the role of epithelial mesenchymal transition in
TRANSCRIPT
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
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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.
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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).
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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
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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.
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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
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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
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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
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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
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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).
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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
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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
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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
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primary human oral keratinocytes (hOK). In addition, we will look for markers of EMT in
a mouse model of PD.
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Figure 1-1. Protein, gene, and morphological markers associated with epithelial mesenchymal transition.
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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.
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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
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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.