upon visual

inspection.

Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry (2D-LC-MS/MS)

Exosome pellets were solubilized in 1M urea/0.2 M tris/HCl buffer pH 7.6.

Lysates were transferred into 0.5 mL Amicon filter units (10 kDa cut-off) and subjected

to filter-aided sample preparation (FASP) digestion procedure with trypsin. 95 Protein

24

amounts were monitored using micro-BCA assay (Pierce, Rockford, IL). Resulting

digests were acidified with trifluoroacetic acid (TFA) and purified by reversed-phase

solid-phase extraction. The digested sample from each exosome isolate contained

approximately 5-10 µg of the peptides (determined by NanoDrop 2000, ThermoFisher).

Digested exosome protein extracts were subjected to 2D-LC-MS/MS analysis as

previously described. 96 Rather than the pair-wise concatenation in the first dimension

of separation, triplet-concatenation was opted for (one minute each) yielding a reduction

to ten first dimension sub-samples. Each of these ten sub-samples was analyzed using

a 90-minute LC-MS run. The resulting ten Mascot Generic File (MGF) spectra files

were sequentially concatenated into a single file for peptide identification and

quantitation.

Protein Identification and Quantitation

Spectra files were converted into MGF format for protein identification by

X!Tandem search algorithm (http://hs2.proteome.ca/tandem/thegpm_tandem_a.html).

The following X!Tandem search parameters were used: 20 ppm and 50 ppm mass

tolerance for parent and fragment ions, respectively; constant modification of Cys with

iodoacetamide; default set post-translational modifications: oxidation of Met, Trp; N-

terminal cyclization at Qln, Cys; N-terminal acetylation, phosphorylation (Ser, Thr, Tyr),

deamidation (Asn and Gln); an expectation value cut-off of Log(e) < –1 for both proteins

and peptides. Protein quantitation was performed by calculating Log(2) of the sum of

intensity of all fragments from MS/MS spectra, which belong to a particular protein.

Protein-level differences between media were then mapped into Z-scores, i.e. the

distance from the population mean in units of standard deviation. Proteins with Z-

25

scores greater than 1.65 or smaller than -1.65 (the outermost 10% of the distribution)

were considered significantly upregulated or downregulated in one media versus

another, respectively. Z-scores were calculated for three pairwise comparisons as

follows: 1) Dentin versus Plastic, 2) Bone versus Plastic, and 3) Bone versus Dentin.

Data Analysis

This collection of proteins was submitted to the Database for Annotation,

Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) for mapping

into Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/)

and Protein Analysis through Evolutionary Relationships (PANTHER,

http://www.pantherdb.org) pathways, as well as to Exocarta (http://www.exocarta.org/)

for classification into exosomes. While there is considerable functional overlap between

KEGG and PANTHER, some important pathways are encoded only in one system and

not the other. To gain broad biological insight into the underlying protein expression

differences, each of the 289 mouse pathways extracted from DAVID was examined,

looking for member proteins in our different sets with Z-score magnitudes greater than

1.65 and enumerated the number of positive and negative threshold proteins per

pathway. This step yielded a valuable overview of protein differences on a pathway

level, but was still somewhat unwieldy. To further get a sense of how the system

behaved, a simple additional layer of data reduction was proposed: for each pathway, a

nine-element vector of the difference between the upregulated and downregulated

protein counts was built, effectively giving a coarse view of "how does this pathway

behave across these different matrices?". This also provided the potential to find

correlations or divergences between the different systems on a pathway level. In

addition, gene ontology enrichment, network analysis, and graphical representation of

26

the data set was carried out using FunRich (www.funrich.org), which is an open access

functional enrichment analysis tool. 97

The experimental design for this project is summarized in Figure 2-1.

Figure 2-1. Experimental design.

27

CHAPTER 3 RESULTS

Cell Culture and Characterization of Clastic Cells

Our cell culture protocol was successful with all of the matrices and allowed us to

generate multinuclear clastic cells that were TRAP positive (Figure 3-1A).

Differentiation of BMSC into mono-nucleated pre-clastic cells occurred within 72 hours.

These cells fused to form multi-nucleated cells by day seven, as generally seen. 53 The

resorbing potential of the cells on dentin and bone was also confirmed by the formation

of actin ring structures at the end of the cell culture period (Figure 3-1B).

Figure 3-1. Mouse marrow-derived cells cultured on plastic, bone, and dentin slices. Multinuclear tartrate-resistant acid phosphatase (TRAP) positive cells (A) and actin ring structures (B, arrows) were evident at the end of the cell culture period.

Proteomic Analysis of Exosomes Extracted from Osteoclasts, Odontoclasts, and Non-resorbing Clastic Cells

Our main goal was to characterize the protein composition of exosomes and to

determine whether or not their protein content depends on the mineralized matrix type.

Our MS/MS analysis yielded 2183 proteins, and 84% of these proteins (1840 proteins)

are listed in the Exocarta database (unpublished data). Thus, we concluded that there

was an actual enrichment for exosomal proteins in our samples. When we focused at

28

day 7, the total number of proteins was 1951. Figure 3-2 shows a Venn diagram

summarizing the overlap between proteins from osteoclasts, odontoclasts, and non-

resorbing clastic cells. The proteomic composition was highly different and the

variability identified here is due to the different matrix type and/or the resorbing state of

the clastic cells. Pairwise comparisons at day 7 of the cell culture (dentin vs plastic,

bone vs plastic, and dentin vs bone) revealed many proteins with Z-scores of statistical

significance (Appendices E1 to E5); thus, we rejected both null hypotheses.

Figure 3-2. Three-way Venn diagram showing the overlap and differences between

exosomal proteins originated from osteoclasts, odontoclasts and non-resorbing clastic cells at day 7 of the cell culture. A total of 102 proteins were found only in odontoclasts’ exosomes, whereas 524 unique proteins were identified in exosomes from osteoclasts. In addition, there was an overlap of 426 proteins between osteoclasts and odontoclasts, while 712 proteins (of which 119 presented with significant Z-scores) were common between all cell types.

29

Pathway Analysis of Significantly Changed Proteins

To place this list of proteins in a more functional context, a pathway analysis was

employed as described in the materials and methods. The analysis allowed us to map

the changes in protein levels to identify biological processes that were modulated by the

matrix type. The results showed the endocytosis and oxidative phosphorylation

pathway proteins appeared to be expressed primarily in exosomes from osteoclasts.

Conversely, the vascular endothelial growth factor (VEGF) signaling pathway proteins

were enriched mostly in exosomes from osteoclasts and odontoclasts, but not in

exosomes from cells cultured on plastic wells. The analysis also revealed that the

complement and coagulation cascades (CCC), the N-glycan biosynthesis and the

vasopressin synthesis pathways were the most enriched in exosomes derived from

odontoclasts (Table 3-1, 3-2, 3-3).

Table 3-1. Molecular pathways and associated proteins that were significantly enriched

(Z > 1.65) in dentin and plastic at day 7 of cell culture. The gene symbols of the proteins are presented.

Odontoclasts (Od) vs Non-resorbing Clasts

Pathway Enriched proteins in odontoclast exosomes

Enriched proteins in exosomes from non-resorbing clasts (plastic)

Endocytosis H2-D1, FGFR3, ARAP1, CSF1R, TSG101, LDLR, SH3GLB1, RUFY1, GIT1, VPS36, VPS4B, VPS28, EHD4

ARAP2, RAB5A, H2-L

Oxidative Phosphorylation

ATP6V0D2, ATP6AP1, ATP6V1F, PPA1

None

VEGF Signaling PIK3CB, MAPKAPK2, MAPK3 NRAS, PRKCB, PPP3CC CCC SERPIND1, C1RA, F8, VWF,

C1QB None

N-Glycan Biosynthesis MAN1A2, MGAT5, B4GALT1 None Vasopressin Synthesis CPE PAM, SPCS3

30

Table 3-2. Molecular pathways and associated proteins that were significantly enriched (Z > 1.65) in bone and plastic at day 7 of cell culture. The gene symbols of the proteins are presented.

Osteoclasts (Oc) vs Non-resorbing Clasts

Pathway Enriched proteins in osteoclast exosomes Enriched proteins in exosomes from non-resorbing clasts

Endocytosis ITCH, H2-D1, ARRB1, VPS4A, GIT2, ARAP1, VPS45, SNF8, CSF1R, USP8, TSG101, SH3KBP1, LDLR, AGAP1, HSPA2, SH3GLB1, RUFY1, GIT1, VPS37C, VPS36, VPS4B, SH3GL1, MVB12A, AP2S1, RAB31, VPS25

ARAP2, H2-L

Oxidative Phosphorylation

ATP5B, ATP6V0D2, NDUFA4, UQCRC2, SDHA, NDUFS1, ATP6AP1, ATP6V0D1, ATP5A1, ATP6V1F, ATP5O, NDUFV1, PPA1

None

VEGF Signaling HSPB1, PIK3CB, MAPKAPK2, PPP3CA, MAPK14, PIK3CG, PLA2G4A

NRAS, PRKCB, PPP3CC

CCC C1RA, F8, VWF, MASP2 C8A, F10, SERPINC1

N-Glycan Biosynthesis None None Vasopressin Synthesis None None

Table 3-3. Molecular pathways and associated proteins that were significantly enriched

(Z > 1.65) in bone and dentin at day 7 of cell culture. The gene symbols of the proteins are presented.

Osteoclasts vs Odontoclasts

Pathway Enriched proteins in osteoclast exosomes Enriched proteins in odontoclast exosomes

Endocytosis ITCH, H2-D1, ARRB1, VPS4A, GIT2, ARAP1, VPS45, SNF8, CSF1R, USP8, TSG101, SH3KBP1, LDLR, AGAP1, HSPA2, SH3GLB1, RUFY1, GIT1, VPS37C, VPS36, VPS4B, SH3GL1, MVB12A, AP2S1

ARAP2, RAB31, H2-L

Oxidative Phosphorylation

ATP6V0D2, NDUFA4, UQCRC2, SDHA, NDUFS1 ATP6V0D1, ATP5A1, ATP5O, NDUFV1

None

VEGF Signaling HSPB1, PIK3CD, PXN, PPP3CA, PIK3CG, PLA2G4A

PIK3CA

CCC MASP2 SERPIND1, C8A, PLAU, C1QB, CFH

N-Glycan Biosynthesis MAN2A2 MAN1A2, MGAT5, B4GALT1

Vasopressin Synthesis CPE PAM, SPCS3,

31

Functional Enrichment Analysis

To better understand the matrix-specific proteomic changes in the exosome

composition, we initially performed functional enrichment analysis of the proteins that

were found exclusively in odontoclast or osteoclast exosomes. Of the 1951 proteins

identified at day 7, only 55 proteins were not successfully mapped to the FunRich

database. Among them, 102 proteins were expressed only in odontoclasts’ exosomes

whereas 524 proteins were unique to osteoclasts’ exosomes. The FunRich analysis of

proteins that were exclusively found in one media but not the other identified unique

underlying biological mechanisms associated with exosomes from odontoclasts and

exosomes from osteoclasts. In the context of biological processes, there were a total of

six biological processes found only in Od exosomes, three found only in Oc exosomes,

and three common between both cells. The six biological processes belonging to

odontoclast exosomes included enzyme linked receptor protein signaling pathway,

aldehyde metabolism, cellular morphogenesis during differentiation, cell recognition,

synaptic transmission, and xenobiotic metabolism. The three processes belonging to

osteoclast exosomes included cell-cell signaling, pyrimidine salvage, and regulation of

translation. The three mutual processes included energy pathways, metabolism, and

protein metabolism. It is interesting to note the mutual biological processes are also the

three processes with the highest percentage of proteins in both Od and Oc exosomes.

Metabolism is associated with the highest percentage of genes in both groups, with

bone at 19.4% and dentin at 15.6% (Figure 3-3A). On the cellular component aspect,

the proteins in both groups were found to be present in similar locations in the cell.

Eleven cellular components were found, and only one of these eleven components was

not found in both groups. This single component, the perinuclear vesicle, was found

32

only in odontoclast exosomes. The other ten cellular components were mutual between

both cells and include Golgi apparatus, axon, cytoplasm, Golgi transport component,

chromosome, lysosome, exosomes, cytosol, centrosome, and nucleolus. Of the eleven

components, the cytoplasm had the highest percentage of proteins present in both

odontoclast (56.2%) and osteoclast (61.2%) exosomes. All differences between the two

groups were less than 9% for each cellular component (Figure 3-3B). As for the

molecular function, a total of twelve were identified by FunRich with four belonging only

to odontoclasts exosomes, one belonging only to osteoclast exosomes, and seven

being present in both cells. The four molecular functions found only in odontoclast

exosomes included aldehyde dehydrogenase activity, protease activator activity,

neurotransmitter receptor activity, and transcription factor binding. The single molecular

function found only in osteoclast exosomes was structural constituent of ribosome. The

seven molecular functions found in both cells’ exosomes included protein

serine/threonine phosphatase, nucleotidyltransferase activity, catalytic activity, ubiquitin

specific protease activity, transporter activity, ligase activity, and RNA binding.

Odontoclast exosomes had the highest percentage of gene expression in ubiquitin

specific protease (4.2%). Osteoclasts’ exosomes had the highest percentage of gene

expression in two separate molecular functions – catalytic activity and transporter

activity – at 6.1%. Of the seven mutual molecular functions, five had higher

percentages in Oc than in Od exosomes (Figure 3-3C).

33

A.

B.

C.

Figure 3-3. Enrichment analysis of exosomes by FunRich. Comparison of A) biological processes, B) cellular components, C) and molecular function of proteins that were exclusively found in one media but not the other (dentin vs bone).

34

It was also of interest to identify biological implications of proteins that were

present in odontoclast and osteoclast exosomes but not in exosomes derived from cells

cultured on plastic, as this set of proteins may have an association with the resorbing

potential of clastic cells. To clarify, the ‘active clastic cell’ group included those proteins

found in dentin only, bone only, or dentin and bone simultaneously (Appendix E1-E3).

A comparison was made to proteins found only in plastic (Appendix E5). Seven

of 108 genes found to be unique to plastic were unmapped in FunRich, and 24 of 1052

genes associated with active cells were unmapped. In addition, six proteins from the

active cell group were not imported into FunRich due to not having a gene symbol. The

FunRich analysis indicated differences between exosomes derived from actively

resorbing clastic cells (dentin and bone) and exosomes derived from inactive clastic

cells (plastic). In the context of biological processes, there were three biological

processes found only in active cells, three found only in plastic, and six common

between the two groups. The three biological processes belonging to active cells were

protein modification, regulation of signal transduction, and peptide metabolism. The

three processes belonging to media from plastic included neurotransmitter metabolism,

wound healing, and hemopoeisis. The six mutual processes included protein

metabolism, metabolism, energy pathways, cell growth and/or maintenance, cell

proliferation, and synaptic transmission. Though mutual pathways, protein metabolism,

metabolism, and energy pathways had the highest percentage of genes for the active

clastic cells with 15.3%, 17.8%, and 17.2%, respectively. Cell growth and/or

maintenance at 15.2% was the highest for plastic. It is interesting to note the four

highest for both active and plastic were mutual processes (Figure 3-4A).

35

A.

B.

C.

Figure 3-4. Enrichment analysis of exosomes by FunRich. Comparison of A) biological processes, B) cellular components, and C) molecular function of proteins found in media from resorbing clastic cells (dentin, bone, and bone + dentin) versus those exclusive to inactive clastic cells (plastic).

36

Regarding cellular component, the proteins in both groups were found to be mutually

present in nine of the eleven locations. The other two components were found to be

unique to plastic, costamere and kinetochore microtubule, while zero were unique to

active cells. The nine mutual components included cytoplasm, lysosome, exosomes,

cytosol, centrosome, ribosome, fibrinogen complex, platelet alpha granule lumen, and

extracellular space. Of the eleven components, cytoplasm had the highest percentage

of proteins present in both active cells (63.8%) and plastic (57.3%) (Figure 3-4B). As for

molecular function, a total of twelve functions were identified by FunRich with two

belonging only to active cells, one belonging only to plastic, and nine being present in

both active and plastic. The two molecular functions unique to active cells included

ligase activity and translation regulator activity. The single molecular function unique to

plastic was spliceosomal catalysis. The nine molecular functions found in both active

and plastic included structural constituent of ribosome, transporter activity, ubiquitin-

specific protease activity, RNA binding, acyltransferase activity, cell adhesion molecule

activity, cytoskeleton protein binding, structural constitute of cytoskeleton, and GTPase

activator activity. Active clastic cells had the highest percentage of gene expression in

transporter activity (6.8%), while plastic had the highest percentage of gene expression

in two separate molecular functions, transporter activity and cell adhesion molecule

activity, at 6.1% (Figure 3-4C).

Screening of Exosomal Proteins as Potential Biomarkers of Dentin Resorption

The protein dataset (unpublished data) was filtered to identify exosomal proteins

that were significantly upregulated in odontoclasts when compared to osteoclasts (Z >

1.65) and identified by at least four peptides in odontoclast media (Table 3-4).

37

Table 3-4. Significantly upregulated proteins in odontoclast exosomes when compared to osteoclasts (Z >1.65) with at least four peptides in odontoclast samples. An asterisk (*) represents two proteins unique to odontoclast exosomes.

Gene Protein name # Peptides

Z-score (D7/B7)

PPP2CA Serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform

10 18.33

SELENBP2 Selenium-binding protein 2 7 18.13 HMCN2 Hemicentin-2 4 16.81 TTN Titin 6 16.69 ALDH1A1 Retinal dehydrogenase 1* 5 16.28 GAS6 Growth arrest-specific protein 6 7 16.05 FAT3 Protocadherin Fat 3 4 15.18 MMP19 Matrix metalloproteinase-19 5 15.09 D1PAS1 Putative ATP-dependent RNA helicase Pl10* 5 14.86 PDIA4 Protein disulfide-isomerase A4 4 14.52 NPM1 Nucleophosmin 6 3.67 MRC1 Macrophage mannose receptor 1 41 3.34 DDX58 Probable ATP-dependent RNA helicase DDX58 4 3.3 NCL Nucleolin 9 3.26 HAL Histidine ammonia-lyase 5 3.15 RAB8A Ras-related protein Rab-8A 4 3.12 HSP90AA1 Heat shock protein HSP 90-alpha 41 3.02 SDK2 Protein sidekick-2 5 2.92 TIE1 Tyrosine-protein kinase receptor Tie-1 4 2.9 UBE2G2 Ubiquitin-conjugating enzyme E2 G2 4 2.84 STAB1 Stabilin-1 63 2.45 NAP1L1 Nucleosome assembly protein 1-like 1 6 2.21 ST3GAL1 CMP-N-acetylneuraminate-beta-galactosamide-

alpha-2,3-sialyltransferase 1 4 2.21

LGALS3 Galectin-3 5 2.19 QPCT Glutaminyl-peptide cyclotransferase 10 2.02 ADAM10 Disintegrin and metalloproteinase domain-

containing protein 10 8 2.02

RUVBL1 RuvB-like 1 7 2 EPB41L2 Band 4.1-like protein 2 4 1.99 IFITM3 Interferon-induced transmembrane protein 3 4 1.98 MMP12 Macrophage metalloelastase 14 1.96 MGAT5 Alpha-1,6-mannosylglycoprotein 6-beta-N-

acetylglucosaminyltransferase A 4 1.93

PRKAR2B cAMP-dependent protein kinase type II-beta regulatory subunit

4 1.93

PLXDC2 Plexin domain-containing protein 2 4 1.93 EIF3A Eukaryotic translation initiation factor 3 subunit A 18 1.92

38

Table 3-4. Continued.

Gene Protein name # Peptides

Z-score (D7/B7)

NRP1 Neuropilin-1 10 1.91 C1QB Complement C1q subcomponent subunit B 6 1.91 NDRG2 Protein NDRG2 4 1.91 COL1A1 Collagen alpha-1(I) chain 7 1.89 PLAU Urokinase-type plasminogen activator 13 1.87 TUBB2A Tubulin beta-2A chain 4 1.86 UMPS Uridine 5'-monophosphate synthase 4 1.85 CFP Properdin 16 1.84 MRC2 C-type mannose receptor 2 10 1.84 ITGA4 Integrin alpha-4 4 1.79 EIF3D Eukaryotic translation initiation factor 3 subunit D 6 1.77 RPLP2 60S acidic ribosomal protein P2 4 1.75 CALR Calreticulin 4 1.75 GALC Galactocerebrosidase 6 1.74 CFH Complement factor H 41 1.72 PTPRD Receptor-type tyrosine-protein phosphatase delta 10 1.71 C1QA Complement C1q subcomponent subunit A 6 1.63 EMR1 EGF-like module-containing mucin-like hormone

receptor-like 1 4 1.63

CNTN1 Contactin-1 7 1.62 FGL2 Fibroleukin 4 1.62 HSP90B1 Endoplasmin 18 1.6 SDCBP Syntenin-1 12 1.3

39

CHAPTER 4 DISCUSSION

Oxidative Phosphorylation Pathway

Results of the present study indicate proteins of the oxidative phosphorylation

pathway are expressed primarily in exosomes from osteoclasts, and to a lesser degree

in exosomes from odontoclasts, but fail to be present in exosomes from non-resorbing

clastic cells (plastic). Energy hemostasis is a requirement for cell survival.

Mitochondrial oxidative phosphorylation (OxPhos) is a vital pathway in metabolism and

energy production, and is the last step of cellular respiration in eukaryotes. 98 OxPhos

works by driving reactions that require energy from those that release energy, and

accounts for a high yield of ATP. The electron transport chain and the ATP synthase

are known to be embedded in mitochondrial membranes. As electrons move through

the electron transport chain, an exergonic energy-releasing process occurs. This

released energy is transferred to the ATP synthase via movement of hydrogen ions

across the membrane resulting in ATP synthesis. ATP is then available for use by the

V-ATPase, which is an enzyme located in the ruffled border of actively resorbing mature

osteoclasts. This energy-dependent V-ATPase is responsible for the pumping of

protons against a high concentration gradient across the membrane into the sealed

extracellular resorbing zone. This proton transport results in a low-pH acidic

environment which allows for degradation of bone matrix. 99,100 Since the activity of the

V-ATPase is strictly dependent on the availability of ATP, a high demand for ATP is

required for bone resorption to occur. 101-103

The mitochondrial OxPhos pathway allows for large amounts of ATP production.

Different cell types differ in the number of mitochondria present, and osteoclasts have

40

been reported as mitochondria-rich cells containing a high expression of oxidative

phosphorylation enzymes. It has been suggested that during RANKL-induced

osteoclastogenesis, there is a metabolic shift towards increased mitochondrial

respiration.99,104 Previous studies have compared immature osteoclasts to mature

osteoclasts. Findings include increased numbers of subunits of the electron transport

chain, a higher mitochondrial oxygen consumption rate (OCR), increased expression of

respiratory chain complexes, and higher levels of intracellular ATP content in mature

osteoclasts.105,106 Since metabolic pathways are tightly regulated to provide responses

to environmental changes, some authors believe the energy-consuming processes of

osteoclastogensis may also be regulated and dictated by changes in metabolic

substrate concentrations and pathway activities, as well as oxygen availability. 99 In

addition, it has been suggested that OxPhos supports osteoclast survival, as previous

reports show hypoxia reduces the viability of osteoclasts. 107,108 These findings,

combined with our own, support the idea of increased OxPhos metabolism to sustain

the high energy-consuming functions and resorptive processes of mature active clastic

cells, including actin reorganization, proton pumping, acid degradation, vesicle

transportation, and enzymatic activities for bone resorption.105

In this study, some of the OxPhos pathway proteins found in exosomes from

resorbing cells include ATP6V0D2, ATP6AP1, and ATP6V1F, which are distinct

subunits of the V-ATPase. ATP6V1F belongs to the peripheral domain of the enzyme

(V1), whereas ATP6V0D2 is a membrane intrinsic subunit (V0). The well-studied V0

and V1 domains form the core of the enzyme and control ATP hydrolysis and proton

translocation, respectively.109 ATP6AP1, on the other hand, is an accessory protein to

41

the V-ATPase, also known as Ac45, whose function has not been entirely clarified. 110

Yang et al.111 reported that ATP6AP1 may play a direct role in guiding lysosomal

trafficking and exocytosis during osteoclastic bone resorption; nevertheless, the V-

ATPase subunits that interact with ATP6AP1 are still unknown. 109

V-ATPase is found in large numbers on the ruffled borders of active clastic cells;

thus, the significant enrichment of V-ATPase subunits observed in the exosomes from

resorbing osteoclasts and odontoclasts is not surprising. Raimondo et al.112 reported

that 34% of plasma membrane proteins are sorted into exosomes; however, the exact

mechanism by which these proteins are loaded into exosomes is still unclear. 113 It is

also possible that the V-ATPase subunits identified in our study come from ectosomes

and not exosomes. These two types of extracellular vesicles are categorized mainly by

origin: while exosomes are secreted by fusion of intracellular multivesicular bodies

(MVB) with the plasma membrane, ectosomes bud directly from the cell membrane. 113

Most studies in the current literature categorize these two type of vesicles under a broad

umbrella term named extracellular vesicles (EVs), since purification methods to

separate them are rarely successful. Regardless, the presence of V-ATPase subunits

in EVs may be explored as a clinical biomarker of mineralized tissue remodeling in

dental maladies, especially because our previous study identified ATPases in gingival

crevicular fluid (GCF). 1 The fact that not all V-ATPase subunits were identified in our

samples is intriguing and may indicate a more controlled peptide-sorting mechanism

during V-ATPase recycling.

Stabilin-1

In this study, we identified an enrichment of Stabilin-1 in exosomes of resorbing

cells, but not in exosomes of non-resorbing clasts. This was the most prominent single

42

marker of active clastic cells compared to inactive clastic cells. Stabilin-1 is a multi-

functional, large, type-1, transmembrane receptor protein originally discovered on bone

marrow sinusoidal endothelial cells. It is currently known that Stabilin-1 is also found on

tissue macrophages and can be recognized as an immune regulator of inflammatory

processes. 114,115 Macrophages are wide-spread mononuclear cells that use

phagocytosis to consume unwanted particles and debris. Their function is to monitor

tissue in their location and act to maintain tissue hemostasis by initiating the innate

immune system and recruit cells for the adaptive immune system. 116,117 On the

contrary, they also release cytokines, which play a role in anti-inflammation, and are

called alternatively activated macrophages (M2s). Macrophages, osteoclasts, and

odontoclasts are blood-borne cells derived form a common ancestor: the monocyte.

Therefore, they are closely related. Some authors even report that macrophages can

fuse on the bone surface to form multinucleated osteoclasts 118 119 while others believe

that osteoclast-like cells develop from a distinct monocyte progenitor. 119

Although there are no studies implicating a direct role of stabilin-1 in mineralized

tissue resorption, current studies highlight the importance of this protein in macrophage

function and soft tissue remodeling. 120,121 Stabilin-1 may play a role in homeostasis,

alternative macrophage activation (M2), angiogenesis, lymphocyte homing, cell

adhesion, cell-cell and cell-matrix interaction, inflammation, tumorigenesis, and receptor

scavenging. Regarding receptor scavenging, Stabilin-1 is variably expressed on

alternatively activated macrophages, has phagocytic capabilities, and has been

reported to endocytose ligands and aid in clearance of apoptotic cells and unwanted-

self components, thereby participating in the anti-inflammatory and healing

43

process.122,123 It is also involved in trafficking between the plasma membrane and early

endosomes and it has been suggested as a link between the extracellular environment

and intracellular vesicular processes. 124 Secreted protein acidic and rich in cysteine

(SPARC) is a regulator of extracellular matrix and tissue remodeling and has increased

expression during wound healing, angiogenesis, and development due to its role in

inflammation as an anti-adhesive. 120 Stabilin-1 was discovered to be a receptor for

SPARC and is considered a mediator for its internalization by alternatively activated

macrophages, clearing SPARC and regulating its concentration in the environment.

Because of this, it has been proposed that alternately activated macrophages are

involved in regulation of extracellular matrix remodeling. 121

An inflammatory response will result in an acidification of the extracellular

environment. In order to protect healthy cells, macrophages must remove apoptotic

cells from acidic inflammatory sites. It has been reported that that the degree of

reduction in inflammatory response is related to the extracellular acidification. 125 Park

et al.125 evaluated the existence of a link between the acidic environment associated

with inflammation and the function of macrophages. The results of this study

demonstrated that in the presence of a low pH environment, Stabilin-1 expression

increased and phagocytic activity of macrophages increased. In addition, by inhibiting

Stabilin-1 via anti-Stabilin-1 antibody, no increase in phagocytosis occurred. 125 This

suggests that an acidic environment will induce up-regulation of Stabilin-1, which

increases the phagocytic capacity of alternatively-activated macrophages increasing

clearance of apoptotic cells, which in turn aids in resolution of inflammation and tissue

hemostasis.

44

Three things may be significant in relation to our work. First, clastic cells and

macrophages share the same lineage and therefore may express similar receptors,

such as Stabilin-1. Second, when clastic cells are activated, a sealing zone is formed

and the cell reorganizes itself and develops a ruffled border membrane. This supports

the transport of protons for acidification of the extracellular resorption compartment and

dissolution of the bone mineral. This low pH acidic environment may be functioning in a

similar way to those previously described in inflammation. 122 Third, debris and

degradation products from the resorption zone are removed via endocytic vesicles and

via transcytosis from the ruffled border to the free membrane to be released via

exocytosis.126,127 Therefore, we may be able to suggest that the extracellular low pH

resorption zone, which is accompanied by increased inflammation and increased hard

tissue debris and apoptotic particles, increases Stabilin-1 expression on the osteoclast

and/or local macrophages. This may lead to clearance of resorption debris and aid in

inflammatory resolution. The present study finding of enriched Stablin-1 expression in

exosomes of resorbing cells, may point that this protein indicates active clastic cells are

present and bone or dentin resorption may be occurring. Therefore, Stabilin-1 could

possibly be a potential biomarker for the detection of resorbing cells, and future

research should look further into this relationship.

Plexin B1

Bone remodeling is an orchestrated process that requires a continuous crosstalk

between osteoblasts and osteoclasts. Plexin B1 is a transmembrane protein located on

osteoblasts that interacts with Semaphorin 4D (Sema 4D), which is a soluble protein

secreted by osteoclasts. Studies show that the Sema4D/Plexin B1 interaction plays an

important role in bone turnover. 128-130 Plexin B1 is involved in the osteoclast

45

differentiation pathway by acting as a transduction factor. It is also known as a receptor

for Semaphorin 4D (Sema4D). Sema4D is a multifunctional protein and is important in

regulation of physiological and pathological immune responses. It is released by

osteoclasts and binds Plexin B1 to inhibit the motility and osteoid formation of

osteoblasts, thereby inhibiting new bone formation. 129 Sema4D is upregulated during

RANKL-induced osteoclastogenesis. The Sema4D and Plexin B1 interaction causes a

change in bone remodeling hemostasis in favor of osteoclastogenesis. In addition, the

binding of Sema4D to Plexin B1 mediates osteoblast production of interleukin-8 (IL-8),

which indirectly activates osteoclasts and stimulates bone resorption. These processes

occur through cytoplasmic signaling cascades of Sema4D – Plexin B1 – RhoA

activation – ending with either IGF1 suppression (inhibition of osteoblasts) or IL-8

production (stimulation of osteoclasts). 130,131 Previous studies report that a deficiency

in Plexin B1 or Sema4D results in high bone mass phenotype, by stimulating the

proliferation of osteoblasts without inhibiting osteoclast function. 131-133 Because of

these findings and the regulatory cell-cell communication mediated by Sema4D and

Plexin-B1 for bone formation and resorption, these molecules and their mechanisms are

becoming of interest as potential targets for treatment of bone diseases and cancer

metastases. 128,129,133

Our results show Plexin B1 to be uniquely present in exosomes released by

odontoclasts. This is indeed an intriguing observation. On one hand, the lack of this

protein in exosomes derived from bone slices makes perfect sense because Plexin-B1

is located in osteoblasts and our cell culture protocol did not employ a co-culture model

of osteoclasts and osteoblasts to promote bone resorption. On the other hand, the high

46

abundance of Plexin B1 in odontoclast exosomes is hard to explain based on our

current understanding of the dentin resorption process. Unlike bone, dentin is structural

in nature and has reduced healing properties. Besides, it is not completely proven that

cell-cell crosstalk is needed for dentin resorption. As such, many questions remain: Is

Plexin B1 expressed in odontoclasts? Is this protein embedded in the dentin matrix,

and therefore the byproducts are being released as a result of dentin breakdown? The

biological and clinical implication of this finding remain to be completely understood;

however, this result represents a relevant piece of evidence indicating functional

differences between osteoclasts and odontoclasts.

Serpin A11

In the present study, Serpin A11 was another protein found to be enriched in

exosomes from odontoclasts only. Serpin A11 is a member of the serpin family, which

is a large family of endogenous protease inhibitors functioning in proteolytic cascades,

tissue remodeling, blood clotting, and inflammatory responses. Serpins are most known

for the ability to irreversibly inhibit their target protease by inducing a structural change.

Specifically, Serpin A11 is a serine protease inhibitor, meaning it inhibits the breakdown

of proteins and peptides. The Uniprot database has classified it as a human molecule in

the extracellular space. 134 Its gene expression is low in breast, breast tumors, and

benign prostatic hyperplasia, while its expression is high in the healthy liver. 135,136

To the best of our knowledge, there is sparse literature regarding the role of

Serpin A11 in mineralized tissue resorption. A recent study 137 used mass spectrometry

techniques to identify proteins with heparin affinity (anticoagulant) in plasma samples

collected from patients with different types of arthritis. The authors found Serpin A11 to

be down-regulated in psoriatic arthritis and up-regulated in rheumatoid arthritis (RA),

47

when compared to non-inflammatory arthritis (osteoarthritis). The synovial tissue in RA

invades the subchondral bone marrow where it erodes the calcified cartilage.

Histological studies of RA joints show that the interface between the calcified cartilage

and pannus is populated by multinucleated cells with morphological features of

‘chondroclasts’. Thus, the increased expression of Serpin A11 in patients in rheumatoid

arthritis should be further validated as potential diagnostic biomarkers of cartilage

destruction. As far as we know, there is no report showing an association between

Serpin A11 and dentin resorption, and this correlation is demonstrated for the first time

in our in vitro experiments.

Limitations and Final Remarks

It must be taken into consideration that the present work analyzed the proteomic

composition of exosomes released from cells, therefore it cannot be conclusive from

this study that the enriched proteins found in exosomes directly correlate to an increase

of the protein expression within the cell. The enrichment of proteins in exosomes may

represent a cellular mechanism to get rid of unnecessary proteins or pathways, as well

as a signaling mechanism for intercellular communication. Thus, the biological

relevance of these findings should be interpreted with caution

.

48

CHAPTER 5 CONCLUSIONS

With continual improvements in orthodontic techniques comes an increase in

patient expectations. Orthodontic treatment is a major contributing factor of root

resorption. Moderate to severe dental root resorption may cause premature tooth loss,

which can have a financial and emotional impact on the patient. Considering the

inadequacy of conventional 2D radiography and the radiation exposure associated with

3D CT scans, it is of benefit to the profession to develop a safe, inexpensive, and

accurate method to diagnose and prevent dental root resorption. The potential use of

biomarkers could provide an easy-to-use method that allows for detection and diagnosis

at an earlier stage of root resorption, and therefore may become the standard technique

used by practitioners. Although many studies report the discovery of potential

biomarkers of dentin resorption, most of them have not been validated and this leads to

a low number of successful market applications. Recent and ongoing research may

lead to the use of oral-fluid derived exosomes as a potential source of biomarkers for

dental maladies. Thus, it is important to determine the existence of odontoclast-specific

exosomal markers, which was the main purpose of this research project. Both null

hypotheses were rejected in the present study; thus, our results support the conclusion

that odontoclasts secrete exosomes in vitro and that these vesicles can be successfully

isolated. In addition, exosomal proteins may serve as markers for the resorbing state of

osteoclasts and odontoclasts. Therefore, the results of our study may serve a

diagnostic purpose, as well as fundamental knowledge for the future development of

non-invasive assays to monitor bone and dentin resorption in vivo.

49

APPENDIX A ISOLATION OF PRIMARY BONE MARROW STEM CELLS AND

OSTEOCLAST/ODONTOCLAST CELL CULTURING FOR EXOSOME ANALYSIS

Bone Marrow Extraction – Day One (2-4 Hours) Prior to Starting:

1. Read the protocol 2. Disinfect biosafety hood 3. Create/gather all needed media and solutions: α-MEM, α-MEM + 10% FBS

(exosome-depleted and non-heat-inactivated), sterile 1X PBS. Place α-MEM and sterile 1X PBS on ice under hood. Place α-MEM + 10% FBS in 37°C water bath

4. Disinfect needed pipettes, conical tubes, surgical instruments, and paper towels and place under hood

5. Fill needed 10mL syringes with α-MEM and attach needles (27 or 30 gauge). Place syringes on ice under hood

6. Place three 60mm dishes under hood. Leave one plate empty, fill one halfway with cold sterile 1X PBS, and fill one with ~10mL α-MEM

7. Gather all other needed items and place in close proximity to hood 8. Turn on centrifuge and set temperature to 4°C

Protocol:

1. Properly euthanize mouse per IACUC regulations. Wet thoroughly with 70% ethanol on paper towels under hood

2. Remove femur and tibia by severing at hip and then ankle. Remove excess tissue and muscle from femur. Place femur in cold sterile 1X PBS in 60mm dish

3. Repeat steps 1 and 2 until all mice are euthanized and all femurs are removed and stored in 1X PBS dish

4. Properly dispose of mice 5. Trim ends of femur to expose interior of marrow shaft 6. Using 27 or 30 gauge needle attached to a 10mL syringe filled with α-MEM, flush

out marrow cells into 60mm dish containing α-MEM from both ends of femur until all marrow is removed (bone should be transparent; usually requires 1-2mL media per femur)

7. Break up clumps in cell/medium mixture by pipetting up and down against bottom of dish

8. Filter mixture through 70µm cell strainer into 50mL conical tube 9. Centrifuge mixture at 1500 rpm for 15 min at 4°C 10. Discard supernatant and re-suspend cell pellet in 5mL α-MEM + 10% FBS 11. To two 150mm tissue culture (TC) dishes, add 40mL α-MEM + 10% FBS and

20ng/ml M-CSF. Split cell re-suspension evenly between the dishes (2.5 mL per dish)

12. Incubate overnight at 37°C with 5% CO2 13. Leave pre-washed bone/dentin slices needed for culturing of cells soaking in a

1:10 mixture of Penicillin-Streptomycin : α-MEM + 10% FBS in a sterile petri dish overnight at 37°C with 5% CO2. The next morning (at least a few hours before day two protocol is started), flip the bone/dentin slices over under the hood to

50

allow both sides exposure to the antibiotic. Place petri dish back into 37°C incubator with 5% CO2 when finished

Culturing of Cells on Bone/Dentin Slices – Day Two (3-5 Hours) Prior to Starting:

1. Read the protocol 2. Disinfect biosafety hood 3. Create/gather all needed media and solutions: α-MEM, α-MEM + 10% FBS,

sterile 1X PBS. Place α-MEM and sterile 1X PBS on ice under hood and α-MEM + 10% FBS in 37°C water bath

4. Disinfect needed pipettes, conical tubes, and other supplies and place under hood.

5. Gather all other needed items and place in close proximity to hood 6. Turn on centrifuge and set temperature to 4°C 7. Bone/dentin slices should have been flipped over for at least a few hours at this

point (see step 13 in day one protocol). Wash bone/dentin slices by emptying petri dish and filling with α-MEM + 10% FBS three times. After third rinse, fill petri dish with α-MEM + 10% FBS and place dish back into 37°C incubator with 5% CO2 until bone/dentin slices are needed

Protocol:

1. To gather the non-adherent cells from the two TC plates, collect the supernatant from each plate and transfer to 50mL conical tubes (1 tube per plate). Very gently rinse each TC plate with 5mL sterile 1X PBS and add the rinsate to the corresponding conical tube (leaves 47 mL per tube). Dispose of TC plates

2. Centrifuge conical tubes at 1500 rpm for 15 min at 4°C 3. Discard the supernatant from each tube and re-suspend each pellet in 7mL α-

MEM 4. To two 15mL conical tubes, add 5mL Histopaque 1083. Add directly to the

bottom of each tube (try to avoid droplets against the sides of tube) 5. Gently overlay the cell re-suspension from each 50mL conical tube onto the

Histopaque layer of one 15mL conical tube (one tube to one tube). Accomplish this by gently tilting the 15mL conical and pipetting the cell-re-suspension against the inside of the tube. Do not allow these layers to mix. Pipetting should be slow enough to take approximately two minutes to empty the pipette of the 7mL of cell re-suspension

6. Gently cap the 15mL conical tubes and carefully transfer to centrifuge. Centrifuge conical tubes at 1500 rpm for 15 min at 4°C

7. Carefully remove tubes from centrifuge and collect the interface layer (opaque layer between the top pink layer and the lighter bottom layer) from each tube and transfer both to one 50mL conical tube. If the overlay in step 5 was done correctly, this interface layer should yield 4-5mL

8. Add enough α-MEM to the 50mL conical tube to bring the volume to 45mL 9. Centrifuge conical tube at 1500 rpm for 15 min at 4°C 10. Discard the supernatant and re-suspend the pellet in 5mL α-MEM + 10% FBS

51

11. Using proper counting technique, count the cells and re-suspend cells at a concentration of 2 x 105 cells per mL in α-MEM + 10% FBS + 20ng/ml M-CSF + 20 ng/ml mRANKL. The cells will be plated at this concentration

12. Transfer bone/dentin slices to wells in 12-well tissue culture plate, making sure the slice rests on the bottom of the well. Bone/dentin slices should cover as much of the bottom of the well as possible with even coverage among wells

13. Mix cell re-suspension and add 2mL to each well in the 12-well plate 14. Incubate cells at 37°C with 5% CO2

Notes:

• From this point, feed cells on days 3 and 7 (today is day 0) by removing ¾ of the media from the wells (1.5mL per well) and adding 2mL fresh α-MEM + 10% FBS + 20 ng/ml M-CSF + 20 ng/ml mRANKL to each well. Pipette gently against the side of the well when removing and adding media to wells to not disrupt the cells. Replacement media should be warmed to 37°C before adding to wells and should be added immediately after removing the existing media. Place plate back in 37°C incubator with 5% CO2 immediately after each feeding. Proceed with the ExoQuick-TC protocol with the conditioned media pulled from the wells

• Using 12-well plates is not mandatory. As long as the correct media proportions are maintained then any size plate can be used (2mL in 12-well plate, 4mL in 6-well plate, etc.). Smaller wells may be a favorable option when bone/dentin is scarce, as smaller pieces can be used to provide larger surface area coverage in the wells

52

APPENDIX B OSTEOCLAST/ODONTOCLAST TRAP STAINING TECHNIQUE

(Adapted from Sigma Aldrich Acid Phosphatase Kit)

Trap Staining – Day Eleven (2-3 Hours) Prior to Starting:

1. Read the protocol 2. Disinfect biosafety hood 3. Gather all needed chemicals/solutions: Acetone, Citrate, Formaldehyde,

Fastgamel Base Solution, Sodium Nitrite Solution, Napthol AS-BI Phosphate Solution, Acetate Solution, Tartrate Solution, Hematoxylin Solution (Gill No. 3)

4. Disinfect needed pipettes, conical tubes and place under hood 5. Gather all other needed items and place in close proximity to hood 6. Warm enough double-deionized water to 37°C in water bath

Protocol:

1. Create Fixative Solution: 65ml acetone + 25ml citrate + 8ml formaldehyde in sterile bottle. Allow to come to room temperature before use. Store at 4°C when finished. May be used repeatedly

2. Create Fastgamel Solution: mix Fastgamel Base Solution + Sodium Nitrite Solution in 1:1 ratio to create enough volume of Fastgamel Solution needed in Step 3. Mix by gently inverting for 30 seconds, then allow to stand for two minutes before use

3. Create Staining Solution (to be done every time): for 13 wells mix 5.85ml dd-water (37.5°C) + 130ul fastgamel solution + 65ul napthol + 260ul acetate + 130ul tartrate

4. Remove media from wells and freeze back at -80°C in temperature-safe conical tube

5. Add 500ul Fixative Solution per well. Incubate for 10 minutes at room temperature

6. Remove and discard fixative solution from all wells 7. Add 1mL Staining Solution to each well and incubate plate in 37°C incubator for

1 hour 8. Remove and discard staining solution from all wells 9. Rinse wells with 1mL double-deionized water two times, discarding rinsate each

time 10. Add 500ul Gill No. 3 Solution (Hematoxylin) and let stand for two minutes at room

temperature 11. Remove and discard Gill No. 3 Solution from all wells. Rinse wells once with

500ul tap water for several minutes, then discard rinsate 12. View wells under microscope to confirm presence of osteoclasts/ odontoclasts

Note: This protocol assumes use of a 12-well culture plate. Measurements given may need to be altered if using a different size plate.

53

APPENDIX C ACTIN RING STAINING TECHNIQUE

(Adapted from Holliday Lab 138)

To detect actin rings, osteoclasts that had been cultured for 11 days were put on coverslips, bone slices, or dentine slices. They were fixed with 2% formaldehyde in PBS for 20 minutes, permeabilized with 1% Triton X-100 for 10 minutes, and then stained with Texas Red-Tagged phalloidin (10 ug/ml) for 30 minutes (Sigma/Aldrich) to detect the actin rings. Actin rings were visualized by epifluorescence microscopy.

54

APPENDIX D EXOSOME ISOLATION FROM OSTEOCLAST/ODONTOCLAST CELL CULTURE

USING EXOQUICK-TC*

(Adapted from ExoQuick-TC Protocol (System Biosciences, Mountain View, CA)) Prior to Starting:

1. Read the protocol 2. Disinfect biosafety hood 3. Create/gather all needed media and solutions and place at proper temperatures 4. Disinfect needed pipettes, tubes, and other items and place under hood 5. Remove conditioned media from wells as instructed in “Isolation of Primary Bone

Marrow Stem Cells and Osteoclast/Odontoclast Culture for Exosome Analysis” and pool in sterile 15mL conical tubes

Protocol: 1. Centrifuge conditioned media at 3,000 × g for 30 minutes at room temperature 2. Using a stripette to measure the volume, transfer the supernatant to sterile 15mL

conical tube 3. Add 1mL ExoQuick-TC per 5mL conditioned media to the conical tube. Mix well

by inversion. Make sure to add at least this ratio of ExoQuick-TC. 4. Incubate tube overnight at 4°C 5. The following day, centrifuge at 1,500 × g for 30 minutes at room temperature

and remove the supernatant. Be careful to not disturb the pellet, as it is loose at this step. Do not get too close to the pellet when pulling off the supernatant – leaving a little supernatant behind is better than removing some of the pellet with the supernatant

6. After removing supernatant, centrifuge again at 1,500 × g for five minutes at room temperature and aspirate remaining supernatant. Avoid aspirating the pellet

7. Re-suspend the pellet in 100uL sterile 1X PBS. Remove two 10uL aliquots of the cell re-suspension and transfer each to a sterile 0.6mL tube. Freeze both at -80°C. One will be used for the EXOCET Exosome Quantitation Kit and the other will be used for transmission electron microscopy

8. Airfuge remaining cell re-suspension for two hours, aspirate supernatant, then freeze the pellet at -80°C

55

APPENDIX E PROTEINS PRESENT DAY 7 BY MATRIX TYPE

Table E-1. Proteins Present in Dentin Day 7, but not Bone Day 7 or Plastic Day 7.

Protein Description (103) Z-Score Dentin7/ Plastic7

Z-Score Bone7/ Dentin7

Actin, cytoplasmic 1 16.51 -23.58 Receptor-interacting serine/threonine-protein kinase 3 13.88 -20.17 Plexin-B1 12.49 -18.37 Insulin-like growth factor-binding protein 6 12.33 -18.15 Serpin A11 12.3 -18.12 Ras-related protein Rab-40C 12.05 -17.79 Histone H3.2 11.68 -17.32 Signal peptidase complex subunit 3 11.63 -17.25 Leucine-rich repeat protein SHOC-2 11.33 -16.85 Coiled-coil domain-containing protein 66 11.28 -16.79 Partitioning defective 3 homolog B 11.17 -16.65 Kinesin-like protein KIF1B 10.93 -16.33 Retinal dehydrogenase 1 10.89 -16.28 Heparin cofactor 2 10.89 -16.28 Arginase-1 10.59 -15.89 Sulfotransferase 1C2 10.46 -15.73 Proteasome subunit beta type-9 10.39 -15.64 Acid-sensing ion channel 2 10.31 -15.53 SH3 and PX domain-containing protein 2B 10.2 -15.38 Protein FAM208B 10.17 -15.35 Serine/arginine-rich splicing factor 2 10.09 -15.25 Chromodomain-helicase-DNA-binding protein 5 10.09 -15.25 UDP-N-acetylhexosamine pyrophosphorylase 10.05 -15.2 Protein FAM179B 10.05 -15.19 Neurofibromin 10.03 -15.17 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform 10.01 -15.14 Protein TALPID3 9.99 -15.11 Collagen alpha-6(VI) chain 9.99 -15.12 Nuclear migration protein nudC 9.97 -15.09 Acidic leucine-rich nuclear phosphoprotein 32 family member A 9.96 -15.08 Amyloid beta A4 protein 9.93 -15.04 Centrosomal protein of 72 kDa 9.91 -15.02 Endothelin-converting enzyme 1 9.91 -15.01 Platelet glycoprotein 4 9.89 -14.99 NAD(P)H dehydrogenase [quinone] 1 9.88 -14.97 SET-binding protein 9.85 -14.93

56

Table E-1. Continued.

Protein Description (103) Z-Score Dentin7/ Plastic7

Z-Score Bone7/ Dentin7

Large proline-rich protein BAG6 9.84 -14.91 Histone-lysine N-methyltransferase, H3 lysine-36 and H4 lysine-20 specific 9.82 -14.9 Fibroblast growth factor receptor 3 9.81 -14.88 Putative ATP-dependent RNA helicase Pl10 9.8 -14.68 Small subunit processome component 20 homolog 9.74 -14.79 Proteasome activator complex subunit 1 9.72 -14.76 Protein enabled homolog 9.72 -14.76 Zinc finger homeobox protein 3 9.67 -14.7 Putative N-acetylglucosamine-6-phosphate deacetylase 9.64 -14.66 Liprin-beta-1 9.62 -14.63 Ring finger protein 26 9.61 -14.62 Latent-transforming growth factor beta-binding protein 4 9.53 -14.52 FK506-binding protein 15 9.5 -14.48 Peptidyl-glycine alpha-amidating monooxygenase 9.49 -14.46 Cardiomyopathy-associated protein 5 9.49 -14.46 Tumor necrosis factor alpha-induced protein 2 9.48 -14.45 Rho GTPase-activating protein 29 9.46 -14.43 Pleckstrin 9.45 -14.41 Meteorin-like protein 9.43 -14.38 Conserved oligomeric Golgi complex subunit 6 9.36 -14.3 Mannosyl-oligosaccharide 1,2-alpha-mannosidase IB 9.34 -14.27 E3 ubiquitin-protein ligase TRIM68 9.33 -14.26 Amyloid-like protein 2 9.32 -14.25 Prostaglandin E synthase 3 9.25 -14.15 Centrosomal protein of 112 kDa 9.24 -14.14 Mitogen-activated protein kinase kinase kinase 4 9.2 -14.09 5'-AMP-activated protein kinase catalytic subunit alpha-2 9.2 -14.09 Zinc finger protein-like 1 9.2 -14.09 Epididymal secretory protein E1 9.14 -14.02 Testin 9.11 -13.97 Queuine tRNA-ribosyltransferase 9.11 -13.97 NudC domain-containing protein 2 9.1 -13.96 Neutrophil cytosol factor 1 9.09 -13.94 Kinesin-like protein KIF23 9.08 -13.93 Intraflagellar transport protein 81 homolog 9.05 -13.89 Alpha-2-HS-glycoprotein 9.03 -18.87 DNA replication licensing factor MCM5 9.03 -13.87 Exosome complex component RRP41 9.03 -13.87 Receptor-type tyrosine-protein phosphatase-like N 9.02 -13.85

57

Table E-1. Continued.

Protein Description (103) Z-Score Dentin7/ Plastic7

Z-Score Bone7/ Dentin7

Branched-chain-amino-acid aminotransferase, mitochondrial 9.01 -13.85 UDP-glucuronosyltransferase 1-7C 8.97 -13.79 Glutathione S-transferase Mu 2 8.93 -13.74 Vacuolar protein sorting-associated protein 28 homolog 8.9 -13.7 Extended synaptotagmin-1 8.9 -13.7 Sortilin-related receptor 8.89 -13.69 Unconventional myosin-XVIIIa 8.89 -13.68 Beta-1,4-galactosyltransferase 1 8.86 -13.65 Peroxisomal N(1)-acetyl-spermine/spermidine oxidase 8.83 -13.61 Long-chain-fatty-acid--CoA ligase 5 8.82 -13.59 Elongation factor G, mitochondrial 8.8 -13.57 Exocyst complex component 2 8.78 -13.55 Oxysterol-binding protein-related protein 11 8.78 -13.54 Adipocyte plasma membrane-associated protein 8.76 -13.52 Non-receptor tyrosine-protein kinase TYK2 8.72 -13.46 Ubiquitin-conjugating enzyme E2 D2 8.71 -13.45 Protein phosphatase 1A 8.62 -13.34 Proto-oncogene vav 8.6 -13.31 Splicing factor 3A subunit 3 8.59 -13.3 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D member 3 8.47 -13.15

Bone morphogenetic protein 10 8.44 -13.1 HHIP-like protein 1 8.43 -13.09 Conserved oligomeric Golgi complex subunit 1 8.39 -13.04 UDP-glucuronic acid decarboxylase 1 8.36 -13 Centromere-associated protein E 8.36 -12.99 Neurexin-1 8.32 -12.95 Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit alpha isoform 8.07 -12.62

Choline-phosphate cytidylyltransferase A 7.98 -12.5

58

Table E-2. Proteins Present in Bone Day 7, but not Dentin Day 7 or Plastic Day 7.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

FERM and PDZ domain-containing protein 1 12.71 20.09 Cathepsin O 11.33 18.05 MI 11.12 17.74 Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform 10.98 17.53 Alanine aminotransferase 1 10.93 17.46 Angiotensin-converting enzyme 10.61 16.98 Lysosomal acid lipase/cholesteryl ester hydrolase 10.54 16.88 Selenium-binding protein 1 10.24 16.44 Beta-actin-like protein 2 10.24 16.44 Lanosterol synthase 9.98 16.06 Uncharacterized protein C5orf42 homolog 9.94 16 Histone H2B type 3-A 9.92 15.97 Sclerostin 9.92 15.97 F-box only protein 30 9.89 15.92 Target of Myb protein 1 9.84 15.85 Dynein heavy chain 2, axonemal 9.75 15.71 Rab GTPase-activating protein 1 9.72 15.68 28S ribosomal protein S15, mitochondrial 9.7 15.64 Ankyrin-2 9.64 15.55 40S ribosomal protein S20 9.53 15.38 CAD protein 9.51 15.37 Beta-enolase 9.51 15.36 40S ribosomal protein S10 9.49 15.33 Tenascin 9.47 15.3 ATP synthase subunit alpha, mitochondrial 9.42 15.22 WD repeat-containing protein 78 9.27 15 Leucine-rich repeat and calponin homology domain-containing protein 1 9.25 14.97 E3 ubiquitin-protein ligase Itchy 9.21 14.92 40S ribosomal protein S26 9.17 14.85 Glycogen [starch] synthase, muscle 9.14 14.82 Acylamino-acid-releasing enzyme 9.14 14.82 Cytochrome c oxidase subunit NDUFA4 9.13 14.8 Beta-centractin 9.11 14.77 Palmitoyl-protein thioesterase 1 9.1 14.76 Serine/threonine-protein kinase MRCK beta 9.1 14.75 Proline-serine-threonine phosphatase-interacting protein 1 9.09 14.74 Ataxin-10 9.08 14.73 Protein phosphatase 1H 9.05 14.68 Biglycan 9.04 14.67 AP-1 complex subunit sigma-2 9.03 14.66

59

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

40S ribosomal protein S14 9.03 14.66 Sialidase-1 9.02 14.64 Keratin, type I cytoskeletal 16 9.02 14.63 Tenascin-N 9.01 14.62 60S ribosomal protein L11 8.97 14.56 Mannan-binding lectin serine protease 2 8.95 14.54 TBC1 domain family member 24 8.95 14.54 5'-AMP-activated protein kinase catalytic subunit alpha-1 8.92 14.49 Protein transport protein Sec24A 8.91 14.48 60S ribosomal protein L21 8.91 14.47 Unconventional myosin-Va 8.91 14.47 Nucleoprotein TPR 8.9 14.45 E3 ubiquitin-protein ligase UBR1 8.88 14.43 Armadillo repeat-containing protein 10 8.87 14.42 Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial 8.87 14.41 Ragulator complex protein LAMTOR1 8.86 14.4 Kinesin-like protein KIF20B 8.86 14.39 Polyadenylate-binding protein-interacting protein 1 8.85 14.38 Aldo-keto reductase family 1 member C13 8.81 14.32 40S ribosomal protein S27 8.79 14.3 Neurolysin, mitochondrial 8.79 14.3 ADP-ribosylation factor 4 8.77 14.27 SPARC 8.75 14.23 E3 ubiquitin-protein ligase HECTD1 8.73 14.21 Eukaryotic translation initiation factor 6 8.73 14.21 Cystatin-C 8.72 14.2 Proteasome subunit beta type-10 8.72 14.2 40S ribosomal protein S17 8.72 14.2 Adipocyte enhancer-binding protein 1 8.72 14.19 Vitronectin 8.71 14.18 60S ribosomal protein L38 8.7 14.16 Rho-associated protein kinase 2 8.7 14.16 N-alpha-acetyltransferase 50 8.7 14.16 Probable E3 ubiquitin-protein ligase HERC4 8.69 14.15 Peroxisomal carnitine O-octanoyltransferase 8.69 14.15 2-oxoglutarate dehydrogenase, mitochondrial 8.68 14.13 60S ribosomal protein L28 8.66 14.1 Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase 8.65 14.09 THO complex subunit 2 8.65 14.09 Ubiquitin carboxyl-terminal hydrolase 24 8.64 14.08 N-terminal kinase-like protein 8.64 14.08

60

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

ATP-dependent RNA helicase DDX3X 8.64 14.07 Copine-3 8.62 14.05 Glutathione S-transferase theta-2 8.62 14.04 Aldose reductase-related protein 1 8.62 14.04 Ketosamine-3-kinase 8.62 14.04 Serine/threonine-protein phosphatase PP1-gamma catalytic subunit 8.62 14.04 Syntaxin-binding protein 3 8.6 14.02 Isocitrate dehydrogenase [NAD] subunit gamma 1, mitochondrial 8.6 14.01 Ubiquitin-protein ligase E3A 8.6 14.01 Coatomer subunit zeta-1 8.59 14 Embigin 8.58 13.99 NADPH--cytochrome P450 reductase 8.58 13.99 60S ribosomal protein L19 8.58 13.98 Msx2-interacting protein 8.58 13.98 Nuclear pore complex protein Nup93 8.55 13.95 BRISC complex subunit Abro1 8.55 13.95 Glycogen synthase kinase-3 beta 8.55 13.95 5'-AMP-activated protein kinase subunit gamma-2 8.53 13.91 Collagen alpha-1(IV) chain 8.53 13.91 Lysozyme C-1 8.53 13.91 Heterogeneous nuclear ribonucleoprotein L-like 8.53 13.91 TBC1 domain family member 13 8.52 13.9 Friend leukemia integration 1 transcription factor 8.51 13.89 Dynactin subunit 3 8.51 13.89 Kelch repeat and BTB domain-containing protein 11 8.51 13.88 Thioredoxin reductase 1, cytoplasmic 8.5 13.87 Dynein light chain Tctex-type 3 8.5 13.87 A-kinase anchor protein 9 8.5 13.86 Protein FAN 8.49 13.85 Lamin-B1 8.49 13.85 Polymeric immunoglobulin receptor 8.49 13.85 Small nuclear ribonucleoprotein Sm D3 8.48 13.84 UPF0160 protein MYG1, mitochondrial 8.48 13.84 Fibulin-2 8.48 13.84 Nucleoside diphosphate kinase B 8.47 13.82 Casein kinase II subunit alpha 8.47 13.82 Ubiquitin carboxyl-terminal hydrolase isozyme L5 8.47 13.82 Striatin 8.46 13.8 Long-chain specific acyl-CoA dehydrogenase, mitochondrial 8.46 13.8 Eukaryotic translation elongation factor 1 epsilon-1 8.45 13.79 Protein phosphatase 1F 8.44 13.78

61

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Activator of 90 kDa heat shock protein ATPase homolog 1 8.43 13.77 Poly(rC)-binding protein 2 8.43 13.77 Sorting nexin-9 8.42 13.75 Heat shock protein 105 kDa 8.41 13.74 Pyrroline-5-carboxylate reductase 3 8.41 13.74 Shootin-1 8.4 13.73 Ribosyldihydronicotinamide dehydrogenase [quinone] 8.4 13.72 Abl interactor 1 8.39 13.71 Caprin-1 8.39 13.7 Coiled-coil and C2 domain-containing protein 1B 8.38 13.69 Guanine nucleotide-binding protein G(q) subunit alpha 8.38 13.69 TBC1 domain family member 15 8.38 13.69 Methionine adenosyltransferase 2 subunit beta 8.38 13.69 Acid trehalase-like protein 1 8.37 13.68 Creatine kinase U-type, mitochondrial 8.37 13.68 Protein C12orf4 homolog 8.37 13.68 Perilipin-3 8.36 13.66 ATP-dependent RNA helicase Dhx29 8.36 13.66 Galectin-9 8.36 13.66 Inactive tyrosine-protein kinase 7 8.35 13.65 Acetyl-CoA acetyltransferase, mitochondrial 8.35 13.65 Autophagy protein 5 8.35 13.64 Chromodomain-helicase-DNA-binding protein 7 8.34 13.62 Phospholipase D4 8.34 13.62 Prolyl 4-hydroxylase subunit alpha-2 8.32 13.6 DNA-directed RNA polymerase III subunit RPC2 8.31 13.59 Syntaxin-7 8.31 13.58 NEDD8 ultimate buster 1 8.31 13.58 Protein-tyrosine kinase 2-beta 8.31 13.58 NAD-dependent malic enzyme, mitochondrial 8.3 13.57 Neutral amino acid transporter B(0) 8.29 13.56 Insulin receptor substrate 1 8.28 13.55 Protein sidekick-1 8.28 13.54 Epididymis-specific alpha-mannosidase 8.28 13.54 Signal recognition particle 54 kDa protein 8.28 13.54 Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 8.27 13.53

Probable ATP-dependent RNA helicase DDX17 8.26 13.51 Protein unc-13 homolog D 8.26 13.51 Cysteine and histidine-rich domain-containing protein 1 8.25 13.5 Vacuolar-sorting protein SNF8 8.25 13.5

62

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Phospholipid hydroperoxide glutathione peroxidase, mitochondrial 8.25 13.5 Importin-4 8.25 13.5 Talin-2 8.25 13.5 Cytoplasmic FMR1-interacting protein 2 8.24 13.48 Dipeptidyl peptidase 9 8.23 13.47 ATP-dependent RNA helicase DDX1 8.23 13.47 Aldehyde oxidase 1 8.22 13.45 Phosphoribosyl pyrophosphate synthase-associated protein 1 8.22 13.45 Pleckstrin homology-like domain family B member 1 8.21 13.44 Phosphotriesterase-related protein 8.2 13.43 Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas 8.2 13.43 Ubiquitin-like modifier-activating enzyme 6 8.19 13.41 Ras-related GTP-binding protein A 8.19 13.41 Exocyst complex component 3 8.18 13.4 Ran-specific GTPase-activating protein 8.18 13.4 Formin-like protein 2 8.18 13.4 Beta-arrestin-1 8.18 13.39 Signal recognition particle subunit SRP68 8.17 13.38 LanC-like protein 1 8.16 13.37 Importin subunit alpha-3 8.16 13.37 Endophilin-A2 8.16 13.36 26S proteasome non-ATPase regulatory subunit 10 8.14 13.34 Myb-binding protein 1A 8.14 13.34 WASH complex subunit strumpellin 8.14 13.33 MMS19 nucleotide excision repair protein homolog 8.13 13.32 1,5-anhydro-D-fructose reductase 8.13 13.32 Hypoxanthine-guanine phosphoribosyltransferase 8.12 13.3 Ras-related protein Rab-9A 8.11 13.29 WD repeat domain phosphoinositide-interacting protein 2 8.11 13.29 Nck-associated protein 1 8.11 13.29 Actin-related protein 2/3 complex subunit 5-like protein 8.11 13.29 V-type proton ATPase subunit d 1 8.1 13.28 Glycerol kinase 8.1 13.28 Neuron navigator 1 8.1 13.27 Heat shock protein beta-1 8.1 13.27 CTP synthase 2 8.09 13.26 Ubiquitin conjugation factor E4 A 8.09 13.26 Interleukin enhancer-binding factor 3 8.09 13.26 Armadillo repeat-containing protein 8 8.08 13.25 Arylsulfatase B 8.08 13.24 Calmodulin 8.08 13.24

63

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Serine/threonine-protein phosphatase 5 8.07 13.23 Elongator complex protein 1 8.07 13.23 Stress-70 protein, mitochondrial 8.07 13.23 Peroxiredoxin-4 8.06 13.22 DNA-directed RNA polymerase I subunit RPA2 8.05 13.21 tRNA (cytosine(34)-C(5))-methyltransferase 8.05 13.21 Protein phosphatase 1 regulatory subunit 21 8.05 13.21 E3 ubiquitin-protein ligase RNF123 8.05 13.21 AP-3 complex subunit beta-1 8.05 13.2 Acid sphingomyelinase-like phosphodiesterase 3a 8.05 13.2 Lys-63-specific deubiquitinase BRCC36 8.05 13.2 Calpastatin 8.04 13.19 Heterogeneous nuclear ribonucleoprotein H 8.04 13.19 Golgi reassembly-stacking protein 2 8.03 13.18 Dehydrogenase/reductase SDR family member 1 8.03 13.18 Aflatoxin B1 aldehyde reductase member 2 8.02 13.16 ATP-binding cassette sub-family F member 1 8.02 13.16 Transient receptor potential cation channel subfamily V member 2 8.01 13.15 Lysosomal thioesterase PPT2 8.01 13.14 C-type lectin domain family 4 member E 8.01 13.14 Mitochondrial import inner membrane translocase subunit Tim13 8 13.13 Histone-lysine N-methyltransferase setd3 8 13.13 E3 ubiquitin-protein ligase RNF213 8 13.13 Histone H1.4 7.98 13.1 Septin-6 7.98 13.1 DnaJ homolog subfamily B member 1 7.98 13.09 Ornithine aminotransferase, mitochondrial 7.98 13.09 Conserved oligomeric Golgi complex subunit 7 7.96 13.07 Collagen alpha-2(VI) chain 7.95 13.06 Small nuclear ribonucleoprotein Sm D2 7.95 13.05 SH3 domain-binding glutamic acid-rich-like protein 3 7.95 13.05 Collagen alpha-1(XVI) chain 7.95 13.05 ATP-dependent (S)-NAD(P)H-hydrate dehydratase 7.95 13.05 Carbonic anhydrase 3 7.94 13.04 Syntaxin-binding protein 5 7.93 13.03 40S ribosomal protein S25 7.92 13.02 Cold shock domain-containing protein E1 7.92 13.01 Long-chain-fatty-acid--CoA ligase 1 7.91 12.99 Choline transporter-like protein 1 7.9 12.98 Protein TSSC1 7.9 12.98 Myosin-10 7.9 12.97

64

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Filamin-binding LIM protein 1 7.9 12.97 Protein-arginine deiminase type-2 7.9 12.97 Glutaredoxin-3 7.9 12.97 CTP synthase 1 7.9 12.97 Inositol-3-phosphate synthase 1 7.89 12.97 Dynein light chain Tctex-type 1 7.89 12.97 Copine-1 7.89 12.97 Aggrecan core protein 7.88 12.96 IST1 homolog 7.88 12.96 UPF0668 protein C10orf76 homolog 7.88 12.96 Dual specificity mitogen-activated protein kinase kinase 3 7.88 12.95 Protein phosphatase methylesterase 1 7.87 12.94 Aspartate aminotransferase, cytoplasmic 7.87 12.93 Toll-interacting protein 7.86 12.92 Uridine phosphorylase 1 7.86 12.91 SUMO-activating enzyme subunit 1 7.85 12.91 COMM domain-containing protein 7 7.85 12.91 Myoferlin 7.85 12.91 mRNA cap guanine-N7 methyltransferase 7.84 12.9 COMM domain-containing protein 9 7.84 12.9 Cyclic AMP-responsive element-binding protein 3-like protein 3 7.84 12.9 Glycogen synthase kinase-3 alpha 7.84 12.89 Unconventional myosin-If 7.84 12.89 Prefoldin subunit 3 7.84 12.89 Histone H1.0 7.83 12.87 Keratin, type I cytoskeletal 42 7.83 12.87 60S ribosomal protein L26 7.82 12.85 40S ribosomal protein S15 7.81 12.85 TBC1 domain family member 22A 7.8 12.84 Zinc finger protein castor homolog 1 7.8 12.84 Nuclear cap-binding protein subunit 1 7.8 12.84 Cytospin-B 7.8 12.84 Nucleolar RNA helicase 2 7.8 12.83 Dedicator of cytokinesis protein 7 7.8 12.83 Histone-lysine N-methyltransferase EHMT2 7.8 12.83 Diphosphomevalonate decarboxylase 7.8 12.83 Anthrax toxin receptor 1 7.79 12.82 Vesicular integral-membrane protein VIP36 7.79 12.82 EPM2A-interacting protein 1 7.79 12.82 Sorting nexin-18 7.79 12.81 26S proteasome non-ATPase regulatory subunit 8 7.79 12.81

65

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Cytochrome b-c1 complex subunit 2, mitochondrial 7.79 12.81 Cystatin-B 7.78 12.8 Elongation factor 1-beta 7.77 12.79 Heat shock-related 70 kDa protein 2 7.77 12.79 Cytosolic phospholipase A2 7.77 12.79 Vacuolar protein sorting-associated protein 45 7.76 12.78 Developmentally-regulated GTP-binding protein 1 7.76 12.78 Small glutamine-rich tetratricopeptide repeat-containing protein alpha 7.76 12.77 Heterogeneous nuclear ribonucleoprotein A1 7.76 12.77 Signal transducer and activator of transcription 5B 7.76 12.77 ATP-dependent 6-phosphofructokinase, muscle type 7.75 12.76 HEAT repeat-containing protein 5B 7.75 12.76 Inositol monophosphatase 1 7.75 12.76 Kinesin light chain 4 7.75 12.76 Isopentenyl-diphosphate Delta-isomerase 1 7.75 12.76 Heat shock 70 kDa protein 4L 7.75 12.75 Sorting nexin-3 7.75 12.75 Mitochondrial antiviral-signaling protein 7.75 12.75 Carbonyl reductase family member 4 7.74 12.74 Protein BRICK1 7.73 12.73 Propionyl-CoA carboxylase alpha chain, mitochondrial 7.72 12.72 Cytosolic 5'-nucleotidase 3A 7.72 12.72 ADP-ribosylation factor-binding protein GGA1 7.72 12.71 Lymphocyte cytosolic protein 2 7.72 12.71 Protein argonaute-2 7.71 12.7 Exocyst complex component 1 7.71 12.69 Golgi phosphoprotein 3 7.71 12.69 Plasma membrane calcium-transporting ATPase 1 7.71 12.69 Ubiquitin carboxyl-terminal hydrolase 8 7.7 12.68 Rab3 GTPase-activating protein catalytic subunit 7.7 12.68 Sphingosine-1-phosphate lyase 1 7.7 12.68 Protein phosphatase 1B 7.7 12.68 WD repeat and FYVE domain-containing protein 2 7.69 12.67 Receptor-type tyrosine-protein phosphatase C 7.68 12.66 Death-associated protein kinase 2 7.68 12.66 Cytochrome c, somatic 7.68 12.66 Zinc-binding alcohol dehydrogenase domain-containing protein 2 7.68 12.66 Guanylate cyclase soluble subunit beta-1 7.68 12.66 Gasdermin-D 7.68 12.65 Leucine-rich repeat and calponin homology domain-containing protein 4 7.67 12.64 GTP-binding nuclear protein Ran, testis-specific isoform 7.67 12.64

66

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Laminin subunit gamma-1 7.66 12.63 WASH complex subunit 7 7.66 12.62 Ribosome biogenesis protein WDR12 7.66 12.62 Nucleolar protein 58 7.66 12.62 Multivesicular body subunit 12A 7.66 12.62 Programmed cell death protein 4 7.66 12.62 Splicing factor 3B subunit 6 7.66 12.62 WD repeat-containing protein 41 7.66 12.62 Wiskott-Aldrich syndrome protein homolog 7.65 12.62 Dual specificity mitogen-activated protein kinase kinase 4 7.64 12.6 Zinc finger protein ZPR1 7.64 12.6 Zinc phosphodiesterase ELAC protein 2 7.64 12.6 Phostensin 7.64 12.6 U2 small nuclear ribonucleoprotein B'' 7.64 12.59 Formin-binding protein 1 7.64 12.59 Macrophage-expressed gene 1 protein 7.63 12.58 Cell cycle and apoptosis regulator protein 2 7.63 12.58 60S ribosomal protein L23a 7.62 12.56 Exocyst complex component 4 7.62 12.56 Phosphatidylinositol 3-kinase catalytic subunit type 3 7.62 12.56 Polypeptide N-acetylgalactosaminyltransferase 2 7.61 12.56 Phospholipase D3 7.61 12.55 Ethanolamine-phosphate cytidylyltransferase 7.61 12.55 Exocyst complex component 5 7.6 12.54 Microtubule-associated protein 1S 7.6 12.54 Polypeptide N-acetylgalactosaminyltransferase 6 7.6 12.53 Phosphoinositide 3-kinase regulatory subunit 4 7.59 12.52 Short-chain specific acyl-CoA dehydrogenase, mitochondrial 7.59 12.52 Actin-related protein 10 7.59 12.52 U5 small nuclear ribonucleoprotein 40 kDa protein 7.58 12.51 Semaphorin-3F 7.58 12.51 Hydroxymethylglutaryl-CoA lyase, mitochondrial 7.58 12.5 Biotinidase 7.58 12.5 Rho-associated protein kinase 1 7.58 12.5 Cyclin-G-associated kinase 7.58 12.5 Alpha-mannosidase 2C1 7.57 12.49 Formin-like protein 1 7.57 12.49 Cdc42-interacting protein 4 7.56 12.48 Metastasis-associated protein MTA3 7.56 12.48 Aldehyde dehydrogenase family 3 member B1 7.55 12.47 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial 7.55 12.47

67

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

AP2-associated protein kinase 1 7.55 12.46 Splicing factor 3A subunit 1 7.55 12.46 Protein 4.1 7.54 12.44 Far upstream element-binding protein 1 7.53 12.44 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform 7.53 12.44 Pyruvate dehydrogenase E1 component subunit beta, mitochondrial 7.53 12.44 Protein DDI1 homolog 2 7.53 12.44 E3 ubiquitin/ISG15 ligase TRIM25 7.53 12.43 Lysine-specific histone demethylase 1A 7.53 12.43 5'(3')-deoxyribonucleotidase, cytosolic type 7.53 12.43 Peptidyl-prolyl cis-trans isomerase D 7.53 12.43 Phosphatidylinositide phosphatase SAC1 7.52 12.42 Protein misato homolog 1 7.52 12.42 Formin-binding protein 1-like 7.52 12.42 WD repeat-containing protein 5 7.51 12.4 Polypeptide N-acetylgalactosaminyltransferase 5 7.51 12.4 Protein unc-45 homolog A 7.5 12.39 Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform 7.5 12.39

Exocyst complex component 6B 7.5 12.39 Vacuolar protein sorting-associated protein 53 homolog 7.5 12.38 p21-activated protein kinase-interacting protein 1 7.5 12.38 Wiskott-Aldrich syndrome protein family member 2 7.49 12.38 E2/E3 hybrid ubiquitin-protein ligase UBE2O 7.49 12.38 U4/U6.U5 tri-snRNP-associated protein 2 7.49 12.37 PX domain-containing protein kinase-like protein 7.49 12.37 Ras-related protein Rap-2b 7.47 12.35 Constitutive coactivator of PPAR-gamma-like protein 1 7.47 12.35 Caspase-8 7.47 12.34 Proteasome assembly chaperone 2 7.47 12.34 Pyruvate carboxylase, mitochondrial 7.47 12.34 4F2 cell-surface antigen heavy chain 7.46 12.33 Prostaglandin F2 receptor negative regulator 7.46 12.32 RNA-binding protein Musashi homolog 2 7.45 12.32 40S ribosomal protein S19 7.45 12.31 SWI/SNF complex subunit SMARCC2 7.45 12.31 U2 small nuclear ribonucleoprotein A' 7.44 12.3 Eukaryotic translation initiation factor 4 gamma 1 7.43 12.29 Polypyrimidine tract-binding protein 1 7.43 12.28 60S ribosomal protein L8 7.42 12.26 C-C chemokine receptor type 1 7.41 12.26

68

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Host cell factor 1 7.4 12.25 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-12 7.4 12.25 Sorting nexin-17 7.4 12.24 Gephyrin 7.4 12.24 F-box only protein 6 7.39 12.22 Brain-specific angiogenesis inhibitor 1-associated protein 2 7.39 12.22 DNA polymerase delta catalytic subunit 7.38 12.21 Methylosome protein 50 7.35 12.17 Signal recognition particle receptor subunit alpha 7.35 12.17 NudC domain-containing protein 1 7.35 12.17 Alpha-mannosidase 2x 7.35 12.17 Protein NDRG1 7.35 12.17 AP-1 complex subunit gamma-like 2 7.35 12.17 Prenylcysteine oxidase 7.35 12.16 Disks large homolog 1 7.35 12.16 Catenin alpha-1 7.35 12.16 Membrane-associated phosphatidylinositol transfer protein 2 7.34 12.15 Integrin alpha-5 7.34 12.15 Neutral alpha-glucosidase C 7.34 12.15 Isoleucine--tRNA ligase, mitochondrial 7.33 12.14 Protein O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase 1 7.32 12.13 E3 UFM1-protein ligase 1 7.32 12.13 Protein-lysine 6-oxidase 7.3 12.09 FACT complex subunit SSRP1 7.3 12.09 TIP41-like protein 7.29 12.08 Nucleolysin TIAR 7.28 12.07 Parkinson disease 7 domain-containing protein 1 7.28 12.07 tRNA (guanine-N(7)-)-methyltransferase non-catalytic subunit WDR4 7.28 12.07 TBC1 domain family member 2B 7.28 12.06 Exocyst complex component 7 7.28 12.06 Docking protein 3 7.27 12.05 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform 7.26 12.03

Prostamide/prostaglandin F synthase 7.26 12.03 Alpha-ketoglutarate-dependent dioxygenase FTO 7.25 12.03 Serine/threonine-protein kinase TBK1 7.25 12.02 Integral membrane protein 2C 7.25 12.02 DNA-directed RNA polymerase II subunit RPB2 7.23 11.99 Carboxypeptidase D 7.23 11.99 Membrane primary amine oxidase 7.23 11.98 Eukaryotic translation initiation factor 5B 7.23 11.98

69

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Proline synthase co-transcribed bacterial homolog protein 7.22 11.97 Translin 7.22 11.97 Coiled-coil domain-containing protein 132 7.22 11.97 COMM domain-containing protein 8 7.21 11.97 E3 ubiquitin-protein ligase HECTD3 7.21 11.96 Protein ITFG3 7.2 11.95 WD repeat-containing protein 82 7.2 11.95 Protein disulfide-isomerase A6 7.2 11.95 Breast carcinoma-amplified sequence 3 homolog 7.18 11.92 Beta-adrenergic receptor kinase 2 7.18 11.92 Cullin-4A 7.18 11.91 Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 1 7.18 11.91 Carnitine O-acetyltransferase 7.17 11.91 UDP-GalNAc:beta-1,3-N-acetylgalactosaminyltransferase 1 7.16 11.89 Phospholipase DDHD2 7.16 11.89 Tether containing UBX domain for GLUT4 7.16 11.88 5'-AMP-activated protein kinase subunit gamma-1 7.16 11.88 Vacuolar protein sorting-associated protein 37C 7.15 11.87 Cell division cycle protein 16 homolog 7.15 11.87 Importin-11 7.14 11.86 Dual specificity protein phosphatase 3 7.14 11.86 Oligophrenin-1 7.13 11.85 Poly(A) polymerase alpha 7.11 11.81 NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial 7.11 11.81 Ragulator complex protein LAMTOR2 7.1 11.8 Medium-chain specific acyl-CoA dehydrogenase, mitochondrial 7.1 11.79 Heterogeneous nuclear ribonucleoprotein A/B 7.1 11.79 Protein PBDC1 7.09 11.79 Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D 7.09 11.79 Quinone oxidoreductase-like protein 1 7.09 11.78 Fibromodulin 7.08 11.76 F-BAR and double SH3 domains protein 2 7.06 11.74 ATP synthase subunit O, mitochondrial 7.05 11.72 Chitinase-like protein 3 7.04 11.71 Serine beta-lactamase-like protein LACTB, mitochondrial 7.04 11.71 Cathepsin L1 7.04 11.71 RNA-binding protein FUS 7.04 11.71 Exosome complex exonuclease RRP42 7.04 11.71 Inactive phospholipase C-like protein 2 7.03 11.69 Uncharacterized protein KIAA0930 homolog 7.02 11.68 Neural cell adhesion molecule 1 7.02 11.68

70

Table E-2. Continued.

Protein Description (524) Z-Score Bone7/ Plastic7

Z-Score Bone7/Dentin7

Ankycorbin 7.01 11.67 Ran-binding protein 9 7.01 11.66 Serine/threonine-protein kinase A-Raf 6.99 11.64 Procollagen galactosyltransferase 1 6.99 11.63 GEM-interacting protein 6.98 11.62 UPF0585 protein C16orf13 homolog 6.98 11.62 AP-5 complex subunit zeta-1 6.97 11.61 COMM domain-containing protein 1 6.97 11.6 Transcriptional activator protein Pur-alpha 6.95 11.58 Vacuolar protein sorting-associated protein 4A 6.95 11.57 Cytoplasmic polyadenylation element-binding protein 4 6.92 11.54 ARF GTPase-activating protein GIT2 6.92 11.53 DNA topoisomerase 3-beta-1 6.9 11.5 Annexin A6 6.89 11.49 EF-hand domain-containing protein D2 6.88 11.47 Propionyl-CoA carboxylase beta chain, mitochondrial 6.87 11.45 Fidgetin-like protein 1 6.86 11.44 SH3 domain-containing kinase-binding protein 1 6.83 11.39 Serine/threonine-protein kinase TAO1 6.82 11.38 Nuclear RNA export factor 1 6.82 11.38 5'-3' exoribonuclease 2 6.81 11.37 Ubiquitin thioesterase otulin 6.8 11.35 Negative elongation factor B 6.77 11.32 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 6.75 11.28

Prohibitin 6.75 11.28 Ubiquitin-conjugating enzyme E2 Z 6.73 11.25 Serine/threonine-protein phosphatase 6 regulatory subunit 3 6.72 11.23 Sequestosome-1 6.67 11.16 TBC1 domain family member 9B 6.65 11.13 Phenylalanine-4-hydroxylase 6.57 11.02 Nuclear factor NF-kappa-B p105 subunit 6.54 10.97 Rho guanine nucleotide exchange factor 2 6.48 10.88 Nicastrin 6.47 10.86 Microtubule-associated protein 4 6.43 10.8 Tubulointerstitial nephritis antigen-like 6.22 10.5 Myotubularin-related protein 9 6.04 10.23

71

Table E-3. Proteins Present in Dentin Day 7 and Bone Day 7, but not Plastic Day 7. Gray indicates a non-significant Z-score.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

CD109 antigen 9.98 11 3.26 Brefeldin A-inhibited guanine nucleotide-exchange protein 2 9.95 10.95 3.24 Eukaryotic translation initiation factor 3 subunit I 8.6 9.36 2.63 Protein SEC13 homolog 8.73 9.44 2.58 Vacuolar protein sorting-associated protein 33A 8.62 9.16 2.3 V-type proton ATPase subunit d 2 9.76 10.15 2.29 Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein 8.22 8.79 2.28

Ubiquitin carboxyl-terminal hydrolase 7 8.93 9.34 2.17 Protein-methionine sulfoxide oxidase MICAL1 8.71 9.08 2.08 Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform 8.8 9.1 1.99

3-ketoacyl-CoA thiolase, mitochondrial 9.32 9.51 1.92 Receptor-type tyrosine-protein phosphatase epsilon 8.84 9 1.79 Ras-related GTP-binding protein C 8.14 8.36 1.76 DNA-directed RNA polymerase II subunit RPB1 8.05 8.24 1.69 FH1/FH2 domain-containing protein 1 9.01 9.06 1.66 Importin subunit alpha-4 9.16 9.16 1.62 Dynactin subunit 4 8.89 8.93 1.62 Kinesin light chain 1 8.71 8.77 1.62 Glyoxylate reductase/hydroxypyruvate reductase 8.45 8.54 1.62 Transcription elongation factor B polypeptide 1 8.53 8.61 1.61 EMILIN-1 7.98 8.07 1.53 Dedicator of cytokinesis protein 8 8.8 8.74 1.45 Semaphorin-4D 8.81 8.73 1.44 Vacuolar protein sorting-associated protein 16 homolog 9.34 9.18 1.42 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform 9.27 9.13 1.42

60 kDa heat shock protein, mitochondrial 9.84 9.58 1.35 UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit 8.99 8.83 1.35

Huntingtin 9.67 9.42 1.33 Squamous cell carcinoma antigen recognized by T-cells 3 8.81 8.66 1.33 Ras-related protein Rap-1A 9.51 9.27 1.32 Serine/threonine-protein kinase SMG1 9.34 9.12 1.32 26S protease regulatory subunit 6B 11.09 10.65 1.31 Tyrosine-protein kinase SYK 9.43 9.19 1.31 Ras-related protein Rab-21 7.99 7.93 1.31 FAD synthase 8.43 8.31 1.3 60S ribosomal protein L27 10.36 10 1.29

72

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Aminoacyl tRNA synthase complex-interacting multifunctional protein 1 9.83 9.53 1.29

Proteasome activator complex subunit 3 10.02 9.68 1.26 Regulator of chromosome condensation 9.36 9.1 1.26 Apoptosis inhibitor 5 9.05 8.83 1.26 Coatomer subunit gamma-2 10.1 9.74 1.25 Tsukushin 8.82 8.61 1.23 40S ribosomal protein S16 11.14 10.64 1.22 Bifunctional epoxide hydrolase 2 10.04 9.66 1.21 Ubiquitin carboxyl-terminal hydrolase isozyme L3 8.32 8.14 1.21 N-acylneuraminate cytidylyltransferase 11.03 10.5 1.17 Methionine synthase 8.99 8.69 1.15 Alpha-aminoadipic semialdehyde dehydrogenase 8.54 8.31 1.15 Histone H2A type 2-A 13.09 12.29 1.13 Tensin-3 9.97 9.54 1.12 60S ribosomal protein L24 9.55 9.17 1.12 Type II inositol 1,4,5-trisphosphate 5-phosphatase 9.13 8.8 1.12 E3 ubiquitin-protein ligase UBR4 10.17 9.71 1.1 Pyridoxal-dependent decarboxylase domain-containing protein 1 9.18 8.83 1.1 Peroxiredoxin-2 10.05 9.6 1.09 ATP-binding cassette sub-family E member 1 9.96 9.51 1.09 Disintegrin and metalloproteinase domain-containing protein 17 10.65 10.11 1.08 Eukaryotic initiation factor 4A-III 9.84 9.4 1.08 Nicotinamide phosphoribosyltransferase 8.73 8.42 1.08 Thioredoxin-like protein 1 8.95 8.61 1.07 Unconventional myosin-Ie 10.86 10.28 1.06 Sorting nexin-1 9.92 9.46 1.06 Sepiapterin reductase 9.55 9.13 1.06 40S ribosomal protein S15a 10.66 10.1 1.05 Protein transport protein Sec31A 10.43 9.9 1.05 Exportin-T 8.93 8.58 1.05 Inorganic pyrophosphatase 9.66 9.21 1.03 C-terminal-binding protein 1 9.41 8.99 1.03 Macrophage colony-stimulating factor 1 receptor 8.74 8.4 1.03 Apoptosis-inducing factor 1, mitochondrial 8.55 8.23 1.03 Sphingomyelin phosphodiesterase 9.52 9.07 1.01 Peptidyl-prolyl cis-trans isomerase B 9.66 9.17 0.98 Isocitrate dehydrogenase [NADP], mitochondrial 10.12 9.57 0.97 Rab3 GTPase-activating protein non-catalytic subunit 8.84 8.45 0.97 V-type proton ATPase subunit F 10.43 9.84 0.96

73

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Trifunctional purine biosynthetic protein adenosine-3 10.14 9.57 0.94 Microtubule-associated protein RP/EB family member 1 8.77 8.36 0.94 ATP-dependent RNA helicase A 10.42 9.81 0.93 Mitotic checkpoint protein BUB3 10.2 9.61 0.93 3-hydroxyacyl-CoA dehydrogenase type-2 9.18 8.71 0.92 Signal-induced proliferation-associated protein 1 9.04 8.58 0.91 Phosphoethanolamine/phosphocholine phosphatase 8.4 8.02 0.91 AP-3 complex subunit delta-1 9.93 9.35 0.9 NEDD8-activating enzyme E1 catalytic subunit 9.6 9.06 0.9 Ribosomal protein S6 kinase alpha-3 9.14 8.66 0.89 Twinfilin-2 9.12 8.63 0.88 Core histone macro-H2A.1 9.84 9.25 0.86 Protein RCC2 10.29 9.64 0.85 Spermidine synthase 10.48 9.8 0.84 Serpin B8 10.37 9.7 0.84 Unconventional myosin-Ic 8.72 8.25 0.84 Phospholipase A-2-activating protein 10.2 9.53 0.81 Mitochondrial fission 1 protein 8.59 8.12 0.81 Tyrosine--tRNA ligase, cytoplasmic 9.79 9.16 0.79 NADH-cytochrome b5 reductase 3 9.56 8.96 0.79 Sorcin 9.88 9.23 0.77 Tyrosine-protein phosphatase non-receptor type 23 9.31 8.72 0.77 UPF0505 protein C16orf62 homolog 9.28 8.7 0.77 Protein MEMO1 9.91 9.25 0.76 Peptidyl-prolyl cis-trans isomerase-like 1 9.3 8.71 0.76 WD repeat-containing protein 61 9.39 8.78 0.75 Histone H2B type 1-M 13.44 12.33 0.74 40S ribosomal protein S7 9.88 9.2 0.74 Importin-9 8.51 8 0.74 Mannose-1-phosphate guanyltransferase beta 10.18 9.46 0.73 Heterogeneous nuclear ribonucleoprotein U-like protein 2 9.27 8.66 0.73 Vam6/Vps39-like protein 9.92 9.23 0.72 Flap endonuclease 1 9.11 8.51 0.72 Glyoxalase domain-containing protein 4 9.09 8.49 0.72 Angiopoietin-related protein 3 8.18 7.69 0.72 Septin-11 11.1 10.25 0.71 Mitogen-activated protein kinase 14 8.84 8.27 0.71 40S ribosomal protein S5 10.9 10.08 0.7

74

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Endophilin-B1 8.93 8.35 0.7 Cytoplasmic dynein 1 intermediate chain 2 10.47 9.69 0.69 Sec1 family domain-containing protein 1 10.36 9.59 0.69 Ubiquitin carboxyl-terminal hydrolase 4 9.13 8.51 0.69 Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 1 8.39 7.87 0.69 Acetyl-CoA carboxylase 1 8.47 7.93 0.68 SUMO-activating enzyme subunit 2 9.47 8.8 0.67 Dedicator of cytokinesis protein 10 9.15 8.51 0.67 Inositol monophosphatase 2 9.38 8.71 0.66 V-type proton ATPase subunit S1 9.34 8.68 0.66 CD82 antigen 10.63 9.8 0.64 Cullin-2 10.11 9.34 0.64 Histone-binding protein RBBP4 10.44 9.62 0.63 Ras GTPase-activating protein-binding protein 1 10.07 9.3 0.63 Ubiquitin carboxyl-terminal hydrolase 14 10.35 9.54 0.62 55 kDa erythrocyte membrane protein 9.59 8.87 0.62 Histone H2A.V 9.23 8.54 0.61 Actin-like protein 6A 9.8 9.04 0.6 Leucine-rich repeat-containing protein 47 9.72 8.97 0.6 Eukaryotic translation initiation factor 2A 9.55 8.82 0.6 MOB-like protein phocein 8.6 7.98 0.6 Translation initiation factor eIF-2B subunit delta 9.38 8.65 0.58 Keratin, type I cytoskeletal 14 9.54 8.79 0.57 Protein VAC14 homolog 10.17 9.34 0.56 BRCA1-A complex subunit BRE 8.99 8.3 0.56 Protein farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha 8.61 7.97 0.56

Methylthioribose-1-phosphate isomerase 10.34 9.47 0.55 Splicing factor 3B subunit 1 10.17 9.33 0.55 Drebrin-like protein 8.93 8.24 0.55 Cytokine receptor-like factor 3 9.62 8.83 0.52 Leukocyte immunoglobulin-like receptor subfamily B member 3 9.1 8.36 0.51 60S ribosomal protein L9 10.42 9.52 0.5 Protein transport protein Sec23A 10.14 9.27 0.5 Vacuolar protein sorting-associated protein 41 homolog 9.75 8.92 0.5 WD repeat-containing protein 91 8.67 7.98 0.5 Switch-associated protein 70 9.93 9.07 0.48 Neurogenic locus notch homolog protein 1 10.93 9.94 0.47 Rho guanine nucleotide exchange factor 1 10.34 9.42 0.46

75

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

DNA-directed RNA polymerase II subunit RPB3 8.53 7.83 0.46 Prostaglandin reductase 1 9.71 8.84 0.43 N-acetylgalactosamine kinase 9.18 8.38 0.43 Beclin-1 8.65 7.91 0.43 26S protease regulatory subunit 4 11.7 10.58 0.41 60S ribosomal protein L17 9.78 8.9 0.41 Dipeptidyl peptidase 2 9.24 8.42 0.41 Cleavage and polyadenylation specificity factor subunit 1 9.2 8.39 0.41 Putative pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 10.68 9.68 0.4

Hsp90 co-chaperone Cdc37 10.37 9.41 0.4 Vacuolar protein sorting-associated protein 4B 10.37 9.4 0.4 cAMP-dependent protein kinase type I-alpha regulatory subunit 9.86 8.95 0.4 40S ribosomal protein S24 9.69 8.81 0.4 Legumain 9.91 8.99 0.39 Putative phospholipase B-like 2 9.8 8.9 0.39 Probable tRNA N6-adenosine threonylcarbamoyltransferase 9.22 8.39 0.39 Eukaryotic translation initiation factor 2 subunit 1 10.71 9.69 0.38 Serine/threonine-protein kinase N1 9.81 8.9 0.38 ADP-ribosylation factor-like protein 1 9.61 8.72 0.38 Ras-related protein Rap-2c 9.58 8.7 0.38 Pre-mRNA-processing-splicing factor 8 7.76 7.09 0.38 GTP-binding protein SAR1b 10.82 9.78 0.37 Proteasome activator complex subunit 4 9.97 9.03 0.37 Vacuolar protein sorting-associated protein 18 homolog 10.05 9.09 0.36 Splicing factor, proline- and glutamine-rich 9.75 8.83 0.35 Deoxyribose-phosphate aldolase 11.2 10.1 0.34 Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial 9.88 8.93 0.33 Peptidyl-prolyl cis-trans isomerase A 11.84 10.64 0.32 Glycylpeptide N-tetradecanoyltransferase 1 10.74 9.68 0.32 60S ribosomal protein L23 10.16 9.17 0.32 Alpha-L-iduronidase 9.39 8.5 0.32 NSFL1 cofactor p47 9.24 8.35 0.31 Translation initiation factor eIF-2B subunit beta 9.09 8.21 0.3 Vacuolar protein sorting-associated protein 26B 8.86 8.01 0.3 Dynactin subunit 2 9.4 8.48 0.29 Eukaryotic peptide chain release factor GTP-binding subunit ERF3A 9.29 8.38 0.29 Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 9.62 8.66 0.28

76

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

AP-1 complex subunit sigma-1A 9.45 8.51 0.28 Pro-cathepsin H 9.08 8.19 0.28 Ras-related protein Ral-B 8.9 8.03 0.28 DnaJ homolog subfamily A member 1 9.7 8.73 0.27 Serine/threonine-protein phosphatase 6 catalytic subunit 9.55 8.6 0.27 Prostaglandin E synthase 2 9.16 8.24 0.25 Heterogeneous nuclear ribonucleoprotein A3 10.14 9.09 0.23 Minor histocompatibility protein HA-1 9.63 8.64 0.23 Stress-induced-phosphoprotein 1 9.4 8.44 0.23 Coiled-coil domain-containing protein 22 10.88 9.73 0.21 DmX-like protein 1 10.69 9.56 0.21 ATP-dependent RNA helicase DDX39A 9.71 8.69 0.21 Bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 2 8.64 7.76 0.21 Glutathione S-transferase Mu 1 11.01 9.82 0.19 Cysteine--tRNA ligase, cytoplasmic 9.64 8.62 0.19 DAZ-associated protein 1 8.92 7.99 0.19 AP-3 complex subunit mu-1 8.87 7.94 0.19 Active breakpoint cluster region-related protein 10.49 9.36 0.18 SEC23-interacting protein 9.4 8.4 0.18 KH domain-containing, RNA-binding, signal transduction-associated protein 1 9.35 8.35 0.17

Plastin-1 9.12 8.15 0.17 Probable ATP-dependent RNA helicase DDX5 11.12 9.9 0.16 Ubiquitin-like modifier-activating enzyme ATG7 10.2 9.09 0.16 Endoplasmic reticulum resident protein 44 9.59 8.56 0.16 Non-POU domain-containing octamer-binding protein 10.61 9.45 0.15 Calcium/calmodulin-dependent protein kinase type II subunit delta 10.05 8.96 0.15 Peroxisomal multifunctional enzyme type 2 9.38 8.36 0.15 Maspardin 10.31 9.17 0.14 Transportin-3 9.58 8.53 0.14 Coactosin-like protein 10.21 9.08 0.13 Protein FAM45A 8.71 7.76 0.12 Mimecan 10.7 9.5 0.11 Cytosolic purine 5'-nucleotidase 9.38 8.34 0.1 60S ribosomal protein L10a 11.32 10.03 0.09 Lambda-crystallin homolog 10.49 9.31 0.09 COP9 signalosome complex subunit 7a 9.85 8.74 0.09 Eukaryotic translation initiation factor 5A-1 9.66 8.57 0.09 Ubiquitin conjugation factor E4 B 9.68 8.57 0.07

77

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

GDP-L-fucose synthase 9.61 8.51 0.07 Thrombospondin-3 9.33 8.26 0.06 Serine/threonine-protein kinase Nek9 9.08 8.03 0.05 Voltage-gated potassium channel subunit beta-2 8.68 7.69 0.05 Rabankyrin-5 11.21 9.89 0.03 AMP deaminase 3 10.81 9.54 0.03 Neutrophil cytosol factor 2 8.99 7.94 0.03 U5 small nuclear ribonucleoprotein 200 kDa helicase 10.28 9.06 0.02 60S ribosomal protein L10-like 9.76 8.61 0.02 DnaJ homolog subfamily B member 11 9.74 8.6 0.02 ATPase Asna1 10.1 8.9 0.01 Vacuolar protein-sorting-associated protein 25 9.09 8.02 0.01 Coronin-7 11.24 9.9 -0.01 Glycolipid transfer protein 9.5 8.36 -0.01 Spermine synthase 9.82 8.64 -0.02 Low-density lipoprotein receptor 9.67 8.51 -0.02 WD40 repeat-containing protein SMU1 9.49 8.35 -0.02 L-xylulose reductase 9.4 8.27 -0.02 40S ribosomal protein S23 9.95 8.75 -0.03 Septin-8 8.91 7.83 -0.03 60S acidic ribosomal protein P1 10.68 9.37 -0.05 Elongation factor 1-delta 10.8 9.47 -0.06 DnaJ homolog subfamily C member 3 9.34 8.19 -0.06 Exocyst complex component 8 9.03 7.92 -0.06 Succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial 11.34 9.94 -0.07 ATP-dependent RNA helicase DDX19A 9.47 8.28 -0.09 H-2 class I histocompatibility antigen, D-B alpha chain 11.33 9.91 -0.1 Regulator of nonsense transcripts 1 9.51 8.31 -0.1 Coiled-coil domain-containing protein 93 9.16 8.01 -0.1 [Protein ADP-ribosylarginine] hydrolase 11.07 9.66 -0.13 Coatomer subunit epsilon 10.96 9.55 -0.15 Transcription elongation factor B polypeptide 2 10.88 9.48 -0.15 Heterogeneous nuclear ribonucleoprotein L 10.39 9.05 -0.15 Dipeptidyl peptidase 1 10.33 9 -0.15 Proteasomal ubiquitin receptor ADRM1 9.99 8.71 -0.15 Conserved oligomeric Golgi complex subunit 3 9.39 8.18 -0.15 C-Jun-amino-terminal kinase-interacting protein 4 9.33 8.12 -0.15 Deoxynucleoside triphosphate triphosphohydrolase SAMHD1 11.03 9.61 -0.16

78

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Transcription intermediary factor 1-beta 10.16 8.84 -0.16 Heterogeneous nuclear ribonucleoprotein U 10.17 8.84 -0.17 40S ribosomal protein S13 9.88 8.59 -0.17 Histidine--tRNA ligase, cytoplasmic 9 7.82 -0.17 C-terminal-binding protein 2 9.91 8.61 -0.18 26S proteasome non-ATPase regulatory subunit 7 11.26 9.79 -0.19 Asparagine synthetase [glutamine-hydrolyzing] 10.58 9.17 -0.21 Tropomyosin alpha-3 chain 10.49 9.1 -0.21 Serine/threonine-protein kinase Nek7 8.86 7.66 -0.21 N-acylglucosamine 2-epimerase 11.37 9.86 -0.22 Syntaxin-binding protein 2 11.11 9.63 -0.22 Atrial natriuretic peptide receptor 3 9.38 8.11 -0.22 SCY1-like protein 2 9.32 8.06 -0.22 COMM domain-containing protein 3 8.87 7.66 -0.22 Farnesyl pyrophosphate synthase 10.3 8.92 -0.23 MAP kinase-activated protein kinase 2 10.23 8.86 -0.23 Gamma-glutamyl hydrolase 11.88 10.3 -0.24 Hsp70-binding protein 1 9.37 8.09 -0.24 COP9 signalosome complex subunit 2 11.17 9.66 -0.26 Selenocysteine lyase 9.52 8.21 -0.26 DNA replication licensing factor MCM2 9.2 7.93 -0.26 Metastasis-associated protein MTA2 8.97 7.73 -0.26 Cyclin-dependent kinase 6 9.32 8.02 -0.27 Small nuclear ribonucleoprotein E 9.3 8.01 -0.27 N-acetylglucosamine-6-sulfatase 10.26 8.85 -0.28 Heterogeneous nuclear ribonucleoproteins A2/B1 9.23 7.94 -0.28 Ubiquitin-40S ribosomal protein S27a 11.3 9.75 -0.29 MOB kinase activator 1A 9.86 8.49 -0.29 Vacuolar protein-sorting-associated protein 36 9.91 8.53 -0.31 Heterogeneous nuclear ribonucleoprotein D0 9.7 8.32 -0.32 Protein PRRC1 9.09 7.79 -0.32 Sorting nexin-5 10.97 9.42 -0.36 Bifunctional polynucleotide phosphatase/kinase 9.66 8.27 -0.37 Late secretory pathway protein AVL9 homolog 9.16 7.82 -0.37 Golgi resident protein GCP60 10.03 8.58 -0.38 Oxidation resistance protein 1 9.8 8.38 -0.38 RUN and FYVE domain-containing protein 1 8.97 7.65 -0.38 Translationally-controlled tumor protein 9.96 8.51 -0.39

79

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Ankyrin repeat domain-containing protein 13A 8.64 7.35 -0.39 Integral membrane protein 2B 10.88 9.3 -0.42 Ras-related protein Rab-31 10.3 8.79 -0.42 Sorting nexin-27 10.51 8.96 -0.44 Vacuolar protein sorting-associated protein 26A 11.41 9.74 -0.45 AMP deaminase 2 9.38 7.94 -0.47 Macrophage migration inhibitory factor 11.03 9.39 -0.48 COP9 signalosome complex subunit 8 10.57 8.97 -0.5 Kinectin 9.45 7.98 -0.5 Receptor-interacting serine/threonine-protein kinase 1 9.49 8.02 -0.51 Serrate RNA effector molecule homolog 8.55 7.18 -0.52 Myotrophin 9.15 7.71 -0.53 N-acetyl-D-glucosamine kinase 10.11 8.54 -0.54 Lysozyme C-2 12.01 10.2 -0.56 Glycine amidinotransferase, mitochondrial 9.62 8.09 -0.56 Tropomyosin alpha-4 chain 9.38 7.87 -0.59 Granulins 11.07 9.34 -0.61 Far upstream element-binding protein 2 8.73 7.28 -0.61 Synaptosomal-associated protein 23 9.03 7.53 -0.63 Nardilysin 8.86 7.38 -0.64 UDP-glucose:glycoprotein glucosyltransferase 1 9.61 8.02 -0.67 Calpain-1 catalytic subunit 11.36 9.55 -0.68 Leupaxin 8.59 7.1 -0.68 Acyl-protein thioesterase 2 10.25 8.56 -0.69 Galactose-1-phosphate uridylyltransferase 9.12 7.57 -0.7 Ras-related protein Rab-32 12.47 10.5 -0.71 Carbonyl reductase [NADPH] 3 10.39 8.68 -0.71 Actin, alpha skeletal muscle 9.74 8.1 -0.71 Protein jagged-1 9.46 7.86 -0.71 Vigilin 10.99 9.2 -0.72 Oxysterol-binding protein 1 10.46 8.73 -0.72 Myc box-dependent-interacting protein 1 9.93 8.27 -0.72 Coagulation factor VIII 9.52 7.9 -0.72 von Willebrand factor 10.82 9.03 -0.74 Nucleobindin-1 9.56 7.92 -0.74 GTPase-activating protein and VPS9 domain-containing protein 1 10.26 8.51 -0.77 Fibrillin-1 9.49 7.84 -0.77 Ubiquitin-conjugating enzyme E2 variant 1 9.47 7.82 -0.77

80

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Serine/threonine-protein phosphatase 2A activator 9.3 7.68 -0.77 Mitochondrial import receptor subunit TOM34 8.96 7.36 -0.79 Nuclease-sensitive element-binding protein 1 9 7.39 -0.8 Glucosamine-6-phosphate isomerase 1 10.32 8.54 -0.81 Heterogeneous nuclear ribonucleoprotein Q 10.26 8.49 -0.81 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 8 9.18 7.54 -0.81 Tyrosine-protein kinase Lyn 9.6 7.9 -0.82 Glutamine synthetase 10.09 8.33 -0.83 Probable aminopeptidase NPEPL1 9.39 7.71 -0.83 Endoplasmic reticulum aminopeptidase 1 9.63 7.92 -0.84 Complement C1r-A subcomponent 11.56 9.61 -0.85 UPF0317 protein C14orf159 homolog, mitochondrial 8.37 6.79 -0.87 Aldehyde dehydrogenase family 8 member A1 10.04 8.25 -0.88 Calcyclin-binding protein 10.44 8.58 -0.91 Glycogenin-1 10.15 8.33 -0.91 Leucine-rich repeat flightless-interacting protein 1 9.58 7.83 -0.91 Extracellular matrix protein 1 12.09 10.02 -0.92 DNA replication licensing factor MCM7 8.28 6.67 -0.92 Fibrocystin-L 10.22 8.38 -0.93 NEDD8-activating enzyme E1 regulatory subunit 9.38 7.6 -0.99 WD repeat-containing protein 81 9.16 7.4 -0.99 CCR4-NOT transcription complex subunit 1 9.41 7.6 -1.03 Rho GDP-dissociation inhibitor 2 10.11 8.2 -1.04 Rho GTPase-activating protein 17 10.82 8.82 -1.05 Versican core protein 8.93 7.16 -1.05 ADP-ribosylation factor-like protein 8A 10.12 8.2 -1.07 WD repeat-containing protein 11 9.62 7.75 -1.08 Macrophage receptor MARCO 13.99 11.58 -1.09 ARF GTPase-activating protein GIT1 10.44 8.45 -1.11 Autophagy-related protein 16-1 13.88 11.46 -1.12 Chromodomain-helicase-DNA-binding protein 4 9.99 8.03 -1.14 Choline/ethanolamine kinase 10.63 8.58 -1.15 Geranylgeranyl transferase type-2 subunit alpha 11.53 9.35 -1.2 Tumor susceptibility gene 101 protein 9.84 7.86 -1.2 Rho guanine nucleotide exchange factor 7 10.56 8.47 -1.22 Ubiquitin fusion degradation protein 1 homolog 9.54 7.54 -1.27 G-protein coupled receptor 98 8.94 7.01 -1.28 Conserved oligomeric Golgi complex subunit 2 9.7 7.66 -1.3

81

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Vacuolar protein sorting-associated protein 13C 9.84 7.79 -1.31 Tetratricopeptide repeat protein 38 10.58 8.43 -1.32 AP-2 complex subunit sigma 11.78 9.47 -1.34 Exosome complex exonuclease RRP44 9.64 7.58 -1.34 DCC-interacting protein 13-alpha 10.02 7.91 -1.35 Neutrophil cytosol factor 4 9.7 7.6 -1.41 Nucleosome assembly protein 1-like 4 10.88 8.61 -1.44 Superkiller viralicidic activity 2-like 2 9.61 7.49 -1.45 Peptidylprolyl isomerase domain and WD repeat-containing protein 1 8.88 6.83 -1.48 DnaJ homolog subfamily A member 2 9.97 7.76 -1.51 Protein kinase C delta type 9.86 7.66 -1.51 Ribosome maturation protein SBDS 10.14 7.9 -1.54 Ubiquitin carboxyl-terminal hydrolase 15 10.28 7.99 -1.57 Fibroleukin 9.78 7.51 -1.62 WD repeat-containing protein 26 9.57 7.34 -1.62 Multiple epidermal growth factor-like domains protein 8 9.05 6.87 -1.62 EGF-like module-containing mucin-like hormone receptor-like 1 10.72 8.34 -1.63 Heterogeneous nuclear ribonucleoproteins C1/C2 9.97 7.64 -1.68 Dedicator of cytokinesis protein 2 9.53 7.24 -1.71 Galactocerebrosidase 11.26 8.73 -1.74 60S acidic ribosomal protein P2 10.37 7.95 -1.75 Eukaryotic translation initiation factor 3 subunit D 10.49 8.05 -1.77 Integrin alpha-4 9.96 7.56 -1.79 Nucleoside diphosphate kinase 3 10.71 8.21 -1.81 Uridine 5'-monophosphate synthase 10.33 7.85 -1.85 Alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase A 10.28 7.75 -1.93

cAMP-dependent protein kinase type II-beta regulatory subunit 10.24 7.72 -1.93 Plexin domain-containing protein 2 9.74 7.28 -1.93 60S ribosomal protein L34 8.7 6.36 -1.93 Protocadherin-12 9.61 7.16 -1.94 E3 ubiquitin-protein ligase UBR2 9.12 6.71 -1.97 Band 4.1-like protein 2 9.66 7.16 -1.99 Vacuolar protein sorting-associated protein 52 homolog 10.81 8.07 -2.15 Galectin-3 12.45 9.49 -2.19 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 1 10.86 8.06 -2.21

Lupus La protein homolog 10.97 8.16 -2.22 Vacuolar protein sorting-associated protein 51 homolog 10.44 7.68 -2.24

82

Table E-3. Continued.

Protein Description (426) - Sorted by Bone7/Dentin7 Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Z-Score Bone7/Dentin7

Eukaryotic translation initiation factor 4 gamma 3 10.09 7.35 -2.26 Stabilin-1 13.86 10.54 -2.45 Ubiquitin-conjugating enzyme E2 G2 10.69 7.5 -2.84 Heterogeneous nuclear ribonucleoprotein M 11.82 8.36 -3.02 Probable ATP-dependent RNA helicase DDX58 11.3 7.72 -3.3 Serine protease HTRA2, mitochondrial 11.3 7.65 -3.41 Nucleophosmin 11.82 7.92 -3.67 HEAT repeat-containing protein 5A 10.78 7.01 -3.68 Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2 13.6 9.38 -3.83 Serine/threonine-protein kinase mTOR 10.79 6.78 -4.03 Nucleolar protein 56 12.57 7.66 -5.04

83

Table E-4. Proteins Present in Dentin Day 7, Bone Day 7, and Plastic Day 7. Gray indicates a non-significant Z-score.

Protein Description (712 Total; 119 Significant Z-Scores) - Sorted by Bone7/Dentin7

Z-Score Dentin7/Plastic7

Z-Score Bone7/ Plastic7

Z-Score Bone7/ Dentin7

Eukaryotic initiation factor 4A-II -1.03 1.07 2.89 60S ribosomal protein L7a -1.29 0.65 2.61 Major vault protein -2.53 -0.48 2.55 Probable ATP-dependent RNA helicase DDX6 0.16 1.79 2.4 Ig mu chain C region -1.48 0.28 2.31 Tubulin-specific chaperone D -3.55 -1.56 2.27 Cathepsin K -1.19 0.47 2.21 LIM and SH3 domain protein 1 -0.87 0.72 2.16 Sorting nexin-8 -1.16 0.39 2.05 Baculoviral IAP repeat-containing protein 6 -2.4 -0.71 2.03 40S ribosomal protein S3a -1.16 0.36 2.02 Sodium/potassium-transporting ATPase subunit beta-3 0.07 1.4 1.95 Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein 2 -0.4 0.96 1.91

Integrin alpha-V -1.12 0.32 1.91 Aldo-keto reductase family 1 member C18 0.36 1.61 1.88 Hexokinase-1 0.11 1.36 1.83 Inosine-5'-monophosphate dehydrogenase 2 -1.07 0.27 1.75 tRNA-splicing ligase RtcB homolog 0.82 1.91 1.74 Integrin beta-3 -0.84 0.43 1.7 Elongation factor Tu GTP-binding domain-containing protein 1 -6.27 -4.35 1.68 Paxillin -0.9 0.36 1.67 Putative helicase MOV-10 0.33 1.43 1.66 Tubulin--tyrosine ligase-like protein 12 0.91 1.94 1.65 Dynamin-like 120 kDa protein, mitochondrial 0.83 1.83 1.59 EGF-like repeat and discoidin I-like domain-containing protein 3 -2.53 -1.13 1.59 Guanine nucleotide-binding protein subunit beta-4 -2.2 -0.99 1.37 60S ribosomal protein L7 -1.91 -0.75 1.34 Acetyl-CoA acetyltransferase, cytosolic -2.09 -0.95 1.28 Matrix metalloproteinase-9 -2.39 -1.23 1.26 Lysosomal protective protein 0.95 1.7 1.25 Ras-related protein Rab-18 1.01 1.73 1.21 ATP synthase subunit beta, mitochondrial 1.05 1.77 1.21 Asparagine--tRNA ligase, cytoplasmic 1 1.68 1.15 Proliferation-associated protein 2G4 1.09 1.75 1.14 Leucine--tRNA ligase, cytoplasmic 1.5 2.02 1 Malate dehydrogenase, mitochondrial 1.58 1.89 0.71 Clustered mitochondria protein homolog 1.36 1.69 0.69

84

Table E-4. Continued. Protein Description (712 Total; 119 Significant Z-Scores) - Sorted by Bone7/Dentin7

Z-Score Dentin7/Plastic7

Z-Score Bone7/ Plastic7

Z-Score Bone7/ Dentin7

Tyrosine-protein phosphatase non-receptor type 11 -2.52 -1.8 0.58 Fructose-bisphosphate aldolase A 2.74 2.8 0.56 26S proteasome non-ATPase regulatory subunit 6 1.58 1.75 0.5 Cytoskeleton-associated protein 5 -3.26 -2.51 0.48 Serine/threonine-protein phosphatase PP1-beta catalytic subunit -2.3 -1.68 0.46 Coatomer subunit gamma-1 1.62 1.72 0.4 Hemoglobin subunit epsilon-Y2 -7.05 -5.9 0.39 Annexin A5 2.39 2.37 0.36 DNA-(apurinic or apyrimidinic site) lyase 1.76 1.79 0.32 Alpha-2-macroglobulin -2.12 -1.66 0.27 Phosphoglucomutase-like protein 5 -1.85 -1.44 0.24 Hsc70-interacting protein -1.86 -1.5 0.16 Serine-threonine kinase receptor-associated protein 1.66 1.56 0.11 Serine/threonine-protein phosphatase PP1-alpha catalytic subunit 2.39 2.15 0.03 EH domain-containing protein 4 1.68 1.47 -0.04 26S proteasome non-ATPase regulatory subunit 2 2.57 2.24 -0.07 Eukaryotic translation initiation factor 3 subunit M 2.45 2.12 -0.09 Aspartyl aminopeptidase 1.83 1.55 -0.12 Annexin A11 -2.49 -2.26 -0.15 ADP-ribosylation factor-like protein 8B 1.83 1.52 -0.16 Rab GDP dissociation inhibitor beta 3.05 2.58 -0.18 Proteasome subunit beta type-8 2.89 2.4 -0.24 Mitogen-activated protein kinase 3 1.66 1.29 -0.28 Tyrosine-protein phosphatase non-receptor type 1 2.09 1.66 -0.29 Serpin B6 3.41 2.82 -0.31 Collagen alpha-1(VI) chain -1.9 -1.91 -0.39 Tubulin alpha-4A chain -3.07 -2.97 -0.44 UDP-N-acetylhexosamine pyrophosphorylase-like protein 1 1.69 1.19 -0.47 Cytosolic non-specific dipeptidase 2.18 1.6 -0.5 Glutathione S-transferase Mu 5 -1.8 -1.99 -0.65 Glycogen phosphorylase, brain form 1.72 1.05 -0.72 TRAF3-interacting protein 1 -2.63 -2.82 -0.78 A disintegrin and metalloproteinase with thrombospondin motifs 13 -1.36 -1.75 -0.86 Rab GDP dissociation inhibitor alpha -1.41 -1.82 -0.89 Peroxiredoxin-1 2.26 1.34 -0.99 Coagulation factor X -1.14 -1.68 -1.03 Eukaryotic initiation factor 4A-I 1.94 1.03 -1.03 Tissue alpha-L-fucosidase -1.12 -1.69 -1.08

85

Table E-4. Continued. Protein Description (712 Total; 119 Significant Z-Scores) - Sorted by Bone7/Dentin7

Z-Score Dentin7/Plastic7

Z-Score Bone7/ Plastic7

Z-Score Bone7/ Dentin7

Bone morphogenetic protein 1 -1.05 -1.66 -1.13 Collagen alpha-2(I) chain -1.14 -1.75 -1.14 Apolipoprotein A-I -1.51 -2.08 -1.15 Cathepsin S 2.91 1.76 -1.21 Apolipoprotein E 1.96 0.9 -1.25 Antithrombin-III -1.28 -1.95 -1.26 Protein disulfide-isomerase -0.95 -1.69 -1.29 Transmembrane glycoprotein NMB 2.6 1.36 -1.39 Fructose-1,6-bisphosphatase isozyme 2 -1.43 -2.3 -1.58 Contactin-1 -0.7 -1.69 -1.62 Collagen alpha-1(XIV) chain 0.3 -0.86 -1.69 Receptor-type tyrosine-protein phosphatase delta -0.74 -1.79 -1.71 Complement factor H -0.13 -1.25 -1.72 Calreticulin -1.64 -2.61 -1.75 Hepatocyte growth factor activator -0.54 -1.66 -1.79 Four and a half LIM domains protein 3 -0.3 -1.47 -1.82 C-type mannose receptor 2 -0.55 -1.7 -1.84 Properdin 0.88 -0.45 -1.84 Tubulin beta-2A chain -0.11 -1.33 -1.86 Urokinase-type plasminogen activator 0.86 -0.49 -1.87 Hepatocyte growth factor-like protein -1.14 -2.25 -1.88 Collagen alpha-1(I) chain -0.35 -1.56 -1.89 Protein NDRG2 -0.66 -1.85 -1.91 Complement C1q subcomponent subunit B 1.99 0.49 -1.91 Neuropilin-1 -0.49 -1.71 -1.91 Eukaryotic translation initiation factor 3 subunit A 0.44 -0.89 -1.92 Macrophage metalloelastase 1.76 0.25 -1.96 Interferon-induced transmembrane protein 3 1.01 -0.43 -1.98 RuvB-like 1 -0.51 -1.78 -2 General vesicular transport factor p115 0.63 -0.77 -2 Disintegrin and metalloproteinase domain-containing protein 10 0.78 -0.66 -2.02 Glutaminyl-peptide cyclotransferase 0.88 -0.57 -2.02 Nucleosome assembly protein 1-like 1 2.03 0.32 -2.21 Bisphosphoglycerate mutase 0.07 -1.5 -2.33 Serotransferrin -1.62 -2.98 -2.33 CD81 antigen 1.76 -0.05 -2.4 Tyrosine-protein kinase receptor Tie-1 0.21 -1.75 -2.9 Protein sidekick-2 -1.57 -3.33 -2.92

86

Table E-4. Continued. Protein Description (712 Total; 119 Significant Z-Scores) - Sorted by Bone7/Dentin7

Z-Score Dentin7/Plastic7

Z-Score Bone7/ Plastic7

Z-Score Bone7/ Dentin7

Heat shock protein HSP 90-alpha 0.09 -1.94 -3.02 Ras-related protein Rab-8A 0.05 -2.04 -3.12 Histidine ammonia-lyase -0.05 -2.16 -3.15 Laminin subunit alpha-2 1.07 -1.2 -3.21 Nucleolin 0.12 -2.08 -3.26 Macrophage mannose receptor 1 1.81 -0.65 -3.34

87

Table E-5. Proteins Present in Plastic Day 7, but not Dentin Day 7 or Bone Day 7.

Protein Description (108) Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Carboxymethylenebutenolidase homolog -9.53 -8.89 BCL-6 corepressor-like protein 1 -9.95 -9.27 Splicing factor U2AF 65 kDa subunit -10.14 -9.43 Serine/threonine-protein kinase 24 -10.18 -9.47 Platelet-activating factor acetylhydrolase IB subunit gamma -10.21 -9.49 5'-nucleotidase domain-containing protein 3 -10.22 -9.5 SURP and G-patch domain-containing protein 1 -10.24 -9.51 G protein-regulated inducer of neurite outgrowth 1 -10.26 -9.54 Methylmalonate-semialdehyde dehydrogenase [acylating], mitochondrial -10.3 -9.57 CLIP-associating protein 2 -10.34 -9.6 GTPase NRas -10.36 -9.62 MAGUK p55 subfamily member 7 -10.45 -9.7 Methyltransferase-like protein 9 -10.46 -9.71 Cellular retinoic acid-binding protein 1 -10.47 -9.72 Protein kinase C beta type -10.48 -9.73 Voltage-dependent L-type calcium channel subunit alpha-1D -10.53 -9.77 Diacylglycerol kinase beta -10.53 -9.77 U3 small nucleolar RNA-associated protein 14 homolog B -10.72 -9.94 Dystrophin -10.72 -9.94 APC membrane recruitment protein 2 -10.78 -9.99 High mobility group protein B1 -10.78 -9.99 Protein PRRC2C -10.79 -10 Serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform -10.82 -10.02 Serine/arginine-rich splicing factor 7 -10.86 -10.06 Macrophage colony-stimulating factor 1 -10.88 -10.08 Lactoylglutathione lyase -10.91 -10.11 Copine-4 -10.95 -10.14 MAP7 domain-containing protein 2 -10.96 -10.15 Nesprin-2 -10.97 -10.16 EMILIN-3 -10.98 -10.17 Choline O-acetyltransferase -11.01 -10.2 Sister chromatid cohesion protein PDS5 homolog A -11.03 -10.21 Supervillin -11.03 -10.21 Thyroid hormone receptor alpha -11.04 -10.22 H-2 class I histocompatibility antigen, L-D alpha chain -11.05 -10.23

88

Table E-5. Continued.

Protein Description (108) Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Serine/threonine-protein kinase WNK3 -11.07 -10.25 Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1 -11.07 -10.25 Retinoblastoma-like protein 2 -11.12 -10.29 Solute carrier family 2, facilitated glucose transporter member 1 -11.14 -10.31 Protein FRG1 -11.14 -10.31 Epiplakin -11.17 -10.34 Ataxin-2-like protein -11.25 -10.4 Unconventional myosin-VIIb -11.27 -10.42 Serpin I2 -11.31 -10.46 Scaffold attachment factor B1 -11.33 -10.47 m7GpppX diphosphatase -11.34 -10.48 Protein transport protein Sec61 subunit beta -11.36 -10.5 Copine-6 -11.36 -10.5 cGMP-gated cation channel alpha-1 -11.37 -10.51 Neurexin-3 -11.4 -10.54 Collagen alpha-1(XI) chain -11.42 -10.55 Lactotransferrin -11.43 -10.57 Glycylpeptide N-tetradecanoyltransferase 2 -11.46 -10.59 Keratin, type I cytoskeletal 19 -11.47 -10.6 Dynein heavy chain 12, axonemal -11.5 -10.62 TBC1 domain family member 17 -11.52 -10.64 Trinucleotide repeat-containing gene 18 protein -11.55 -10.66 Dehydrogenase/reductase SDR family member 11 -11.57 -10.69 E3 ubiquitin-protein ligase RBBP6 -11.6 -10.71 TATA element modulatory factor -11.64 -10.75 Cytochrome P450 2B19 -11.65 -10.76 Zinc finger RNA-binding protein -11.74 -10.84 Sedoheptulokinase -11.78 -10.87 Fibrinogen-like protein 1 -11.79 -10.88 Protein Z-dependent protease inhibitor -11.79 -10.88 WD repeat-containing protein 64 -11.82 -10.9 Keratin, type I cytoskeletal 17 -11.82 -10.9 Quinone oxidoreductase -11.9 -10.98 Abnormal spindle-like microcephaly-associated protein homolog -11.92 -10.99 GATA-type zinc finger protein 1 -11.97 -11.04 Inositol 1,4,5-trisphosphate receptor type 2 -12.01 -11.08 Complement C1q tumor necrosis factor-related protein 3 -12.01 -11.08 Telomere-associated protein RIF1 -12.1 -11.15

89

Table E-5. Continued.

Protein Description (108) Z-Score Dentin7/Plastic7

Z-Score Bone7/Plastic7

Laminin subunit beta-1 -12.16 -11.2 Adenylate kinase 4, mitochondrial -12.22 -11.25 Protocadherin Fat 2 -12.24 -11.27 Zinc finger protein ZFPM1 -12.26 -11.29 Fibrinogen alpha chain -12.26 -11.29 Creatine kinase M-type -12.32 -11.34 Pro-neuregulin-3, membrane-bound isoform -12.33 -11.35 Fibroblast growth factor receptor 1 -12.36 -11.38 DNA annealing helicase and endonuclease ZRANB3 -12.38 -11.4 Disks large-associated protein 1 -12.4 -11.42 Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 2 -12.43 -11.45 Insulin-like growth factor I -12.45 -11.46 pre-mRNA 3' end processing protein WDR33 -12.47 -11.47 Mitogen-activated protein kinase kinase kinase 9 -12.51 -11.51 Rho guanine nucleotide exchange factor 28 -12.58 -11.57 Plakophilin-4 -12.63 -11.62 Brefeldin A-inhibited guanine nucleotide-exchange protein 1 -12.66 -11.64 Insulin-like growth factor-binding protein 1 -12.7 -11.68 Nebulin-related-anchoring protein -12.72 -11.7 Xin actin-binding repeat-containing protein 2 -12.88 -11.84 ATP-binding cassette sub-family A member 13 -12.89 -11.85 Piezo-type mechanosensitive ion channel component 2 -13.09 -12.02 Structural maintenance of chromosomes protein 1B -13.22 -12.13 Down syndrome cell adhesion molecule-like protein 1 homolog -13.26 -12.17 Ubiquitin-60S ribosomal protein L40 -13.26 -12.17 Kinetochore-associated protein 1 -13.59 -12.46 Prickle-like protein 1 -13.66 -12.52 Phosphatidylcholine-sterol acyltransferase -13.7 -12.55 Nuclear receptor corepressor 2 -13.89 -12.72 Protein FAM184B -13.91 -12.74 Serum albumin -13.98 -12.8 Signal-induced proliferation-associated 1-like protein 1 -14.11 -12.91 Tumor necrosis factor alpha-induced protein 3 -14.55 -13.31 Histone H2B type 1-B -14.88 -13.59 Histone H2A type 1-F -15.78 -14.39

90

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BIOGRAPHICAL SKETCH

Alyssa Kathleen Emory-Carter was born in Sherman, Texas to her parents

Michael and Carolyn Emory. She was raised in the small town of Bells, Texas. In 2007,

she moved to Stillwater, Oklahoma to attend Oklahoma State University. Alyssa earned

a Bachelor of Science in physiology and a minor in general business, and subsequently

began dental school at Baylor College of Dentistry in Dallas, Texas in 2010. In 2014,

after completing her Doctor of Dental Surgery degree, she moved to Gainesville, Florida

and began the Graduate Orthodontic Residency Program at the University of

Florida. Alyssa and her husband, Cody H. Carter, enjoy traveling, outdoor activities,

sports, and spending time with their families and friends.