THE ROLE OF EPITHELIAL MESENCHYMAL TRANSITION IN PERIODONTAL DISEASE

By

LINDSEY PIKOS ROSATI

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 Lindsey Pikos Rosati

To my husband, Sam, for his unconditional love and support

4

ACKNOWLEDGMENTS

I would like to thank God and my family for their continued love, guidance and

support over the years. They have helped me develop into the person that I am today. I

am eternally grateful for the opportunities that have been bestowed upon me. I would

also like to thank my colleagues and mentors at the University of Florida who have had

a tremendous impact on my education.

5

TABLE OF CONTENTS page

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

LIST OF TABLES ............................................................................................................ 6

LIST OF FIGURES .......................................................................................................... 7

LIST OF ABBREVIATIONS ............................................................................................. 8

ABSTRACT ..................................................................................................................... 9

CHAPTER

1 INTRODUCTION .................................................................................................... 11

The Periodontium in Health .................................................................................... 11

Periodontal Tissue Destruction ............................................................................... 14 Long Junctional Epithelium ..................................................................................... 17 Epithelial Mesenchymal Transition ......................................................................... 22

EMT and Periodontal Disease ................................................................................ 25

2 MATERIALS AND METHODS ................................................................................ 28

Primary Oral Epithelial Cell Culture ........................................................................ 28

TGF-Beta2 Stimulation ........................................................................................... 28

Protein Extraction ................................................................................................... 28 Western Blot ........................................................................................................... 29 RNA Extraction and PCR Array .............................................................................. 29

3 RESULTS ............................................................................................................... 30

E-Cadherin and Smooth Muscle Actin Protein Expression Does Not Change Following TGFβ2 Stimulation of Oral Epithelial Cells .......................................... 30

Snail1 Protein Expression is Up-Regulated Following TGFβ2 Stimulation of Oral Epithelial Cells ..................................................................................................... 30

Treatment of Human Oral Epithelial Cells with TGFβ2 does Result in Morphological Changes ....................................................................................... 31

Treatment of Human Oral Epithelial Cells with TGFβ2 Induces Regulation of Key Genes Involved in EMT ................................................................................ 31

4 DISCUSSION ......................................................................................................... 37

LIST OF REFERENCES ............................................................................................... 42

BIOGRAPHICAL SKETCH ............................................................................................ 46

6

LIST OF TABLES

Table page

3-1 TGFβ Induced Gene Expression ............................................................................. 35

7

LIST OF FIGURES

Figure page 1-1 Protein, gene, and morphological markers associated with epithelial

mesenchymal transition ...................................................................................... 27

3-1 TGFβ2 does not induce any changes in E-Cadherin. ............................................. 33

3-2 TGFβ2 does not induce any changes in smooth muscle actin expression. ............ 33

3-3 TGF2 induces Snail1 expression in human primary oral epithelial cells in a dose dependent manner ..................................................................................... 34

3-4 Morphological changes in oral epithelial cells with TGF2 treatment induces a mesynchymal phenotype .................................................................................... 34

8

LIST OF ABBREVIATIONS

EMT epithelial mesenchymal transition

HOK human oral keratinocytes

IL-1

MMP

Interleukin-1-beta

Matrix metalloproteinases

OPG osteopotegrin

PD periodontal disease

PDL periodontal ligament

RANK

RANKL

Receptor activator of nuclear factor kappa-B

Receptor activator of nuclear factor kappa-B ligand

TGFβ

TNFα

Transforming growth factor beta

Tumor necrosis factor alpha

9

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

THE ROLE OF EPITHELIAL MESENCHYMAL TRANSITION IN PERIODONTAL

DISEASE

By

Lindsey Pikos Rosati

May 2017

Chair: Kevin McHugh Major: Dental Sciences-Periodontics

Periodontal disease (PD) is characterized by a chronic state of inflammation in

response to the presence of bacteria and their byproducts. The inflammatory state

involves poorly organized hyperplastic gingiva, which then invades the supporting

periodontium. Consequently, alveolar bone resorption occurs, leading to attachment

loss and eventually tooth loss. Epithelial mesenchymal transition is a process by which

epithelial cells de-differentiate, allowing them to change from a polarized epithelial cell

to a mesenchymal cell phenotype where they lose their polarity and their cell-cell

adhesion properties. This phenotype also includes enhanced migratory capacity,

invasiveness, and greater production of extracellular matrix components leading to the

degradation of the underlying basement membrane and the formation of a

mesenchymal cell that has the ability to migrate away from the epithelial layer. TGF

family pathway signaling induces EMT in numerous systems including normal

development and typical healing processes; however, it also occurs in cancer.

We hypothesize that a similar pathway, or portions of the EMT pathway, are

involved in bone loss and tooth loss in PD. Our studies show terminally differentiated

10

gingival epithelial cells undergo a partial transition to a mesenchymal-like phenotype.

We propose that gingival epithelial cells can undergo an EMT-like process in

periodontitis whereby they secrete mediators, which recruit osteoclasts to invade the

periodontium and cause alveolar bone resorption.

11

CHAPTER 1 INTRODUCTION

The Periodontium in Health

The periodontium serves as the foundation for the dentition. It is comprised of

gingiva, periodontal ligament, cementum and alveolar bone (Nanci and Bosshardt

2006). The primary role of the periodontium is to support the teeth during regular

function, maintain surface integrity of masticatory mucosa and provide protection

against bacterial infiltration. In the absence of inflammation, the periodontium is able to

accomplish these tasks, withstanding trauma from function and repelling bacterial

infection(Nanci and Bosshardt 2006). Numerous studies have documented the

dimensions of the dentogingival apparatus. Typically, the epithelial attachment to the

tooth occurs approximately 0.67-1mm apical to the cementoenamel junction (CEJ).

This epithelium is approximately 1mm in length. Moving apically down the tooth

surface, the connective tissue is the next layer, comprised primarily of collagen,

fibroblasts, and ground substance. This ground substance is made up of water,

glycosaminoglycans, proteoglycans, and glycoproteins. The connective tissue layer is

approximately 1mm in length. The term “biologic width” is defined as the sum of the

epithelial and connective tissue layers that are attached to the tooth above the level of

the crestal bone (Gargiulo et al. 1961). In a large cadaver study, Gargiulo et al found the

distance established by the epithelial and connective tissue attachment to be

approximately 2.04mm (Gargiulo et al. 1961). In order to prevent inflammation and

attachment loss, it is imperative to respect biologic width. Biologic width can vary from

person to person, from tooth to tooth and from surface to surface on the same tooth

(Maynard and Wilson 1979). The alveolar bone crest marks the underlying layer below

12

the connective tissue. The periodontal ligament (PDL) joins the tooth root surface with

the alveolar bone (Maynard and Wilson 1979). The individual components of the

periodontium will be elaborated in greater detail in the next sections.

The gingiva is the periodontium’s initial line of defense against bacterial

penetration through its mechanical barrier function. In the absence of inflammation, the

gingiva forms a tight seal around the teeth, protecting the underlying layers of the

periodontium from bacterial infiltration. This seal can be disrupted by the host’s

inflammatory response, which can be induced by trauma or the presence of bacterial

biofilms and plaque. In periodontal disease, the barrier function of the gingiva is

disrupted, bacterial invasion occurs and subgingival biofilms develop. The bacteria in

the subgingival biofilm community thrive and continue to exponentially reproduce, until

the biofilm mineralizes into a substance known as calculus. Calculus is a mineralized

matrix composed of inorganic crystals of calcium phosphate, brushite, octa calcium

phosphate, hydroxyapatite and whitlockite (Lang et al. 2008). The subgingival calculus

serves as a nidus of infection and leads to further progression of the disease in an

apical direction (Bernimoulin 2003). This progression is likely to continue until the

calculus is mechanically disrupted and eradicated (Oshrain et al. 1971).

The PDL is a very vascular and cellular connective tissue surrounding the tooth

roots, joining the root cementum to the alveolar bone. The PDL protects vessels and

nerves, transmits occlusal forces, attaches the tooth to bone and performs formative

and remodeling functions. Secondary functions include providing somatosensory

information and nutrition to the local cells (Perera and Tonge 1981). The average width

of the PDL is 0.25mm, with the greatest width occurring at the apex and narrowest

13

occurring in the middle. The terminal ends of the PDL fibers are known as Sharpey’s

fibers and they insert into the cementum on the tooth side and the periosteum on the

bone side. Collectively, the cementum, alveolar bone, and PDL comprise the

attachment apparatus. The PDL space is essential for tooth mobility and allows

distribution and resorption of forces by the alveolar process via the alveolar bone proper

(Lang and Lindhe 2015). Within the PDL are progenitor cells that can differentiate into

osteoblasts and provide maintenance to the surrounding alveolar bone.

Cementum is the outer layer covering the root surface. It attaches the PDL

Sharpey’s fibers to the root and aids in root surface repair. It lacks blood lymph vessels

as well as innervation. Cementum is continuously deposited throughout life and does

not undergo resorption or remodeling. It is composed primarily of inorganic

hydroxyapatite (65%), and approximately 35% water and organic material, making it

softer than enamel or dentin. There are four types of cementum: acellular afibrillar

cementum (located along the cervical portion of enamel), acellular extrinsic fiber

cementum (derived from PDL Sharpey’s fibers and located along the coronal and

middle portions of the root connecting the tooth with bundle bone), cellular mixed

stratified cementum (located along the apical third of the root and furcation regions),

and cellular intrinsic fiber cementum (produced by cementoblasts and located in

resorption lacunae) (Avery 2001).

Teeth reside in sockets of the alveolar bone proper and are connected to the

bone via Sharpey’s fibers of the PDL. Alveolar bone is a mineralized substance

primarily composed of hydroxyapatite and collagen and is similar to cementum in that

the levels of hydroxyapatite are less than both enamel and dentin. By weight, it is

14

composed of 60% inorganic material, 25% organic material, and 15% water. Alveolar

bone consists of cortical bone, cancellous trabecular bone and the alveolar bone proper

(which lines the tooth socket). Alveolar bone plays a role in calcium metabolism;

however, the primary role is to protect and support the teeth during function (Kornman

et al. 1997). Masticatory forces can be as large as 600-750N. In response to functional

demands, alveolar bone undergoes constant remodeling. Through this bone

multicellular unit, osteoclasts are involved in resorption while osteoblasts partake in

bone formation (Lang and Lindhe 2015). Despite the PDL contributing to the

management of the functional forces, the alveolar bone receives the vast majority of the

forces. The result of long term periodontal disease can cause destruction of this bony

housing and greatly compromise the support and stability of the teeth.

Periodontal Tissue Destruction

According to the data collected during the 2009 and 2010 National Health and Nutrition

Examination Surveys, the prevalence of periodontitis is 47.2% among U.S. adults ages

30 and older. This is further categorized as mild (8.7%), moderate (30%), and severe

(8.5%) cases. Among the adults ages 65 and older, 64% had either moderate or severe

periodontal disease (Eke et al. 2012). Periodontal disease was demonstrated to be

highest in men, Mexican Americans, individuals lacking a high school education,

individuals below the Federal Poverty Level, and current smokers. Risk factors for

periodontal disease include tobacco use, poor oral hygiene, diabetes and genetics.

Smokers are 2.7 times more likely to have periodontitis than non-smokers (Grossi

2000). Poor oral hygiene such as refraining from preventive practices leads to various

rates of periodontal destruction (Loe et al. 1986). Three times more bone loss and

attachment loss is seen in diabetics compared to non-diabetics (Grossi et al. 1997). In a

15

twin study, it was determined that genetics accounts for 50% of an enhanced risk for

periodontitis (Michalowicz et al. 1991).

Periodontal disease can only be properly diagnosed by conducting clinical and

radiographic examinations. Clinical parameters that are gathered during this exam

include: probing depth, bleeding on probing, recession, furcation involvement, and

mobility. Typically, probing depth and recession are the pertinent measurements in

formulating a periodontal diagnosis. In conditions of health, probing depths typically

ranges from 2-3mm with the probe not penetrating beyond the epithelial attachment

(Anderson et al. 1991). Recession is a measurement of the distance between the free

gingival margin and the CEJ of the tooth. Attachment loss is calculated by adding the

probing depth and recession values together. Attachment loss is the extent of apical

migration of the periodontium from its normal level and is the result of long-term

periodontal disease. The attachment apparatus described above should begin

approximately 1-2mm from the CEJ. This can be measured radiographically by

examining the level of the interproximal bone.

Presently, the most commonly used classification system is the one introduced

during the International Workshop for a Classification of Periodontal Diseases and

Conditions (Armitage 1999). Periodontal disease classification is based on the amount

of attachment loss and categorizes the severity of the disease as: 1-2mm Slight, 3-4mm

Moderate, and >=5mm Severe. The disease may manifest as generalized (>30% of

sites are involved) or localized (up to 30% of sites are involved). Furthermore, the type

of disease can be further classified as chronic or aggressive (Armitage 1999).

16

The main distinguishing factor between chronic and aggressive periodontal

disease is the rate of destruction. Chronic periodontal disease tends to progress at a

rate of approximately .2-.25mm per year, though there can be a wide variation among

various individuals (Waerhaug 1977). Additionally, chronic periodontal disease does

not tend to follow any specific pattern of bone loss. Conversely, aggressive periodontal

disease tends to progress approximately 3-4 times as quickly as chronic. Also, in the

case of localized aggressive periodontal disease, bone loss tends to occur around the

first molars and incisors. Similar to chronic periodontitis, aggressive periodontitis can

be further categorized into generalized and localized cases (Califano 2003). Localized

cases tend to have an absence of large plaque and calculus accumulations while

generalized cases have an abundance of local factors (Califano 2003). Localized cases

exhibit interproximal attachment loss on an at least two permanent molars and incisors

with loss on no more than two teeth other than first molars and incisors, while

generalized cases have interproximal attachment loss on at least three teeth that are

not first molars and incisors (Armitage 1999). Aggregatibacter actinomycetemcomitans

tends to be involved in localized aggressive cases while Porphyromonas gingivalis and

Treponema denticola tend to be prevalent in generalized cases. Localized aggressive

cases tend to affect children and adolescents while generalized aggressive cases tend

to affect adolescents and young adults (Califano 2003). The prevalence of localized

aggressive periodontitis was found to be 0.53% with a greater occurrence in African

Americans (possibly due to defects in neutrophil function) while the prevalence of

generalized aggressive periodontitis was found to be 0.13%(Loewenthal 1991) and (Loe

and Brown 1991). Non-surgical therapy involving scaling and root planing in addition to

17

oral hygiene instruction is always the first intervention for the treatment of either chronic

or aggressive periodontal disease. When deep residual periodontal pockets remain,

surgical therapy is often the next treatment modality.

Long Junctional Epithelium

Histological studies have shown that various types of surgical periodontal

procedures result in different types of healing. These include: repair, new attachment,

regeneration and reattachment. The majority of periodontal wound healing involves

repair; however, it is imperative to formally define and distinguish between the various

terms involved in periodontal healing. According to the American Academy of

Periodontology, repair is defined as “healing of a wound by tissue that does not fully

restore the architecture or function of the part” (AAP 2012). Healing via repair involves

the formation of a thin layer of long junctional epithelium extending apically between the

root surface and the gingival connective tissue (Caton and Nyman 1980) and (Listgarten

and Rosenberg 1979). Connective tissue repair (new attachment) is defined as “the

union of connective tissue or epithelium with root surface that has been exposed to

periodontal disease or otherwise deprived of its original attachment apparatus” (AAP

2012). In contrast, regeneration is the reproduction or reconstitution of a lost or injured

part and is characterized by de novo cementum formation, a functionally oriented

periodontal ligament, alveolar bone and gingiva (Caton and Nyman 1980) and

(Listgarten and Rosenberg 1979). Similar to new attachment, regeneration can only be

proven through histology. The final term, reattachment, means to literally attach again.

This occurs when epithelium and connective tissues are reunited with a root surface

after an incision or injury (AAP 2012). Following reconstruction of the periodontal

apparatus, these biologic outcomes tend to occur together and are not distinct entities.

18

For example, periodontal regeneration entails both new attachment and epithelial

attachment (Polimeni et al. 2006).

Many studies have been conducted to evaluate the factors that determine

whether regeneration or repair occurs following a periodontal procedure. Any surgical

therapy creates a wound, and after flap closure, the wound healing cascade begins.

The four phases of healing include: hemostasis, inflammation, proliferation and tissue

remodeling (Guo and Dipietro 2010). A lack of mechanical stability of the wound is a

main determining factor in the formation of long junctional epithelium (Linghorne and

O'Connell 1950). The formation of a fibrin clot onto the root surface serves as a barrier

that prevents apical migration of the gingival epithelium (Hiatt et al. 1968). During

wound healing, if this fibrin clot is disrupted, the formation of long junctional epithelium

will likely occur, thus compromising periodontal wound healing and impairing the

regenerative process. Consequently, wound stability is imperative for the establishment

of a new connective tissue attachment to a root surface deprived of its periodontal

attachment (Polimeni et al. 2006).

Often times, non-surgical therapy alone is not sufficient when deep residual

periodontal pockets are present. Surgical procedures attempting to reduce or even

eliminate these pockets include techniques such as open flap debridement and osseous

recontouring. These procedures involve flapping an area in order to gain access, root

plane diseased root surfaces, and recontour boney defects to create positive boney

architecture and an environment that is amenable to proper oral hygiene by the patient.

These procedures involve healing via periodontal repair, and thus the formation of a

19

long junctional epithelium. The significance of this is that the original architecture and

function of the periodontium is not restored.

The main difference between repair and regeneration is a lack of a new

connective tissue attachment to the root. Novel cementum with inserting collagen fibers

cannot form on a root covered by epithelial cells (Bosshardt et al. 2015). Thus,

periodontal regeneration is the preferable treatment outcome when possible. Surgical

methodologies such as guided tissue regeneration and bone grafting materials can

provide an environment conducive to undisturbed wound healing and thus regeneration.

The biomaterials stabilize the blood clot and encourage the growth of the PDL into the

defect area. Thus, the PDL progenitor and stem cells can give rise to a new periodontal

attachment apparatus (Bosshardt et al. 2015).

In regards to regeneration, tissues originating from the periodontal ligament

serve as a source for periodontal regeneration by giving cells the capacity to

differentiate into cementoblasts, fibroblasts and osteoblasts. Studies by Karring and

Nyman elucidated that cells from the PDL have the capacity to regenerate the

periodontium while cells from gingival connective tissue and alveolar bone do not

(Karring et al. 1993). Melcher concluded that if preference is given to PDL cells,

periodontal regeneration might consistently occur (Melcher 1976). Occlusion of gingival

epithelial cells by means of membranes or tissue barriers, known as guided tissue

regeneration techniques, is imperative in attaining periodontal regeneration.

In 1976, Page and Schroeder described the pathogenesis of periodontal disease.

The disease process follows a certain sequence of events: initial lesion, early lesion,

established lesion and advanced lesion. The initial lesion forms within 2-4 days of

20

bacterial plaque accumulation and is characterized by acute vasculitis in the

surrounding tissues, which can lead to breakdown of the perivascular collagen. This

may be a result of the release of chemotactic and antigenic substances released from

the biofilm. The early lesion develops around days 4-10 and is characterized by the

recruitment of lymphocytes and other mononuclear cells by the host’s immune system.

There is also increased destruction of the adjacent connective tissue and pathologic

alteration of the local fibroblasts. Within 2-3 weeks, the early lesion transforms into the

established lesion, characterized predominantly by the presence of plasma cells in the

absence of significant bone loss. This lesion can remain stable for months to years

before progressing to the final lesion, the advanced lesion. In this lesion, plasma cells

continue to predominate; however, the main difference between the two lesions is that

there is significant loss of alveolar bone and PDL along with disruption of the tissue

architecture with fibrosis. Gingivitis is characterized by the initial, early, and established

lesions, while periodontitis does not develop until the advanced lesion is present (Page

and Schroeder 1976).

Periodontitis is a more advanced form of infection that destroys the tooth-

supporting periodontal tissues. Once the junctional epithelium has been breached,

bacteria are able to penetrate to the underlying connective tissue attachement and the

PDL. Various cytokines, prostaglandins and proteolytic enzymes released by the host

immune system in response to bacterial infection cause degradation of the connective

tissue, leading to inflammation, pocket formation and bone resorption (Ebersole et al.

2013). Although this response is beneficial in destroying bacteria or at least impeding

their proliferation, it also has the negative consequence of causing periodontal tissue

21

destruction. This destruction forms the basis for the loss of attachment observed in

periodontal disease (Kornman et al. 1997). Bacteria and their pro-inflammatory

mediators can rapidly travel throughout periodontal tissues due to the extensive

vascular network of the periodontium (Kornman et al. 1997). The subsequent release of

inflammatory mediators such as Interleukin-1-beta (IL-1β) and Tumor necrosis factor

alpha (TNFα), in addition to matrix metalloproteinases (MMP) results in the tissue

destruction associated with periodontal disease. This destruction occurs rapidly in

aggressive periodontal disease or over long periods of time in chronic periodontal

disease.

Inflammation, pocket formation, and bone resorption are the hallmarks of

periodontal disease (Bosshardt et al. 2015). Bone resorption is performed by

osteoclasts while bone formation is performed by osteoblasts. Normal bone remodeling

involves a balance between bone formation and bone destruction. Osteoblasts present

Receptor activator of nuclear factor kappa-B ligand (RANKL) to osteoclastic precursors

containing Receptor activator of nuclear factor kappa-B (RANK), which activates

osteoclasts and leads to bone resorption. Osteoprotegrin (OPG), produced by

osteogenic cells and certain fibroblasts, inhibits this interaction by acting as a soluble

decoy receptor for RANKL that competes for binding to RANK, thus preventing bone

resorption (Bosshardt et al. 2015).

In periodontal disease, chronic inflammation of the gingival epithelium leads to

extension of the junctional epithelium and periodontal pocket formation. This can

progress to loss of the PDL, bone and even teeth. Bone loss is focal in periodontal

disease. Gingival epithelial cells seem to undergo EMT and assume a phenotype that

22

causes them to have a migratory capacity to invade the periodontium. Once invasion

has occurred, they potentially secrete mediators that recruit osteoclasts to enter and

cause focal bone resorption.

Epithelial Mesenchymal Transition

EMT is a process by which epithelial cells de-differentiate, allowing them to

change from a polarized epithelial cell to a mesenchymal cell phenotype where they

lose their polarity and their cell-cell adhesion properties. This phenotype also includes

enhanced migratory capacity, invasiveness, increased resistance to senescence and

apoptosis and greater production of extracellular matrix components (Kalluri and

Weinberg 2009) (Figure 1-1). EMT completion is signaled by the degradation of the

underlying basement membrane and the formation of a mesenchymal cell that has the

ability to migrate away from the epithelial layer that it was derived from (Kalluri and

Weinberg 2009).

Transforming growth factor family pathway signaling induces epithelial

mesenchymal transition in numerous systems. EMT occurs in normal developmental

processes such as mesoderm formation and neural tube formation in addition to typical

wound healing processes; however, it also occurs in cancer. Additionally, EMT is

involved in cancer cell motility, cancer invasion into adjacent tissues, osteolysis

associated with metastasis to bone and fibrosis (Lamouille et al. 2014). Key

transcription factors such as Snail mediate this switch in cellular differentiation and

behavior. Snail and other transcription factors are modified at the transcriptional,

translational and post-translational levels. Reprogramming of gene expression in EMT is

initiated and controlled by signaling pathways such as transforming growth factor family

that responds to extracellular cues (Lamouille et al. 2014).

23

There are numerous molecular processes that initiate EMT. These include

activation of transcription factors, expression of cell specific surface proteins,

reorganization and expression of cytoskeletal proteins, production of extracellular matrix

degrading enzymes and changes in micro-RNA expression (Kalluri and Weinberg

2009). Most cells have intact junctional complexes and epithelial polarity. During EMT,

cells downregulate the expression of epithelial proteins, specifically those part of cell

junctional complexes (Huang et al. 2012). Their gene expression is also re-directed to

promote changes in the cytoskeleton structure, encourage adhesion with mesenchymal

cells and change the cell interactions with the extracelluar matrix (Yilmaz and Christofori

2009). In order to initiate EMT, growth factors activate membrane receptors, causing

changes in the actin cytoskeleton remodeling and loss of apicobasal polarity. The DDR1

complex activates RhoE, which weakens actomyosin contractility in areas of cell-cell

contact (Hidalgo-Carcedo et al. 2011). TGFβ receptors localized in tight junctions trigger

non-canonical pathways, leading to RhoA ubiquitylation and degradation, and the

destabilization of cortical actin microfilament-associated tight junctions. The activation of

transcriptional repressors, such as Snail and Serpent (Srp/GATA), downregulates

genes encoding junctional proteins, including E-cadherin, claudins and occludin, thus

compromising epithelial integrity (Burk et al. 2008), (Whiteman et al. 2008), and (Lim

and Thiery 2011).

The downregulation of E cadherin is a hallmark of EMT. This reinforces the

destabilization of adherens junctions in EMT. In addition, claudin, occludin, desmoplakin

and plakofilin are repressed, causing disintegration of tight junctions and desmosomes

(Huang et al. 2012). Collectively, these changes in gene expression prevent the

24

formation of new epithelial cell-cell junctions, resulting in a loss of epithelial cell barrier

function (Peinado et al. 2007). The down regulation of E-Cadherin is balanced by an

increased expression of mesenchymal neural cadherin (N cadherin), which causes

transitioning cells to lose their affinity for epithelial cells and acquire one for

mesenchymal cells through N-cadherin interactions (Wheelock et al. 2008). EMT also

induces the expression of neural cell adhesion molecule that interacts with N cadherin

to regulate the expression of the SRC family tyrosine kinase FYN to facilitate the

production of focal adhesions, migration and invasion (Lehembre et al. 2008).

Gene expression changes that contribute to the repression of an epithelial

phenotype and activation of a mesenchymal phenotype involve master regulators such

as Snail, TWIST and zinc-finger-E-box-binding transcription factors. These transcription

factors are very different, and their influence on EMT depends on the tissue and cell

types and the signalling pathways involved in initiating EMT (De Craene and Berx

2013).

Snail1 and Snail2 activate EMT during development, fibrosis and cancer. They

repress epithelial genes by bonding to E-box DNA segments through their carboxy-

terminal-zinc-finger domains (Barrallo-Gimeno and Nieto 2005). Snail represses gene

expression through binding of the promoter region of E cadherin. Snail also activates

genes that contribute to the expression of the mesenchymal phenotype (Batlle et al.

2000). Numerous signalling pathways are involved in the initiation and progression of

EMT, and they often activate the expression of Snail1. TGFβ2 and WNT family proteins,

Notch and growth factors that act through receptor tyrosine kinases, all activate Snail1

expression depending on the context (Peinado et al. 2007). Snail cooperates with other

25

transcription regulators to influence gene expression. For example: Snail cooperates

with ETS1 to activate expression of matrix metalloproteinases (Jorda et al. 2005). It also

cooperates with the SMAD-3 and SMAD-4 complex to cause the TGFβ-mediated

repression of E-Cadherin and occludin expression (Vincent et al. 2009). In regards to

the cytoskeletal changes associated with EMT, formation of actin stress fibers (smooth

muscle actin) that attach to focal adhesion complexes, begin to promote cell migration.

EMT and Periodontal Disease

Periodontal disease is characterized by a chronic state of inflammation in

response to the presence of bacteria and their byproducts. The inflammatory state

involves poorly organized hyperplastic gingiva, which then invades the supporting

periodontium. Consequently, alveolar bone resorption occurs, leading to both

attachment and tooth loss. In periodontitis, gingival epithelial cells potentially undergo a

transition to a motile mesenchymal-like phenotype (similar to EMT in cancer) that

possesses the ability to invade the periodontium and cause alveolar bone resorption.

When gingival epithelial cells undergo EMT, they potentially secrete mediators that

recruit osteoclasts to invade and cause focal bone resorption. Gingival invasion and

bone loss in periodontal disease is focal and is similar to the focal bone loss seen in

cancer.

The overarching hypothesis of the project is that in periodontal disease, gingival

epithelial cells undergo a transition to a motile mesenchymal-like phenotype that, like

EMT in cancer, promotes tissue invasion and facilitates bone resorption specifically in

PD, invasion into the periodontal space and resorption of alveolar bone. The project will

look for molecular and phenotypic similarities between classic EMT and TGF2 induced

26

primary human oral keratinocytes (hOK). In addition, we will look for markers of EMT in

a mouse model of PD.

27

Figure 1-1. Protein, gene, and morphological markers associated with epithelial mesenchymal transition.

28

CHAPTER 2 MATERIALS AND METHODS

Primary Oral Epithelial Cell Culture

Human oral epithelial primary cells were purchased commercially from

Celprogen. These cells positively express EpCAM, simple epithelial markers

cytokeratins 7,8, and 18, stratified epithelial markers cytokeratins 5 and 13, amylase,

claudin-1 and 3, kallikrein, and vimentin. Cells were maintained in human oral epithelial

primary cell culture complete growth medium with serum (Celprogen) and passaged

every 24-48hrs onto human oral epithelial primary cell culture extracelluar matrix. For

experiments, cells were plated on 6-well extracellular matrix coated tissue culture plates

at 5 X 105 cells/well. The cells were imaged under brightfield microscopy at 20X every

24 hours.

TGF-Beta2 Stimulation

Human oral epithelial primary cells were washed once with fresh medium and

treated with recombinant human TGF2 (R&D Systems) diluted in fresh medium at 5,

10, 20, and 40ng/ml. Cells were harvested at 24, 48, 72, and 96 hours.

Protein Extraction

A commercially available detergent-based cell extraction buffer (ThermoFisher)

supplemented with a protease cocktail inhibitor (Roche) and 1mM PMSF (Abcam) was

used to lyse cells and extract proteins from the epithelial cell culture. Lysates were

collected every 24 hours.

29

Western Blot

Cell lysates were assayed by Western blot analysis to determine E-cadherin,

smooth muscle actin, and SNAIL1 protein levels. Samples were boiled in Laemmli

buffer and electrophoresed under reducing conditions on 10% Tris-Glycine TGX gels

(Bio-Rad). Proteins were transferred to PVDF membranes (Bio-Rad), and blots were

blocked with 5% BSA. Antibodies against E-cadherin (Cell Signaling Technology),

smooth muscle actin (CST), and Snail1 (CST) were diluted 1:500, and blots were

incubated overnight at 4C. An anti-rabbit HRP-conjugated antibody (CST) was used as

a secondary for 2 hours at room temperature followed by detection with SuperSignal

West Pico Chemiluminescent Substrate(ThermoFisher). -actin (CST) was used as a

loading control, and secondary antibody and chemiluminescent detection were

completed as above.

RNA Extraction and PCR Array

Total RNA was harvested from tissue culture wells using an RNeasy extraction

kit (Qiagen). Samples were stored at -80C until PCR could be performed. For EMT PCR

array assays, cells were treated with 40ng/mL TGF2 and cultured as above for 96

hours prior to RNA isolation. cDNA synthesis was carried out using the RT2 First Strand

kit (Qiagen) including a gDNA elimination step. Real-time PCR assays were completed

with the RT2 Profiler PCR Array for Human Epithelial to Mesenchymal Transition

(Qiagen). Results were analyzed using the GeneGlobe Data Analysis Center on the

Qiagen website

30

CHAPTER 3 RESULTS

E-Cadherin and Smooth Muscle Actin Protein Expression Does Not Change Following TGFβ2 Stimulation of Oral Epithelial Cells

In order to determine if TGFβ2 could induce changes in proteins known to

contribute to the epithelial mesenchymal transistion (EMT) in human oral keratinocytes,

HOK were treated with TGFβ2 at concentrations of 0, 20, and 40ng/mL over a time

course of 96 hours. After which changes in E-Cadherin (ECad) and smooth muscle

actin (αSMA) expression were evaluated by western blot, whereby decreased

expression of ECad and increased expression of αSMA would be indicative of EMT.

Western blot analysis revealed there were no evident modifications of ECad protein

expression following TGFβ2 at any time point evaluated (Figure 3-1). Western blot

analysis did suggest some changes in the expression of the pro-form of E-cadherin over

the time course of cell culture, but these changes were independent of TGF2 treatment

(Figure 3-1). Furthermore, smooth muscle actin could not be detected in HOK at rest or

following TGF2 treatment (Figure 3-1). Importantly, western blot analysis of β-actin

demonstrated equal protein loading in all Western blots performed. (Figure 3-1; Figure

3-2). These data do not suggest an induction of an EMT by TGFβ2.

Snail1 Protein Expression is Up-Regulated Following TGFβ2 Stimulation of Oral Epithelial Cells

As an additional measure of whether TGFβ2 could induce changes in proteins

known to contribute to EMT in HOK, Western analysis for the transcription factor Snail1

was performed whereby an increase in Snail 1 would be indicative of EMT. Here

Western blot analysis demonstrated an up-regulation of Snail 1 by 10n/g and 40ng/ of

TGF2. In addition, this upregulation could be observed as early 24 hours and remained

31

elevated at 96 hours post treatment (Figure 3-3). Again, western blot analysis of β-actin

demonstrated equal protein loading in all Western blots performed (Figure 3-3).

Contrary to the ECad and αSMA expression, these data would suggest an induction of

an EMT by TGFβ2.

Treatment of Human Oral Epithelial Cells with TGFβ2 does Result in

Morphological Changes

In order to determine if TGFβ2 could induce morphological changes known to

contribute to EMT in HOK, cells were again treated with TGFβ2 at concentrations of 0,

20, and 40ng/mL over a time course of 96 hours and their morphological characteristics

visualized. Here untreated cells exhibit the characteristic cobblestone-like morphology

that is characteristic of HOK (Figure 3-4). Following 3 days of treatment with TGF2, the

cells have become elongated and become progressively spindle-shaped, a

morphological feature characteristic of cells undergoing EMT (Figure 3-4). In particular,

this morphology is often associated with a mesenchymal phenotype that accompanies

increased motility and requirement for tissue invasion. Cell morphology changes in oral

epithelial cells with TGF2 treatment are similar to classic EMT. This morphological

change is often associated with a mesenchymal phenotype that accompanies increased

motility and is required for tissue invasion. Again, these data would suggest an induction

of EMT by TGFβ2.

Treatment of Human Oral Epithelial Cells with TGFβ2 Induces Regulation of Key Genes Involved in EMT

In order to better characterize the expression profile of EMT associated

molecules by HOK following TGFβ2 treatment, an EMT PCR array was employed. Here

HOK, cells were treated with 40ng/ml of TGFβ2 for 96 hours and the RNA expression

32

profile was compared to untreated cells cultured for the same time frame (Table 3-1).

Here the majority of the genes evaluated that demonstrated a change in expression as

defined by >3.0 fold change in either direction were genes that were upregulated in

response to TGFβ2 treatment. While there is a significant amount of data to be mined, it

is of note that genes associated with mesenchymal transition such as FN1 (fibronectin

1) and ITGA5 (alpha polypeptide of integrins) were significantly up-regulated (7.09 and

31.04 fold respectively) (Table 3-1). In addition, molecules normally associated with

invasion of the tissues such as MMP3 and MMP9 were also found to be upregulated

(3.83 and 19.24 fold respectively) (Table 3-1). Finally, the most highly upregulated gene

MST1R (macrophage stimulating 1 receptor) with a 67.45 fold increase in expression

(Table 3-1), is normally observed during cancer development and has been

demonstrated to confer oncogenic potential like many EMT-associated markers

(Danilkovitch-Miagkova et al. 2003). Together these data would support the induction of

an EMT in HOK by TGFβ2.

33

Figure 3-1. TGFβ2 does not induce any changes in E-Cadherin (135kD) expression.

Figure 3-2. TGFβ2 does not induce any changes in smooth muscle actin expression

(42kD).

34

Figure 3-3. TGF2 induces Snail1 expression in human primary oral epithelial cells in a dose dependent manner (A). Protein extracts were blotted for Snail1 (30kD) or β-actin (42kD).

Figure 3-4. Morphological changes in oral epithelial cells with TGF2 treatment induces a mesynchymal phenotype. Scale bar, 10μm.

35

Table 3-1. TGFβ Induced Gene Expression

Gene Fold-Regulation

Description

ITGAV -14.66 Integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51)

WNT5B -2.68 Wingless-type MMTV integration site family, member 5A

AKT1 -1.9 V-akt murine thymoma viral oncogene homolog 1

CTNNB1 -1.85 Catenin (cadherin-associated protein), beta 1, 88kDa

TSPAN13 -1.78 Tetraspanin 13

DESI1 -1.75 PPPDE peptidase domain containing 2

TGFB2 -1.69 Transforming growth factor, beta 2

CAV2 -1.65 Caveolin 2 CALD1 -1.45 Caldesmon 1

KRT7 -1.34 Keratin 7 HPRT1 -1.28 Hypoxanthine phosphoribosyltransferase 1

IGFBP4 -1.24 Insulin-like growth factor binding protein 4

BMP1 -1.23 Bone morphogenetic protein 1

TWIST1 -1.2 Twist homolog 1 (Drosophila)

FOXC2 -1.18 Forkhead box C2 (MFH-1, mesenchyme forkhead 1)

TIMP1 -1.15 TIMP metallopeptidase inhibitor 1

CAMK2N1 -1.13 Calcium/calmodulin-dependent protein kinase II inhibitor 1

STAT3 1.11 Signal transducer and activator of transcription 3 (acute-phase response factor)

CDH2 1.12 Cadherin 2, type 1, N-cadherin (neuronal)

WNT11 1.14 Wingless-type MMTV integration site family, member 11

KRT14 1.16 Keratin 14 FZD7 1.17 Frizzled family receptor 7

VPS13A 1.19 Vacuolar protein sorting 13 homolog A (S. cerevisiae)

ZEB2 1.21 Zinc finger E-box binding homeobox 2

GSK3B 1.24 Glycogen synthase kinase 3 beta

ZEB1 1.26 Zinc finger E-box binding homeobox 1

PTP4A1 1.28 Protein tyrosine phosphatase type IVA, member 1

TMEFF1 1.28 Transmembrane protein with EGF-like and two follistatin-like domains 1

SNAI3 1.31 Snail homolog 3 (Drosophila)

ESR1 1.32 Estrogen receptor 1

TGFB1 1.41 Transforming growth factor, beta 1

ACTB 1.45 Actin, beta

NUDT13 1.48 Nudix (nucleoside diphosphate linked moiety X)-type motif 13

AHNAK 1.53 AHNAK nucleoprotein

MAP1B 1.58 Microtubule-associated protein 1B

VIM 2.26 Vimentin MSN 2.27 Moesin SMAD2 2.32 SMAD family member 2

STEAP1 2.51 Six transmembrane epithelial antigen of the prostate 1

RGS2 3.45 Regulator of G-protein signaling 2, 24kDa

MMP3 3.83 Matrix metallopeptidase 3 (stromelysin 1, progelatinase)

ERBB3 4.65 V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)

ILK 5.23 Integrin-linked kinase

FN1 7.09 Fibronectin 1

TCF4 12.34 Transcription factor 4

GEMIN2 16.63 gem nuclear organelle associated protein 2

36

Table 3-1. Continued

Gene Fold-Regulation

Description

MMP9 19.24 Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase)

TCF3 28.17 Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)

ITGA5 31.04 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)

SERPINE1 34.2 Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type1), member 1

RAC1 54.41 Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1)

MST1R 67.45 Macrophage stimulating 1 receptor (c-met-related tyrosine kinase)

37

CHAPTER 4 DISCUSSION

Periodontal disease is characterized by a chronic state of inflammation in a

susceptible host in response to the presence of bacteria and their byproducts. The

inflammatory state involves poorly organized hyperplastic gingiva, which then invades

the supporting periodontium. As a result, alveolar bone resorption occurs, leading to

both loss of attachment and the dentition. In periodontitis, gingival epithelial cells

potentially undergo a transition to a motile mesenchymal-like phenotype that possesses

the ability to invade the periodontium and create the resorption of alveolar bone

observed. While EMT is well characterized in other diseases including cancer, the

concept of gingival EMT and invasion contributing to bone loss in periodontal disease is

novel. Here our hypothesis is supported by the fact that the focal gingival hyperplasia

and local bone resorption in periodontal disease is at least histologically similar to the

focal bone loss seen in some osteosarcomas. We propose that gingival epithelial cells

have the potential to undergo an EMT-like process whereby by they would secrete

mediators that result in extra-cellular matrix (ECM) degradation and the recruitment of

osteoclasts allowing for both the invasion and destruction of the periodontal space and

focal alveolar bone resorption. Indeed the preliminary data generated in this study

continue to lend support to this hypothesis. Specifically, in this study, we evaluated

molecular and phenotypic similarities between classic EMT and TGF2 induced EMT in

primary human oral keratinocytes (HOK).

It is important to note that most EMT studies are performed in endothelial cell

populations and no studies evaluating EMT have been performed using human primary

ORAL epithelial cells. Thus, our initial experimental design was informed by historical

38

findings in murine endothelial cell studies (Medici et al. 2011). For instance, we did find

that treatment of HOK with TGF2 does induce morphological changes associated with

classical EMT. Terminally differentiated epithelial cells typically exhibit a cobblestone-

like morphology. We observed that following 3 days of treatment with TGF2, HOK

became elongated and become progressively spindle-shaped as is characteristic of

EMT (Figure 3-4). Whereby this morphological change is often associated with a

mesenchymal phenotype that accompanies increased motility and is required for tissue

invasion. Interestingly, the EMT array data supported this finding whereby several

mediators associated with ECM destruction, tissue invasion, cell motility, and

oncogenesis were upregulated, including MMP3, MMP9 and MST1R (Table 3-1).

Similarly, our Western blot analysis demonstrated a significant upregulation in the

mesenchymal marker SNAIL1 (Figure 3-3), while our PCR array analysis demonstrated

significant upregulation in the mesenchymal markers ITGA5 and FN1 (Table 3-1),

supporting the transition of HOK into a more mesenchymal-like state following TGF2

treatment.

There are several proteins involved in regulating EMT, whereby we only

evaluated three of these: Snail, E-cadherin and smooth muscle actin. These particular

markers of EMT were chosen as they are representative of changes in the early, mid-,

and later phases of EMT, respectively, but again whether the process of EMT in HOK is

similar to the classic process described is still unclear. For instance, at the molecular

level while we did demonstrate that the transcription factor Snail is up regulated

following treatment with TGF2, however at the protein level we do not see degradation

of the epithelial junction protein E-cadherin. In addition, HOK did not seem to express

39

smooth muscle actin under any circumstances evaluated. Thus, it is plausible that EMT

occurs in a non-conical fashion in HOK.

Snail1 and Snail family members are key transcriptional repressors that have been

found to mediate repression of epithelial markers and adherence proteins such as E-

cadherin. Down-regulation of E-cadherin disrupts cell-cell adhesion in classic EMT,

allowing for migration and invasion. Thus, while the up-regulation of Snail1 in HOK cells

indicates a similarity to classic EMT demonstrating potential for parallel pathways in

HOK, the lack of E-cadherin downregulation may suggest that Snail1 works on other

adhesion molecules in HOK or uses a different time course in its actions. We believe

that it is most likely that other Snail transcriptional repression targets are regulated in

HOK cells. Overall our data does suggest that TGF2 can at least in part can induce an

EMT-like pathway in HOK, but whether this is a contributor to disease pathogenesis in

periodontal disease is still to be elucidated.

We propose that in addition to the focal gingival hyperplasia, the development of

long junctional epithelium may also be a consequence of epithelial mesenchymal

transition. Indeed, the development of long junctional epithelium is a consequence of

the body healing via repair rather than regeneration. The American Academy of

Periodontology defines repair as “healing of a wound by tissue that does not fully

restore the architecture or function of the part.” Healing via repair involves the formation

of a thin layer of long junctional epithelium extending apically between the root surface

and the gingival connective tissue (Caton and Nyman 1980) and (Listgarten and

Rosenberg 1979). In contrast, regeneration is “reproduction or reconstitution of a lost or

injured part” and is characterized by de novo cementum formation, a functionally

40

oriented periodontal ligament, alveolar bone and gingiva (Caton and Nyman 1980) and

(Listgarten and Rosenberg 1979). The formation of long junctional epithelium

compromises the periodontal wound healing process and impairs the regenerative

process. In addition, we know that TGFβ can have contradictory roles in the healing and

regenerative processes depending on the other growth, tissue and immune factors

present (Fujio et al. 2016).

Unlike a strong, regenerated connective tissue attachment, long junctional

epithelium is easily penetrated and is susceptible to bacterial and plaque invasion. In

periodontal disease, chronic inflammation of the gingival epithelium leads to extension

of the junctional epithelium and formation of periodontal pockets. Thus, while TGF2 is

known to alter human oral epithelial cell biology, leading to phenotypes associated with

the development of long junctional epithelium, whether additional growth, tissue and

immune factors are also capable of inducing these phenotypes, protein and gene

changes has not been evaluated here. In addition, it is most likely that it is a

combination of factors, which ultimately lead to these transitions and changes.

Thus future directions from this study are to evaluate the long term effects of TGFβ2

on HOK cell biology as it relates to EMT as well as potential in vivo models to evaluate

the contribution of this particular induction of EMT to periodontal disease pathogenesis.

Long term goals of this research are to identify additional growth, tissue and immune

factors which can act instead of, synergistically and finally antagonistically with TGFβ2

to either induce or inhibit EMT respectively.

In summary, the cellular and molecular mechanisms involved in gingival invasion

in periodontal disease have never been considered in the light of EMT, and the signals

41

mediating periodontal invasion by gingival epithelial cells and those mediating alveolar

bone resorption have not been yet been identified. On the other hand, EMT in cancer is

well described, whereby data from this study support our hypothesis that the EMT

phenotype in periodontal disease, at least in part, resembles that observed in cancer.

Thus, we postulate that both result in cell transformation, motility, invasion into adjacent

tissues and osteolysis (esp. under the condition of metastasis to bone). Thus

confirmation of and expansion on our results could be transformative in the study of

periodontal disease whereby novel targets for the treatment of periodontal disease

could emerge.

42

LIST OF REFERENCES

Glossary of periodontal terms. 2012. http://members.perio.org/libraries/glossary?ssopc=1: American Academy of Periodontology; [accessed January 5, 2017].

Anderson GB, Caffesse RG, Nasjleti CE, Smith BA. 1991. Correlation of periodontal

probe penetration and degree of inflammation. Am J Dent. 4(4):177-183. Armitage GC. 1999. Development of a classification system for periodontal diseases

and conditions. Ann Periodontol. 4(1):1-6. Avery J. 2001. Oral development and histology. USA: Thieme. Barrallo-Gimeno A, Nieto MA. 2005. The snail genes as inducers of cell movement and

survival: Implications in development and cancer. Development. 132(14):3151-3161.

Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, Garcia De Herreros

A. 2000. The transcription factor snail is a repressor of e-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2(2):84-89.

Bernimoulin JP. 2003. Recent concepts in plaque formation. J Clin Periodontol. 30

Suppl 5:7-9. Bosshardt DD, Stadlinger B, Terheyden H. 2015. Cell-to-cell communication--

periodontal regeneration. Clin Oral Implants Res. 26(3):229-239. Burk U, Schubert J, Wellner U, Schmalhofer O, Vincan E, Spaderna S, Brabletz T.

2008. A reciprocal repression between zeb1 and members of the mir-200 family promotes emt and invasion in cancer cells. EMBO Rep. 9(6):582-589.

Califano JV. 2003. Position paper: Periodontal diseases of children and adolescents. J

Periodontol. 74(11):1696-1704. Caton J, Nyman S. 1980. Histometric evaluation of periodontal surgery. I. The modified

widman flap procedure. J Clin Periodontol. 7(3):212-223. Danilkovitch-Miagkova A, Duh FM, Kuzmin I, Angeloni D, Liu SL, Miller AD, Lerman MI.

2003. Hyaluronidase 2 negatively regulates ron receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus. Proc Natl Acad Sci U S A. 100(8):4580-4585.

De Craene B, Berx G. 2013. Regulatory networks defining emt during cancer initiation

and progression. Nat Rev Cancer. 13(2):97-110.

43

Ebersole JL, Dawson DR, 3rd, Morford LA, Peyyala R, Miller CS, Gonzalez OA. 2013. Periodontal disease immunology: 'Double indemnity' in protecting the host. Periodontol 2000. 62(1):163-202.

Eke PI, Dye BA, Wei L, Thornton-Evans GO, Genco RJ, Cdc Periodontal Disease

Surveillance workgroup: James Beck GDRP. 2012. Prevalence of periodontitis in adults in the united states: 2009 and 2010. J Dent Res. 91(10):914-920.

Fujio K, Komai T, Inoue M, Morita K, Okamura T, Yamamoto K. 2016. Revisiting the

regulatory roles of the tgf-beta family of cytokines. Autoimmun Rev. 15(9):917-922.

Gargiulo AW, Wentz FM, Orban B. 1961. Dimensions and relations of the dentogingival

junction in humans. Journal of Periodontology. 32(3):261-267. Grossi S. 2000. Smoking and stress: Common denominators for periodontal disease,

heart disease, and diabetes mellitus. Compend Contin Educ Dent Suppl. (30):31-39; quiz 66.

Grossi SG, Skrepcinski FB, DeCaro T, Robertson DC, Ho AW, Dunford RG, Genco RJ.

1997. Treatment of periodontal disease in diabetics reduces glycated hemoglobin. J Periodontol. 68(8):713-719.

Guo S, Dipietro LA. 2010. Factors affecting wound healing. J Dent Res. 89(3):219-229. Hiatt WH, Stallard RE, Butler ED, Badgett B. 1968. Repair following mucoperiosteal flap

surgery with full gingival retention. J Periodontol. 39(1):11-16. Hidalgo-Carcedo C, Hooper S, Chaudhry SI, Williamson P, Harrington K, Leitinger B,

Sahai E. 2011. Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by ddr1 and the cell polarity regulators par3 and par6. Nat Cell Biol. 13(1):49-58.

Huang RY, Guilford P, Thiery JP. 2012. Early events in cell adhesion and polarity during

epithelial-mesenchymal transition. J Cell Sci. 125(Pt 19):4417-4422. Jorda M, Olmeda D, Vinyals A, Valero E, Cubillo E, Llorens A, Cano A, Fabra A. 2005.

Upregulation of mmp-9 in mdck epithelial cell line in response to expression of the snail transcription factor. J Cell Sci. 118(Pt 15):3371-3385.

Kalluri R, Weinberg RA. 2009. The basics of epithelial-mesenchymal transition. J Clin

Invest. 119(6):1420-1428. Karring T, Nyman S, Gottlow J, Laurell L. 1993. Development of the biological concept

of guided tissue regeneration--animal and human studies. Periodontol 2000. 1:26-35.

44

Kornman KS, Page RC, Tonetti MS. 1997. The host response to the microbial challenge in periodontitis: Assembling the players. Periodontol 2000. 14:33-53.

Lamouille S, Xu J, Derynck R. 2014. Molecular mechanisms of epithelial-mesenchymal

transition. Nat Rev Mol Cell Biol. 15(3):178-196. Lang NP, Kinane DF, Lindhe J, Sanz M, Tonetti MS. 2008. Sixth european workshop on

periodontology of the european academy of periodontology at the charterhouse at ittingen, thurgau, switzerland. J Clin Periodontol. 35(8 Suppl):1-2.

Lang NP, Lindhe J. 2015. Clinical periodontology and implant dentistry. Lang NP,

Lindhe J, editors. USA: Wiley Blackwell. Lehembre F, Yilmaz M, Wicki A, Schomber T, Strittmatter K, Ziegler D, Kren A, Went P,

Derksen PW, Berns A et al. 2008. Ncam-induced focal adhesion assembly: A functional switch upon loss of e-cadherin. EMBO J. 27(19):2603-2615.

Lim J, Thiery JP. 2011. Alternative path to emt: Regulation of apicobasal polarity in

drosophila. Dev Cell. 21(6):983-984. Linghorne WJ, O'Connell DC. 1950. Studies in the regeneration and reattachment of

supporting structures of the teeth; soft tissue reattachment. J Dent Res. 29(4):419-428.

Listgarten MA, Rosenberg MM. 1979. Histological study of repair following new

attachment procedures in human periodontal lesions. J Periodontol. 50(7):333-344.

Loe H, Anerud A, Boysen H, Morrison E. 1986. Natural history of periodontal disease in

man. Rapid, moderate and no loss of attachment in sri lankan laborers 14 to 46 years of age. J Clin Periodontol. 13(5):431-445.

Loe H, Brown LJ. 1991. Early onset periodontitis in the united states of america. J

Periodontol. 62(10):608-616. Loewenthal B. 1991. Periodontal disease--a growing challenge. Dent Assist. 60(3):9-12. Maynard JG, Jr., Wilson RD. 1979. Physiologic dimensions of the periodontium

significant to the restorative dentist. J Periodontol. 50(4):170-174. Medici D, Potenta S, Kalluri R. 2011. Transforming growth factor-beta2 promotes snail-

mediated endothelial-mesenchymal transition through convergence of smad-dependent and smad-independent signalling. Biochem J. 437(3):515-520.

Melcher AH. 1976. On the repair potential of periodontal tissues. J Periodontol.

47(5):256-260.

45

Michalowicz BS, Aeppli D, Virag JG, Klump DG, Hinrichs JE, Segal NL, Bouchard TJ, Jr., Pihlstrom BL. 1991. Periodontal findings in adult twins. J Periodontol. 62(5):293-299.

Nanci A, Bosshardt DD. 2006. Structure of periodontal tissues in health and disease.

Periodontol 2000. 40:11-28. Oshrain HI, Salkind A, Mandel ID. 1971. An histologic comparison of supra and

subgingival plaque and calculus. J Periodontol. 42(1):31-33. Page RC, Schroeder HE. 1976. Pathogenesis of inflammatory periodontal disease. A

summary of current work. Lab Invest. 34(3):235-249. Peinado H, Olmeda D, Cano A. 2007. Snail, zeb and bhlh factors in tumour progression:

An alliance against the epithelial phenotype? Nat Rev Cancer. 7(6):415-428. Perera KA, Tonge CH. 1981. Metabolic turnover of collagen in the mouse molar

periodontal ligament during tooth eruption. J Anat. 133(Pt 3):359-370. Polimeni G, Xiropaidis AV, Wikesjo UM. 2006. Biology and principles of periodontal

wound healing/regeneration. Periodontol 2000. 41:30-47. Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanell J, Pietras K, Virtanen I,

Philipson L, Leopold PL et al. 2009. A snail1-smad3/4 transcriptional repressor complex promotes tgf-beta mediated epithelial-mesenchymal transition. Nat Cell Biol. 11(8):943-950.

Waerhaug J. 1977. Subgingival plaque and loss of attachment in periodontosis as

evaluated on extracted teeth. J Periodontol. 48(3):125-130. Wheelock MJ, Shintani Y, Maeda M, Fukumoto Y, Johnson KR. 2008. Cadherin

switching. J Cell Sci. 121(Pt 6):727-735. Whiteman EL, Liu CJ, Fearon ER, Margolis B. 2008. The transcription factor snail

represses crumbs3 expression and disrupts apico-basal polarity complexes. Oncogene. 27(27):3875-3879.

Yilmaz M, Christofori G. 2009. Emt, the cytoskeleton, and cancer cell invasion. Cancer

Metastasis Rev. 28(1-2):15-33.

46

BIOGRAPHICAL SKETCH

Dr. Lindsey Pikos Rosati studied integrative biology at the University of Florida,

Gainesville, where she graduated in 2010. After which, she attended dental school at

the University of North Carolina, Chapel Hill, where she graduated in 2014. Dr. Rosati

completed her training in periodontology at the University of Florida in 2017.