ASSESSMENT OF PERIODONTAL REGENERATION IN 1-WALL DEFECTS WITH BIOLOGICAL FACTORS IN DOGS

By

CAMILLE MEDINA-CINTRON

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

2018

© 2018 Camille Medina Cintron

To my family who has been the pillars of love, support and guidance in my life

4

ACKNOWLEDGMENTS

I thank my parents for their unconditional love, support, and because they

instilled in me determination, enthusiasm, and passion to fulfill my goals. I am grateful to

Dr. Rodrigo Neiva for providing me the opportunity to become part of the Periodontics

program and mentoring me during this project. I am also grateful to Dr. Paulo Coelho

who was the creator of this project and led all the surgeries and data analysis. I thank

Dr. L Kesavalu, Dr. Roberta Pileggi, for being part of my committee and helping me

complete this thesis. Finally, and without hesitation I thank my Co-residents Gaby,

Christian and my significant other Frankie for all their motivation, encouragement and

for all the joyful moments we had during the past three years. Lastly, I would like to

express my gratitude to faculty of the Department of Periodontology for their guidance

and continued advancement of my education and clinical abilities.

5

TABLE OF CONTENTS

page

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

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

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

LIST OF ABBREVIATIONS ........................................................................................... 10

ABSTRACT ................................................................................................................... 12

CHAPTER

1 INTRODUCTION .................................................................................................... 14

Terms and Definitions ............................................................................................. 16 Biology of Periodontal Healing and Regeneration .................................................. 17 Guided Tissue Regeneration .................................................................................. 17

Clinical Approaches ................................................................................................ 19 Clinical Indications .................................................................................................. 21

Biological Modifiers and Growth Factors................................................................. 23 Enamel Matrix Derivative.................................................................................. 25

Platelet-Derived Growth Factor ........................................................................ 26 Bone Morphogenetic Proteins .......................................................................... 27

Platelet-Rich Protein/ Plasma-Rich Fibrin......................................................... 28 Transforming Growth Factor-ß ......................................................................... 29 Parathyroid Hormone ....................................................................................... 30

Fibroblast Growth Factor .................................................................................. 30 Insulin-Like Growth Factor ................................................................................ 30 Brain-Derived Neurotrophic Factor ................................................................... 31

Evolution in Periodontal Regeneration .................................................................... 33 Aim.......................................................................................................................... 33 Primary Outcome and Null Hypothesis ................................................................... 34 Secondary Outcomes ............................................................................................. 34

2 MATERIALS AND METHODS ................................................................................ 38

Canines ................................................................................................................... 38 Surgical Procedures ............................................................................................... 38

Pre-Clinical In Vivo Model ................................................................................ 38

CT and µCT 3D Reconstructions ...................................................................... 39 Histologic Processing and Histomorphometric Analyses .................................. 41

Statistical Analysis .................................................................................................. 42

6

3 RESULTS ............................................................................................................... 52

CT and µCT 3D Reconstructions ............................................................................ 52

Histomorphometric Analysis ................................................................................... 53

4 DISCUSSION ......................................................................................................... 64

LIST OF REFERENCES ............................................................................................... 69

BIOGRAPHICAL SKETCH ............................................................................................ 77

7

LIST OF TABLES

Table page 1-1 Secondary outcomes of the study. ..................................................................... 35

2-1 Pre-surgical and surgical medications. ............................................................... 44

2-2 Randomization scheme. A statistician performed the randomizations of the different testing groups across the one-wall-defects in the mandible ................. 44

2-3 List of products applied to the defects. ............................................................... 45

8

LIST OF FIGURES

Figure page 1-1 Phases of healing ............................................................................................... 35

1-2 Stages of periodontal wound healing .................................................................. 36

1-3 Mechanism for BDND ......................................................................................... 37

2-1 Study Design Timeline. ....................................................................................... 45

2-2 Schematic positions of the six surgically created 1-wall infrabony defects ......... 46

2-3 Surgical protocol for the test sites. ...................................................................... 47

2-4 Three-dimensional rendering of CT image sets .................................................. 48

2-5 Regions of interest .............................................................................................. 48

2-6 Representative images of bone formation through the different time points ....... 49

2-7 Three-dimensional rendering .............................................................................. 50

2-8 Bone height measurement at the center of the defect and adjacent to tooth ...... 51

3-1 Percent bone formation within defect quantified through CT reconstructions ..... 56

3-2 Percent bone height maintenance ...................................................................... 57

3-3 Histologic sections .............................................................................................. 58

3-4 Histologic high magnification micrographs depicting PDL .................................. 58

3-5 Histologic images of the areas of interest observed in the study. ....................... 59

3-6 Histomorphologic features utilized for cementum regeneration quantification .... 59

3-7 Mineralized Cementum ....................................................................................... 60

3-8 Cementum and Cementoid ................................................................................. 60

3-9 Histomorphologic features to classify PDL regeneration .................................... 61

3-10 PDL Regeneration .............................................................................................. 61

3-11 Functional PDL ................................................................................................... 62

3-12 Cementum regeneration and bone percentage .................................................. 62

9

3-13 Cementum + cementoid regeneration and mean percentages ........................... 63

3-14 Periodontal ligament regeneration ...................................................................... 63

10

LIST OF ABBREVIATIONS

AAP American Academy of Periodontology

ANOVA Analysis of Variance

β-TCP Beta-Tricalcium Phosphate

BDNF Brain-Derived Neurotrophic Factor

BMP Bone Morphogenetic Proteins

BMP-2 Bone Morphogenetic Proteins-2

CAL Clinical Attachment Level

CT Computerized Tomography

EMD

FDA

Emdogain

US Food and Drug Administration

FGFs

FGG

Fibroblast Growth Factors

Free Gingival Graft

GEM

GEE

Growth Factor Enhanced Matrix

Generalize Estimating Equations

GTR Guided Tissue Regeneration

HMW-HA High-Molecular Weight- Hyaluronic acid

IGFs

JE

Insulin-Like Growth Factors

Junctional Epithelium

L-PRF Leukocyte and Platelet-Rich Fibrin

µCT Micro-Computed Tomography

MWF Modified Widman Flap

OFD Open Flap Debridement

PD Probing Depth

PDGFs Platelet-Derived Growth Factors

11

PDL Periodontal Ligament

PRF Platelet-Rich Fibrin

PRP Platelet-Rich Proteins

PTH Parathyroid Hormone

rhBMP-2

ROI

Recombinant Human Bone Morphogenetic Protein-2

Region of Interest

SRP Scaling and Root Planning

TGF- ß Transforming Growth Factor-Beta

VEGF Vascular Endothelial Growth Factor

12

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

ASSESSMENT OF PERIODONTAL REGENERATION IN 1-WALL DEFECTS WITH

BIOLOGICAL FACTORS IN DOGS

By

Camille Medina-Cintron

May 2018

Chair: Rodrigo Neiva

Major: Dental Sciences–Periodontics

Regenerative surgical procedures are regarded as an appropriate method for

attempting to restore lost periodontal structure and functional attachment through the

regeneration of cementum, periodontal ligament, and alveolar bone. Periodontal

regeneration in infrabony defects has been successfully attempted with a variety of

different approaches and products. Multiple growth factors have been identified to

promote periodontal regeneration in contained defects, but there is still a lack of

evidence concerning bone regeneration in challenging one-wall periodontal defects.

This study was to histologically observe the regenerative effect of Brain-Derived

Neurotrophic Factor (BDNF) combined with collagen sponge and placed in one-wall

infrabony defects in canine models.

Contralateral “Critical size” periodontal defects were created in fourteen Beagle

dogs to simulate a clinical situation. A randomization scheme was utilized to fill the

defects with a combination of three different collagen/BDNF products and Straumann®

Emdogain. The control defect remained unfilled to allow for consistency. Computerized

tomography (CT) and microcomputed tomography (µCT) evaluations were conducted to

13

determine the bone regeneration in the defects. The animals were euthanized after 8

weeks, and block sections were obtained. Histomorphometrical and radiographic

analyses revealed the effects of the test treatment in periodontal regeneration through

statistical analysis.

The results indicated a trend of gradual bone volume increase for all groups

along with defect filling and partial volumetric regeneration. The groups containing

BDNF demonstrated regeneration of the entire periodontal tissue. These results

suggest that the use of BDNF in combination with a scaffold collagen sponge is an

effective treatment option to regenerate the periodontium.

14

CHAPTER 1 INTRODUCTION

Periodontitis and dental caries are the two principal dental diseases that affect

the human population at high prevalence rates worldwide. 1 Recent data indicate that a

prevalence of Periodontitis in adults in the United States estimates that 47.2%, or 64.7

million American adults have the mild, moderate, or severe form of the disease. 2

Around 10% of patients with periodontal disease progress into the severe

category.3 Hence, the prevention and treatment of periodontitis is of utmost importance,

particularly in the field of dentistry. The periodontium is a complex structure that must

remain in biologic harmony in order to maintain a healthy state. During the

inflammatory process of periodontal disease this harmony is interrupted, and

commensal bacteria begin to take advantage. Periodontitis is an aggressive pathology

of concern since it alters the integrity of the periodontal system, eventually involves the

destruction of the periodontal ligament and alveolar process, which when left untreated

can ultimately lead to tooth loss .4,5

The goal of periodontal therapy is to provide patients with a dentition that is

functionally healthy and pain free for the rest of their lives. The preservation of the

natural dentition may be achieved by reducing or controlling the tissue inflammation

induced by bacterial plaque and/or by correcting the defects that lead to bone

resorption. Nonetheless, in order to preserve the remaining dentition of patients with

periodontitis, the disease has to be contained. Clinical intervention via non-surgical

and/or surgical approach is necessary to reestablish healthy periodontal tissues.

Intervention in non-surgical periodontal therapy includes: oral hygiene and plaque

control, scaling and root planning (SRP), maintenance and adjunctive use of

15

chemotherapeutic agents. Periodontal phase 1 therapy may be sufficient to eliminate

the signs and symptoms of mild periodontitis. However, many cases or sites with

moderate to severe disease often continue to progress and show signs of inflammation

in spite of extensive the non-surgical approaches. In such cases, surgical treatment is

inevitable. 6 The various surgical approaches implemented in the secondary phase are

open flap debridement (OFD), resective flap surgery, mucogingival surgery and

regenerative surgery.7-9

During the last three decades there has been a shift in goals when treating

periodontal disease. Now the goal not only aims at containing the disease but also

regenerating the supporting tissues that were lost as a consequence of inflammatory

disease progression. 6,10 More than half a century ago resective surgery via open flap

debridement and osseous re-contouring of diseased bone was the primary approach,

but often resulted in un-esthetic and uncomfortable root exposure. As wound healing

and etiology became better understood the field’s philosophy advanced as well.

Correction was not the only objective, but also, via regenerative therapy, to restore what

was lost. 11- 13 The current techniques for regeneration encompass the utilization

of a wide variety of surgical approaches: OFD7- 9, the use of bone grafting materials,

barrier membranes,14 the use of biologic modifiers, other osteoconductive/inductive

materials or protein mixtures, exogenous growth factors, cell-based technologies, and

genes from recombinant technology. Among these approaches, periodontal tissues

regeneration has achieved a significant success following the use of guided tissue

regeneration

(GTR) strategies.

16

Terms and Definitions

Typically, in resective therapy, wound healing occurs by tissue repair, which is

characterized by the formation of a long junctional epithelium adjacent to the previously

diseased root surface. According to the AAP Glossary of Periodontal Terms, repair is

defined as the “healing of a wound by tissue that does not fully restore the architecture

or function of the parts”. Regeneration is a dynamic process that aims to recreate the

tissues to their original structure and function.15 Periodontal regeneration involves a

“reconstitution of the PDL along with new formation of alveolar bone and

cementum”; in other words, it involves a return to the original, completely functional

periodontium. 16,17 The majority of periodontal wound healing involves repair, although

regeneration is desired.18

When periodontal regeneration is accomplished, new attachment is obtained.

From a histological perspective, it is defined as: “the union of epithelium or

connective tissue with a root surface that has been deprived of its original attachment

apparatus. This new attachment may be epithelial adhesion and/or connective tissue

adaptation or attachment and may include new cementum.” 16

Bone fill is another concept described by the AAP that at times has been

mistakenly interchanged with regeneration of the periodontium. It is the clinical

restoration of bone tissue in a treated periodontal defect.1 Bone fill may be assessed by

bone sounding, subtraction radiography or by open probing which would involve a

reentry to the surgical site. In regeneration, histologic analysis is the sole reliable

method for its detection, as it allows for the observation of the formation of new

cementum, PDL fibers, and bone. It is important to note that the occurrence of bone is

17

not equivalent to regeneration. Previous studies have shown that it is possible to have

bone fill without regeneration of the tooth attachment apparatus.19

Biology of Periodontal Healing and Regeneration

Healing of periodontal surgical wounds differs from other wounds since it has

several unique features. It involves a higher degree of complexity and multiple factors

such as the presence of specialized cell types (cementoblast, osteoblasts, fibroblasts

ie.) and attachment complexes (i.e., Junctional Epithelium (JE)), diverse microbial flora,

and avascular tooth surfaces that complicate the process of periodontal

regeneration.20,21 A better understanding of these special factors involved in the

periodontal wound healing process would allow for more predictable treatment

outcomes following regeneration procedures.

For periodontal regeneration to occur, formation of a functional epithelial seal,

insertion of new connective tissue fibers into the root, reformation of a new acellular

cementum on the tooth surface, and restoration of alveolar bone are required.22 The

complexity in this treatment approach involves recruiting locally-derived progenitor cells

that can differentiate into PDL cells, mineral-forming cementoblasts, or bone-forming

osteoblasts. 23

Guided Tissue Regeneration

Prior to any scientific research proving evidence of periodontal regeneration,

Melcher in 1976 suggested that there are four distinct connective tissue compartments

in the periodontium: the gingival corium, PDL, cementum, and bone cells.18 He

described that selective cellular repopulation of defects is the primordial way to enhance

reformation of these original parts. In other words, the cells in each compartment

represented various phenotypes capable of repopulating and influencing the

18

regenerative response obtained. Periodontal regeneration is acknowledged as resulting

from Melcher’s work in which cell exclusion was proposed as the most forward-thinking

concept. 18

GTR was first developed in the early 1980s by Nyman and Karring as they

staged the first in vivo study of this novel concept, although the term itself was later

coined by Gottlow et al. in 1986.24-26 Nyman’s group performed GTR on a mandibular

incisor using a Millipore filter, and after three months of healing, histologic analysis

revealed the formation of new cementum with inserting collagen fibers on the previously

denuded root surface.27 These researchers conducted a series of studies involving the

formation of new connective tissue attachment that led to the innovative concept of

employing barrier membranes in guided tissue regeneration procedures.24-27,

After establishing GTR as the periodontal therapeutic modality that aims to fully

restore the supporting tissues of the periodontium, the concept accelerated and gained

popularity. It is based on the fact that particular cells contribute to the formation of the

different specific tissues. This procedure asserts that by isolating the periradicular bone

wound from faster proliferating epithelial cells and other connective tissue cells, it grants

the opportunity for the periodontal ligament to repopulate the blood coagulum that forms

between the alveolar bone and root.

Regeneration is accomplished by the migration, proliferation, and differentiation

of residual stem cells that are present in the PDL. In order to achieve this cascade, the

epithelial, CT, and bone cells need to be excluded from the site of interest.18 Numerous

studies have revealed that neither bone nor gingival cells induce the formation of new

CT attachment. These studies were done utilizing transplanted disease-affected root

19

surfaces into bone and most cases resulted in ankylosis and root resorption. The

authors concluded that bone and connective tissue cells lacked the capacity to induce

regeneration24,28 The current understanding seems to suggest that their origin

may be due to both bone and PDL cells, with the majority of evidence revealing PDL

cells as the major source.29

Clinical Approaches

As with most relatively new concepts in the field of Periodontics, the way in which

GTR is performed and understood remains dynamic and ever evolving. Even prior to

obtaining histological validation, clinicians have been utilizing GTR with promising

clinical results. Ellegaard et al. 1974 attempted to retard the apical migration of the

epithelium by using free gingival grafts (FGG) with autogenous bone over intrabony

defects.30 Another pioneer of the epithelial exclusion concept was Prichard,31 who in

1977 performed a surgical technique which excised all the soft tissue in the

interproximal region, leaving bare bone. Later surgical reentry and volumetric analyses

revealed that half of the defects had significant decrease in defect volume. The

utilization of a barrier membrane soon became the “gold standard” treatment approach

in GTR due to both clinical and histological validation. Subsequently, the use of different

types of barrier materials for cellular occlusion evolved. Early studies utilizing non-

absorbable material such as Millipore filter27,32and Polytetrafluoroethylene

(ePTFE)

membranes33,34 in intrabony defects proved to have noticeable regenerative

results. Since non-absorbable material requires a second surgery to retrieve the

membrane, these have commonly been substituted with biodegradable membranes.35

Additional rationale behind the use of absorbable materials is that they have shown to

20

be more biocompatible with soft tissues and have led to less membrane exposure and

fewer postsurgical infections.36 Research behind the use of polylactic acid and collagen

membranes have reported clinical improvements similar to those achieved with non-

resorbable membranes, including ePTFE.37-39

Collagen membranes have been shown to be very effective in promoting

periodontal regeneration. Collagen is the main macromolecule in the body and the

primary protein in alveolar bone and connective tissue, which provides structural

support for the tissues. 40 Another positive property of collagen is that it helps with the

formation and stabilization of a clot by stimulating platelet attachment and fibrin linkage,

not to mention its chemotactic property for PDL fibroblasts.41 Similar to the situation with

non-resorbable membranes, the addition of bone grafting materials with collagen

membranes appears to improve the clinical results for periodontal defects.42 Due to the

ideal structural properties of collagen, several applications have been developed for

regeneration of the periodontal tissues using collagen devices as scaffolds for tissue

engineering. Studies comparing the effectiveness of resorbable membranes vs.

nonresorbable membranes for GTR showed no clinically significant difference among

the

groups in furcations and intrabony defects.37,43

Many of the studies surrounding GTR have successfully demonstrated the

implementation of bone graft material together with a membrane. Combination therapy

for periodontal regeneration is the use of bone replacement grafts (autografts, allografts,

xenografts, and alloplasts) together with a membrane for treating defects.44 The ultimate

goal is that after serving their purpose, the graft materials are completely replaced by a

21

patient’s own bone. 45,46 Reynolds et al. 44 conducted a systematic review evaluating the

efficacy of bone replacement grafts. Their study concluded that utilizing bone

replacement grafts led to better clinical outcomes in periodontal osseous defects

compared to surgical debridement alone. Yet, there was controversy regarding the

effectiveness of performing GTR procedures while only using a graft, only a membrane,

or a combination of both a graft and a membrane. Various research studies have

shown no significant differences in gain of clinical attachment after GTR utilizing graft

materials with or without a membrane. Meanwhile, other studies have demonstrated

data supporting a significant clinical difference .47 In a systematic review of the

literature by Murphy and Gunsolley in 2003, it was proven that there is a significant

difference between GTR with a graft and membrane vs. GTR with just a graft. The

membrane groups showed improved clinical results for furcation defects. However, for

infrabony defects, there were no significant differences between the groups.48

Clinical Indications

In order to obtain successful outcomes in GTR, careful assessment and selection

of each clinical situation needs to be made. Clinicians must discriminately evaluate the

case for the correct treatment option, based on select defect criteria, to increase the

treatment success. Periodontal bone loss results in different types of defects. These

can be classified based on the morphology as suprabony, intrabony and craters. 49

Intrabony pocketing involves vertical bone resorption and may exhibit various forms in

relation to the affected tooth. It is dependent on location and number of osseous walls

remaining. Rateitschack50 described intrabony defects as 1,2,3- walled and crater

defects. Three-wall defects are bordered by one tooth surface and three osseous

surfaces. Two-wall defects, also known as interdental craters, are bordered by two tooth

22

surfaces and two osseous surfaces. One-wall defects are bordered by two tooth

surfaces, one osseous surface, and a soft tissue component. Crater, or cup defects, are

a combined form of a pocket surrounded by several surfaces of a tooth and several of

bone, in which the defect surrounds the tooth. Zero-walled defects, also known as

circumferential defects, occur when no osseous surfaces remain. 50 One- wall and zero-

walled defects are commonly found in diseased periodontal dentition, and since they

have little to no regenerative potential, they tend to not be treated via the GTR

approach. Due to the limited treatment options surrounding one and zero-wall defects,

there is currently a great deal of research underway, attempting to develop surgical

techniques and dental biomaterials to be used in restoring such defects.

Biologically and clinically, the greater the number of walls remaining, the better

the prognosis. An explanation of the healing patterns has been related to the fact that

the number of bony walls of the defect will influence, among other factors, the source of

osteoprogenitor cells. With the presence of more walls, the graft is better held in place

and the membrane is prevented from collapsing over the defect. 51 Another clinically

important factor regarding the increased number of walls is the exponential increase in

blood supply. Blood supply is a key element to regeneration, as it provides nutrients and

transports the mesenchymal cells required in returning to a healthier periodontium.

An ideal osseous defect for regeneration was described by Tonetti, Cortellini, et

al. Their findings suggested that the narrower and deeper the defect, the greater the

regenerative potential and prognosis. Conversely, the shallower and wider the defect,

the lower the amount of regeneration expected.51This study, among others, is

commonly used by clinicians to help designate which defects may most successfully

23

take advantage of GTR procedures, thus resulting in the usage of GTR on 2 or 3-walled

defects and not on 1-walled or 0- walled defects.

Several studies have evaluated the use of GTR techniques in the treatment of

furcation defects. 48,52 Most studies reported favorable results in Class II mandibular

furcations since they possess the greatest regenerative potential due to adequate

defect depth and ample blood supply from the remaining furcal bone. 53,54 On the other

hand, Class I and Class III furcations are not ideal regenerative sites. Class I furcations

are too shallow to adequately hold the graft material in place, thus preventing the graft

containment. Class III furcations lack an adequate blood supply due to the lack of

surrounding bone.55 A series of studies performed by Pontoriero et al. proved that

mesial and distal Class II furcations and all Class III furcations had poor responses to

regenerative therapy.53,55

Other clinical applications of GTR include the treatment of recession,

dehiscence, and fenestration defects. According to numerous studies, GTR has

provided significant improvements in probing depths and clinical attachment levels,

along with evidence of regeneration of a new periodontal attachment in these types of

defects.56,57

Biological Modifiers and Growth Factors

For periodontal regeneration to occur, the appropriate cells, signals, blood

supply, and scaffold need to orchestrate together in order to correct the defect site. All

these elements play a role in the healing process and in the reconstruction of the lost

tissue. Endogenous growth factors perform an indispensable role by modulating the

cellular activity and providing stimuli to the necessary cells to differentiate and produce

24

matrix for the developing tissue. Vascularization and scaffold structure are also

essential elements to help facilitate the process.58

Growth Factors are biologically diffusible active polypeptides that function as the

key regulators for coordinating major cellular biological events in tissue regeneration.

They affect the proliferation, chemotaxis and differentiation of cells from epithelium,

bone, and connective tissue and are released/activated when cell division is needed. If

mesenchymal cells from the PDL or perivascular region of the bone proliferate and

colonize the root surface, regeneration occurs.59 These proteins express their action by

binding in concert to specific cell-surface receptors to form intricate molecular

arrangements in cells involved in the periodontium (osteoblasts, cementoblasts, and

periodontal ligament fibroblasts). Growth factors are believed to have the potential to

accelerate the healing process and, therefore, enhance regeneration in challenging

clinical scenarios.60 For that reason, the focus in research has been to discover how to

harness the body’s natural growth factors to accelerate and direct the healing event into

one that will lead to predictable and successful periodontal regeneration.

In order to understand how growth factors function in normal wound healing, one

needs to have a full understanding of the process and the its intricate phases. All wound

healing commences with a coagulation and hemostasis formation. The clot has two

main functions: to temporarily protect the denuded tissues and serve as a provisional

matrix for cell migration. Beyond the blood clot formation, the process has been divided

into stages: an inflammatory stage that leads to proliferation of the fibroblast, followed

by granulation, and finalizing with a maturation phase.61 (Figure 1-1)62

25

Optimal periodontal healing requires different processes in a sequential manner.

After the initial coagulation phase, inflammatory reaction, and granulation tissue

formation events, progenitor cells involved in tissue regeneration are locally recruited

and mediate the availability of growth factors (Figure 1-3). 63 Neovascularization of the

tissues is of the utmost importance as it is an essential step in providing nutrients to the

wound and helping maintain the granulation bed.

Platelet-derived growth factors (PDGFs), Vascular endothelial growth factor

(VEGF), Insulin-like growth factors (IGFs), Fibroblast growth factors (FGFs), Parathyroid

hormone (PTH), Transforming growth factor- ß (TGF- ß), and Bone morphogenetic

proteins (BMPs) are some of the best characterized growth factors, all of which have

been extensively reviewed via animal model studies. The efficacy of exogenous growth

factors to regenerate the periodontium has been reviewed exhaustively both for clinical

and for pre-clinical applications. All these growth factors exert their biologic effect on

mitogenesis, chemotaxis, differentiation, and metabolism.

Enamel Matrix Derivative

Enamel matrix proteins are secreted by Hertwig’s epithelial root sheath during

the development of the root and helps stimulate the formation of acellular cementum.

This ultimately stimulates the formation of PDL fibers and novel alveolar bone. The

commercially available product is Emdogain®, which consists of enamel matrix proteins

derived from porcine tooth buds. EMD is composed of enamel matrix proteins (primarily

amelogenin), water, and a propylene glycol alginate (PGA carrier). Emdogain® is

intended as an adjunct to periodontal surgery as a topical application onto exposed root

surfaces. Studies have shown that Emdogain® induces PDL fibroblast proliferation and

growth while inhibiting epithelial cell proliferation, and it has been suggested that a cell

26

occlusive membrane is not required when utilizing EMD. Emdogain® also stimulates the

production of osteoprotegrin, which promotes osteoblastic activity while inhibiting

osteoclastogenes. Also has a chemotactic effect on endothelial cells, improving wound

healing. 64 -66 The use of EMD has been FDA approved for treatment of intrabony

defects and to optimize tissue height in esthetics zones.67 Earlier split mouth design

studies performed by Heijil et al. 68 demonstrated that modified Widman flap (MWF) and

EMD had significantly greater radiographic bone fill and CAL gain in comparison to

MWF only. Supporting these findings, other human clinical trials on the use of EMD for

periodontal defects compared with OFD showed significantly greater improvements

than OFD at 10 years (Sculean et al. 2008).69 Previous studies by Grusovin and

Esposito et al. in 2009 in which a clinical trial result suggested EMD provided no

additional benefits in the treatment of deep and wide infrabony defects. 70 Although

extensive research has been done on the clinical applications of EMD on periodontal

defects, conflicting results have been reported in the literature.

Platelet-Derived Growth Factor

PDGF is an endogenous growth factor stored in the alpha granules which is

extravasated upon injury and hemorrhage. After platelet exposure, PDGF directly

recruits and activates neutrophils and monocytes then subsequently serves to activate

mesenchymal cells essential to the proliferative phase, including endothelial cells and

smooth muscle cells. In the maturation phases of wound healing, PDGF also stimulates

secretion of the collagenase and extracellular matrix by associated fibroblasts. 71

GEM 21S (growth factor enhanced matrix) is the FDA commercially approved

form of PDGF. This enhanced matrix combines the bioactive protein (highly purified

recombinant platelet derived growth factor) with beta-tricalcium phosphate (β-TCP), an

27

osteoconductive scaffold. After placement, PDGF is released from the β-TCP matrix into

the neighboring environment. PDGF then binds to cell surface receptors to stimulate a

cascade of intracellular signaling pathways. The resulting effect is chemotaxis and

proliferation of osteoblasts, PDL cells, and cementoblasts, as well as stimulation of

angiogenesis. This in turn leads to increased matrix synthesis and the formation of

novel alveolar bone, PDL, and cementum. 72 Nevins et al. in 2005 completed a

multicenter human clinical trial on 180 patients with an intrabony defect utilizing b-TCP

plus rhPDGF at different concentrations (0.3 mg/mL rhPDGF-BB versus 1.0 mg/mL

rhPDGF-BB) and a control group with b-TCP only. His findings reported that using 0.3

mg/mL of rhPDGF-BB resulted in greater bone gain and defect fill at 6 months, as well

as faster CAL gain than controls. In additional to several other studies, Nevins et al.

demonstrated that PDGF possesses mitogenic and chemotactic effects on osteoblasts,

cementoblasts and PDL cells. 73

Bone Morphogenetic Proteins

BMPs are members of the transforming growth factor family first described by

Urist after observing ectopic bone formation from implanted devitalized cadaver bone in

a rat model. 74 Subsequent research led to the purification of bone and the isolation and

cloning of several BMPs using recombinant technology. BMPs have been shown to be

osteoinductive proteins that stimulate bone formation from mesenchymal cells in several

animal models of clinically relevant bone defects.75 Recombinant human bone

morphogenetic protein-2 (rhBMP-2) is the most actively studied of the recombinant

human proteins.76

BMPs have an anabolic effect on periodontal tissues through stimulation of

osteoblastic differentiation in human PDL cells.77 Some studies suggested that BMP-2

28

could help induce periodontal regeneration; however this was not proven, as their

results found adverse healing events, such as ankylosis and root resorption, leading to

a questionable treatment option for GTR.78,79 BMP-2 is FDA approved and commercially

available as an alternative for autogenous bone grafting during sinus augmentation

procedures or alveolar socket grafting. 76

Platelet-Rich Protein/ Plasma-Rich Fibrin

The use of platelet concentrates to improve healing has been an ongoing topic of

research over the last decade. Platelets contain quantities of high quality desirable

growth factors, such as PDGF, TGF- ß, and VEGF. These growth factors are able to

stimulate cell proliferation, matrix remodeling, and angiogenesis. 80 All of these factors

are highly beneficial to valued regenerative therapy.

PRP has been defined as an autologous concentration of human platelets, three

to five times greater than physiologic concentration of thrombocytes in the whole blood.

81 PRP, consisting of uncoagulated blood, centrifuged to isolate and concentrate human

platelets and leukocytes within a fibrin scaffold, remains a source of growth factors.82

Preparation protocol requires collection of blood with anticoagulant and centrifugation in

two steps. This allows the separation of the blood into layers. Finally, the obtained

platelet concentrate is applied to the surgical site, together with thrombin and/or calcium

chloride, to elicit platelet activation and fibrin polymerization. 80 The use of PRP in dental

surgery has become quite popular due to its claim to enrich the natural blood clot and

hasten wound healing and bone regeneration. Piemontese et al., 2008

attempted

regeneration in 60 human intrabony defects using PRP plus DFDBA versus DFDBA

alone. The results demonstrated greater PD reductions and CAL gains at 12 months,

yet no difference in hard-tissue outcomes. 83 However, other studies suggest the use of

29

PRP failed to improve the results obtained with GTR and bone substitutes. Therefore,

no conclusions can be drawn regarding the bone regenerative effect of PRP, based on

the available current literature.84,85

PRF is another platelet concentrate that was more recently developed in France

by Choukroun et al. Similar to PRP, PRF contains high amounts of bioactive growth

factors to enhance wound healing through increased chemotaxis, proliferation,

differentiation, and angiogenesis.86 It involves a simpler technique, eliminating the need

for an anticoagulant or jellifying agent. Natural blood is centrifuged sans the addition of

additives, allowing a natural coagulation process. This produces a leukocyte and

platelet-rich fibrin (L-PRF) clot highly concentrated in platelets and leucocytes. 80 The

incorporation of PRF in GTR procedures is under significant review. Sharma et al.

conducted a randomized controlled clinical trial for the treatment of 3-wall intrabony

defects utilizing PRF and reported a statistically significant improvement in pocket depth

reduction and bone fill in test groups compared with controls. Although clinical

improvements were noticed, actual regeneration, as in most of the human clinical trials,

could not be assessed because no histology was obtained.87

Unlike the PRPs, L-PRF does not dissolve quickly after application. Other

benefits of utilizing PRF include its ability to serve as a dense autologous membrane, it

employs a simpler process to prepare, low cost, and easy handling for clinicians. The

topical use of platelet concentrates is still a relatively recent discovery and its efficacy

remains controversial.

Transforming Growth Factor-ß

The role of TGF- ß as mediator in wound healing has been evaluated and has

repeatedly demonstrated the ability to enhance wound strength, connective tissue

30

matrix density, synthesis of matrix protein, and migration of macrophages and

fibroblasts into the healing site. It has been shown to increase the differentiation of

osteoblasts, osteoblast precursors, and extracellular matrix formation, such as type I

collagen. Furthermore TGF- ß stimulates the proliferation of gingival fibroblastic cells,

the formation of blood vessels, and the formation of granulation tissue during the

healing of periodontal tissue.88

Parathyroid Hormone

PTH is an endogenous hormone with both anabolic and catabolic properties in

bone. Animal experiments have documented an anabolic effect on both cancellous and

cortical bone. Its application has been applied to the treatment of osteoporosis, although

current research has focused on investigating PTH function in periodontal

applications.89 Bashutski et al. completed a randomized human clinical trial on 40

patients with intrabony defects who were supplemented with daily injections of 20mg

PTH in conjunction with periodontal surgery. His results revealed that PTH had greater

infrabony defect resolution, CAL gain, and PD reduction.90

Fibroblast Growth Factor

Preclinical evidence suggests that FGF-2 may promote periodontal regeneration

since it displays potent angiogenic activity and mitogenic ability on mesenchymal cells.

Several animal model studies of artificial defects support its role as a regenerative

agent. Kitamura in 2008 carried a multicenter clinical trial showing greater increase in

alveolar bone height when treating infrabony defects.91-93

Insulin-Like Growth Factor

IGF-1 is a polypeptide growth factor that helps regulate DNA and protein

synthesis in bone matrix formation. It is shown to increase osteoblast mitogenesis in

31

cultured bone cells when combined with other growth factors. In-vivo, studies have

demonstrated IGF-1 acts as a local messenger between cells in the wound

environment.94 Although IGF-1 alone has minimal effects on wound healing, when

combined with PDGF they interact synergistically to enhance regeneration of the

periodontal attachment apparatus. 95

Brain-Derived Neurotrophic Factor

Many research efforts have focused on developing new products and techniques

to accomplish regeneration of the periodontal tissues. Recently, experiments have

been conducted with different growth factors such as BDNF to understand its function

and modulation of the wound healing process in periodontal therapy. BDNF is a growth

factor essential for the development and function of the central and peripheral nervous

system. 96 It is involved in the survival and differentiation of neurons by directly affecting

them to prevent apoptosis. They form part of the neurotrophins family and are the most

abundant in the CNS. 97,98 Imbalances in BDNF have been reported to provoke

neurological and psychiatric disorders, since the expression level of BDNF mRNA

significantly decreases under stress induction. 99 It is not only expressed in the brain,

but also evidence suggests it is found at a tissue level in tooth germ, bone, cartilage,

heart, spleen, placenta, prostate, and kidney. BDNF has also been allocated at the

cellular level in dental mesenchymal cells such as osteoblasts, cementoblasts, PDL,

and endothelial cells.100, 101 Due to the multiple roles played by BDNF in several of the

tissues, it has been suggested as a candidate for regeneration in different complex

clinical scenarios.

Recent studies have suggested that the expression of different bone and

cementum related genes is upregulated by activation of the MAPK-ERK1/2 signaling

32

pathway. Kajiya et al. (2008) describes the molecular mechanism by which BDNF

regulates the functions of cementoblasts. BDNF regulates the mRNA expression of

bone/cementum-related proteins such as alkaline phosphatase (ALP), osteopontin

(OPN), and BMP-2 in cultures of immortalized human cementoblast-like cells. This

occurs when BDNF molecules bind to tyrosine kinase B (trkB) receptors on the surface

of a cementoblast, which proceeds to activate the ERK1/2-Elk-1 signaling pathway via

phosphorylation of the c-Raf Protein (Figure1-3). Kajiya’s findings prove for the first

time that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the

BDNFinduced mRNA expression of specific growth factors that influence the

differentiation of osteoblast and cementoblast like cells. Activation of these specific

growth factors also leads to gingival cell apoptosis, supporting periodontal regeneration.

BDNF stimulates the expression of VEGF and Tenascin X which enhances proliferation

in endothelial cells thus supporting angiogenesis.102,103

Investigations concerning BDNF’s use in periodontal tissue regeneration have

been performed in a non-human primate model. Jimbo et al. (2014) used alginate

material to induce a chronic periodontitis furcation-like defect situation, and attempted

the regeneration with BDNF at different concentrations, and HA scaffold material:

{Group A: BDNF (500 mg/ml) in high-molecular weight- hyaluronic acid (HMW-HA;

Group B: BDNF (50 mg/ml) in HMW-HA; Group C: HMW-HA acid only; Group D: empty

defect; Group E: BDNF (500 mg/ml) in saline}. Only Group A, the higher concentration

product, regenerated the entire periodontal tissue, i.e., alveolar bone, cementum and

periodontal ligament. This demonstrated that BDNF is solely dose dependent.

Although Group E was given the same concentration as Group A, without the presence

33

of a proper carrier, the effect of BDNF cannot be exerted and be altered by other growth

factors existing in the bodily fluid. 104, 105 This, among other studies performed in the

animal model, suggests a promising future for the usage of BDNF in periodontal

defects.

Evolution in Periodontal Regeneration

There are hundreds of growth factor proteins found in the human body. Those

discussed above are just a few of the main growth factors with research-based literature

to support their use and efficacy. Regenerative medicine and tissue-engineering

innovations have greatly advanced periodontology over the past decade. With such a

promising field, and so little information and research available, the potential for

significant improvements and advances is virtually unlimited. In vitro studies with

growth factors have successfully demonstrated the potential for regeneration of

mesenchymal cells of the periodontium. Yet, a void remains between in vitro and human

experiments. There is a need for further research to answer questions regarding long

standing growth factors delivered to the periodontium without quick dilution. Currently,

growth factor delivery mechanisms are one of the main challenges encountered in GTR.

The need for further study is profound and hopefully the next decade fulfills the promise

that growth factors present.

“The future of periodontal reconstruction techniques will depend on the

emergence of new products, which will lead to a predictable positive outcome when

used in proper combination in select defects.”- Carranza 2006 106

Aim

The aim of the present study was to evaluate if collagen/BDNF combinations

work better when compared with Straumann® Emdogain.

34

Primary Outcome and Null Hypothesis

The primary outcome is that the application of BDNF combinations to one-wall

critical size periodontal defects, irrespective of type of collagen sponge used for

application, demonstrates superior histologic cementum regeneration when compared

to Emdogain, and no treatment. The null hypothesis is that the application of BDNF

and collagen sponge to one-wall critical size periodontal defects, irrespective of type of

collagen sponge used for application, does not demonstrate superior histologic

cementum regeneration when compared to Emdogain, and no treatment.

Secondary Outcomes

The secondary outcome of the study is to investigate periodontal regeneration

through μCT Examination, histological and histometric analyses on the parameters

listed on Table 1-1.

35

Table 1-1. Secondary outcomes of the study.

Secondary Outcome Variables

μCT Examination- Bone Formation

Histological Analyses Histometric Analyses

Cementum coverage/ morphology Defect Height

Residual biomaterial and associated tissue reaction(s)

Connective tissue attachment

Root resorption / ankylosis Root resorption / ankylosis

Bone regeneration (lamellar and woven bone)

Inflammatory Status

Periodontal Ligament (PDL) orientation/density

Epithelial attachment

Vascularization Bone regeneration (height)

Exuberant bone formation

Figure 1-1. Phases of healing. Adapted from 62

36

Figure 1-2.. Stages of periodontal wound healing. Adapted from 63

37

Figure 1-3. Mechanism for BDND. Adapted from 102

38

CHAPTER 2 MATERIALS AND METHODS

Canines

The study protocol was approved by Institutional Animal Care and Use NYU

Committee (protocol #: 16-DS-002). Following approval of the ethics committee for

animal studies, a total of fourteen healthy beagle dogs (Canis Lupus Familiaris,

approximately 1.5 years of age and an average body weight of 12-15kg) were acquired

and acclimatized to the animal facility for a week prior to the first surgical intervention.

For identification purposes, each dog received a serial number marked with ear

tattoos. The design of the study is presented in Figure 2-1. All dogs were fed with

commercially available canine diet and filtered tap water. A soft diet was applied during

the entire experiment.

Surgical Procedures

Pre-Clinical In Vivo Model

Surgical protocol is described with surgical photos in Figure 2-3. For all surgical

interventions, the animals were premedicated with a cocktail mix of Ketamine

/Midazolam by intramuscular injection (IM), and subsequently received general

anesthesia with Isoflurane IM (Table 2-1).

Bilateral atraumatic extractions of mandibular first molar and third premolar was

performed. Oral prophylaxis was implemented in conjunction with the extractions. The

control and experimental groups were assigned according to a randomization scheme,

resulting in a balanced study design (Table 2-2). Eight weeks after healing, buccal and

lingual mucoperiosteal flaps were elevated to create unilateral, critical-size, “box-type”

(4 x 5 mm; width x height) one-wall intrabony defects at the mesial aspect of the second

39

molar and distal aspect of second and fourth pre-molar teeth in the right and left

mandibular quadrants using a 701/703 drill. Schematic positions of the defects in the

mandible of the beagle dog are presented in Figure2-2. Curettes were used for root

planning the root surface and to remove the PDL and root cementum. Root

conditioning was performed with 14-18% EDTA (pH 7.0) for 2 min for all the defects

except for the Emdogain receiving sites. After pre-conditioning the root, thorough

rinsing with sterile saline was done for all defects to avoid contamination of the root

surface with saliva or blood after last rinse. Following the preparation of the surgical

site, the defects were assigned to a group and treated with the BDNF combinations.

Straumann® Emdogain and Sham group, which remained unfilled (Table 2-3).

Each defect site was filled to the level of the alveolar crest with the different

investigational products covering the denuded root surface. The same surgical defect

was created for the control sites without any additional material placement. In all cases,

the mucogingival flaps were sutured using a non-absorbable suture (PROLENE®

Polypropylene Suture, Ethicon Inc, Somerville, NJ, USA). After surgery, dogs were

subjected to a soft diet in order to prevent incision line opening. Post-operative

medication included antibiotics (penicillin, 20,000 UI/kg) and anti-inflammatory

(ketoprophen, 1 mL/5 kg) for a period of 48 hours. Euthanasia was performed eight

weeks after the experimental surgical procedure using an overdose of pentobarbital

sodium (90-120 mg/kg; i.v.).

CT and µCT 3D Reconstructions

Computerized tomography (CT) (GE NXI Pro (GE MedicalSystems, Madison, WI)

and microcomputed tomography (µCT 40, Scanco Medical, Basserdorf, Switzerland)

was utilized to evaluate the bone fill within defects.

40

CT scans were performed at baseline and at 2, 4, 6, and 8 weeks

postintervention. AMIRA software (version 6.3.0; FEI Company, Berlin Germany) was

used for three-dimensional rendering of the image data using a standard “bone mask”

(bluescale – baseline; grayscale – subsequent time points). The same software was

used to realign the mandibles along the physiological axes and anatomic landmarks

were used to ensure that the scans at different time points were all aligned in an

identical fashion (Figure 2-4). The region of interest (ROI) was defined by a box-shaped

contour of the intact bone from the baseline scan and was fixed within as well as for

each defect for evaluation at 2, 4, 6 and 8 weeks (Figure 2-5). A uniform threshold for

bone was determined across all samples and the bone defect ROIs. Bone volume (BV)

to total volume (TV) ratio was determined on the CT images for all time points. To

quantify the bone formation through different time points, the percent of pristine bone

was subtracted from the percent of new bone.

For the purpose of obtaining higher resolution data regarding bone healing in this

model, µCT imaging was also used to analyze BV/TV ratio in the defect space. The

Xray energy level used was 70 kVp and the current level 114 A. All data were

exported in a DICOM-format and imported into Amira software (Mercury Computer

Systems, Chelmsford, MA, USA) for 3D reconstruction and quantification. The same

protocol of image superimposition was used to realign the mandible µCT scans to the

baseline CT data (Figure 2-7). Bone height regeneration measurement at the center of

the defect and adjacent to tooth was also performed through superimposition of the

baseline CT and final µCT reconstructions (Figure 2-8). The % bone height

41

maintenance relative to baseline was calculated at the center of the defect and adjacent

to the tooth for all samples.

Histologic Processing and Histomorphometric Analyses

After completion of the radiographic reconstructions, the specimens were fixed in

70% ethanol and reduced to blocks containing one defect each. The blocks were

gradually dehydrated in a series of alcohol solutions ranging from 70 to 100% ethanol.

Following dehydration, the samples were embedded in a methacrylate-based resin

(Technovit 9100, Heraeus Kulzer GmbH, Wehrheim, Germany). The blocks were then

cut into slices (~300 μm thick) centering the tooth according its long axis with a

precision diamond saw (Isomet 2000, Buehler Ltd., Lake Bluff, IL) and glued to acrylic

plates with an acrylate-based cement (Technovit 7210 VLC, Heraeus Kulzer GmbH,

Wehrheim, Germany), and a 24-hour setting time was allowed prior to grinding and

polishing. The sections were generated vertically in a mesial distal direction within the

defect. The sections were then reduced to a final thickness of ~80 μm by means of a

series of silicon carbide (SiC) abrasive papers (600, 800, 1200, and 2500; Buehler Ltd.,

Lake Bluff, IL) in a grinding/polishing machine (Metaserv 3000, Buehler Ltd., Lake Bluff,

USA) under water irrigation. Subsequently, the samples were stained with Stevenel’s

Blue and Van Giesons’s Picro Fuschin (SVG) and digitally scanned via an automated

slide scanning system, and a specialized computer software (Aperio Technologies,

Vista, CA, USA).

After delimitation of the defect margins for each sample (region encompassing

the denuded root areas and their respective adjacent structures), percent bone

formation within defect (from bone height prior to defect creation) was quantified by

42

means of computer software (ImageJ, NIH, Bethesda, USA). Quantitative linear

measurements for defect dimensions:

mineralized cementum

mineralized cementum + cementoid

periodontal ligament (insertion of fibers in bone and mineralized cementum and/or cementoid)

functional periodontal ligament (insertion of fibers in bone and mineralized cementum)

were performed along the adjacent tooth. Following linear measurement acquisition,

% mineralized cementum

% mineralized cementum and cementoid

% periodontal ligament (PDL)

% functional PDL

were calculated as a function of defect size (length) and as a function of the length

where full periodontal ligament regeneration was possible (between tooth and

regenerated bone).

Following linear measurements, the number of samples for each group that

presented 100% regeneration of mineralized cementum, mineralized cementum and

cementoid, PDL, and functional PDL were considered full regeneration to group the

different variables. All samples that presented no or partial regeneration were

considered non-full regeneration.

Statistical Analysis

Statistical evaluation of percent bone formation within defect through CT, µCT,

and 2D histomorphometric quantification was performed using linear mixed model

ANOVA and post hoc test for multiple comparisons with fixed factors of time for CT (2-,

43

4-, 6- and 8-week in vivo) and group for CT, µCT, and histomorphometry linear

measurements (E, H, R, S, Tp and Ts). Crosstabulated non-full and full regeneration

histologic data was assessed by generalized estimating equations (GEE) with a logistic

link to estimate effects of group on each tissue outcome. This approach is conceptually

similar to logistic regression but optimized for nested within subject observations. In all

analyses, post-hoc comparisons were based on estimated confidence limits using the

pooled estimate of the standard error for the mixed model analysis of variance and

Wald estimates for the GEE model. All statistical analyses of data were

performed using IBM SPSS (v23, IBM Corp., Armonk, NY). Statistical significance was

given at a value of p<0.05.

44

Table 2-1. Pre-surgical and surgical medications.

Drug Name Indication Dosage Route Frequency

Ketamine HCL PreMeds 10-12 mg/kg IM Pre-Med

Midazolam PreMeds 0.1 0.3 mg/kg IM Pre-Med

Isoflurane Anesthesia 2-5% Intubation General

Table 2-2. Randomization scheme. A statistician performed the randomizations of the different testing groups across the one-wall-defects in the mandible.

Dog Nr. P1 P2 P3 P4 P5 P6 1 Sham Collagen x

+ GF[4] Collagen 1

+ GF Emdogain Collagen 3

+ GF Collagen 2

+ GF

2 Collagen 1

+ GF Emdogain Collagen 3

+ GF Collagen x

+ GF[4] Collagen 2

+ GF Sham

3 Collagen 3

+ GF Collagen 1

+ GF Emdogain Sham Collagen

[4] Collagen 2

+ GF 4 Sham Collagen 3

+ GF Collagen x

+ GF [4] Emdogain Collagen 1

+ GF Collagen

5 Collagen 3

+ GF Emdogain Sham Collagen Collagen 2

+ GF Collagen 1

+ GF 6 Collagen 2

+ GF Collagen x

+ GF [4] Collagen 3

+ GF Collagen 1

+ GF Sham Emdogain

7 Sham Collagen 2

+ GF Collagen 1

+ GF Emdogain Collagen 3

+ GF Collagen x

+ GF [4] 8 Emdogain Collagen 1

+ GF Sham Collagen 2

+ GF Collagen 3

+ GF Collagen x

+ GF [4] 9 Collagen 3

+ GF Collagen 2

+ GF Collagen x

+ GF [4] Sham Collagen 1

+ GF Emdogain

10 Emdogain Collagen 2

+ GF Collagen 1

+ GF Collagen x

+ GF [4] Sham Collagen 3

+ GF 11 Collagen 1

+ GF Sham Emdogain Collagen 2

+ GF Collagen x

+ GF [4] Collagen 3

+ GF 12 Collagen x

+ GF[4] Emdogain Collagen 2

+ GF Collagen 3

+ GF Collagen 1

+ GF Sham

13 Collagen 2

+ GF Collagen 3

+ GF Sham Collagen 1

+ GF Emdogain Collagen x

+ GF [4] 14 Collagen x

+ GF[4] Sham Collage 2

+ GF n Collagen 3

+ GF Emdogain Collagen 1

+ GF

45

Table 2-3. List of products applied to the defects.

Group Investigational Product

E Emdogain®

H Heliplug + BDNF 2

R RCP+ BDNF 2

T p Teraplug + BDNF 2

T s

Teraplug + BDNF 1 - old

S Sham

Figure 2-1. Study Design Timeline. All the surgical procedures took part during the experimental phase including the teeth extractions. Then after 8 weeks of healing, the defects were created and the application of the GF products was done. Biweekly μCT analysis taken at baseline, 2, 4, 6 and 8 weeks. At week 16, the animals were euthanized and the evaluation phase consisted of histological evaluation and data analysis.

46

Figure 2-2. Schematic positions of the six surgically created 1-wall infrabony defects in the mandible of the beagle dog.

47

Figure 2-3. Surgical protocol for the test sites. (A) Initial Photos (B) After bilateral mandibular M1 and PM3 extractions. (C) and (D) After 8 weeks of healing, full thickness flap was elevated in previous extraction site and surgical defects were created on the distal of PM2 and PM4 and M2 in the R and L jaw. (E), (F) and (G) Application of the products with a collagen sponge (H) Passive primary closure of the flap. Photos courtesy of Dr. Rodrigo Neiva.

48

Figure 2-4. Three-dimensional rendering of (A) baseline (bluescale) and (B) 8 weeks (grayscale) CT image sets. (B) Mandible realignment

Figure 2-5. Regions of interest. A) and (B) are regions of interest (ROI) defined by a box-shaped contour involving full defect volume (standardized through different time-point superimpositions). (C) Three-dimensional reconstruction of baseline (blue) and 8 weeks (pink).

49

Figure 2-6. Representative images of bone formation through the different time points. (A) Three-dimensional rendering and superimposition of baseline and (1) 2 weeks, (2) 4 weeks. (3) 6 weeks, (4) 8 weeks CT image sets, and (5) µCT scan. (B) Three-dimensional reconstruction and superimposition of baseline (blue) and (1) 2 weeks, (2) 4 weeks. (3) 6 weeks, (4) 8 weeks (pink) CT image sets, and (5) µCT image (yellow).

50

Figure 2-7. Three-dimensional rendering. (A) baseline CT and (B) 8 weeks µCT image sets. (B) Superimposed CT and µCT scans. (C) and (D) Region of interest (ROI) defined by a box-shaped contour involving full defect volume (standardized through different time-point superimpositions). (E) Threedimensional reconstruction of baseline (blue) and 8 weeks (yellow) superimposition.

51

Figure 2-8. Bone height measurement at the center of the defect and adjacent to tooth through superimposition of the baseline CT and final µCT reconstructions.

52

CHAPTER 3 RESULTS

Overall, no complications in either surgical interventions or post-operative times

were observed in any of the dogs regarding infections and/or other clinical procedure.

The dogs maintained healthy along the completion of the experimental phase and no

weight loss was observed. Additionally, following euthanasia, dissection of the mandible

defects did not reveal any sign of inflammation and/or infection.

CT and µCT 3D Reconstructions

From a temporal perspective, CT 3D reconstructions evidenced a trend of

gradual bone volume increase for all groups along with defect filling and partial

volumetric regeneration (Figure 3-1). At 2 weeks, the S group presented the highest

mean bone formation and R group the lowest estimated mean (significantly different,

p<0.05). E, H, Tp and Ts presented intermediate values with no statistical significant

difference relative to any other experimental group (Figure 3-1a). E group presented

significantly lower percentage of new bone relative to other groups at 4 weeks

(p=0.018), except R (p=0.163) (Figure 3-1b). At 6 and 8 weeks in vivo, statistical

analysis depicted the lowest bone regeneration values for E group compared to others

(p<0.04). Furthermore, S, Tp and Ts groups presented the highest mean percent bone

formation (non-significant between each other), with Ts values significantly higher than

E, H and R (p<0.025). H and R groups presented intermediate estimated mean with no

statistical significant difference between each other (Figure 3-1c-d).

The µCT 3D reconstruction and image thresholding of defects also depicted bone

formation for all experimental groups with partial volumetric regeneration after 8 weeks

in vivo. Regarding experimental group effect on percent bone formation, group E

53

demonstrated the lowest estimated mean relative to others (p=0.047), except for R

(Figure 3-1e). Percent bone height regenerated calculated as a function of initial bone

height depicted that only Tp versus E group comparison presented borderline statistical

insignificance at the center of the defect (p=0.052), in which Tp presented higher mean

values than E (Figure 3-2a). On the other hand, percent bone height adjacent to tooth

depicted statistically significant higher mean values for Tp and S groups relative to R

(p=0.047). All other group comparisons were statistically homogeneous with,

repeatedly, Tp and E groups comparison demonstrating borderline statistical

significance (p=0.054) (Figure3-2b).

Histomorphometric Analysis

Histologic representative sections were obtained for each defect (Figures 3-3 and

3-4). Root surgical damage at the mesial or distal tooth surfaces was easily identified

resulting in dentin exposure toward the defect. Areas not surgically treated evidenced

typical anatomic features of periodontal tissue (Figure 3-5).

No noticeable differences in the bone healing characteristics was observed

among groups, and no considerable inflammatory cell remnants were observed for any

of the experimental groups. Histomorphology after 8 weeks in vivo demonstrated partial

alveolar bone regeneration relative to original defect dimensions for all groups. Such

newly formed bone, distinct from the native alveolar bone, was predominantly woven

bone (Figures 3-3 and 3-4). Ankylosis was not observed in any defect. Additionally, in

concurrence with 3D reconstruction quantification values, the histometric evaluation of

bone formation within defect indicated that S, Ts, Tp and H groups presented the

highest mean percentage values (not significantly different between each other),

followed by R and E groups. While the R mean values were significantly lower than S

54

group (p<0.050), group E presented significantly lower percentage of new bone relative

to S and Ts groups (p<0.02) (Figure 3-1f).

Regarding cementum regeneration, regions presenting no cementum formation,

cementoid tissue over dentin, and mineralized cementum (cellular, acellular, and mixed

cellular + acellular) were observed for all groups (Figure 3-5a-c). Histologic

representative images depicted the presence of cementoblasts along with an

unmineralized pre-cementum on the root’s surface (cementoid), and the morphologic

similarity between regenerated mineralized cementum and typical cementum (Figure 3

6a-c).

No significant differences were confirmed between groups regarding the

percentage of mineralized cementum per defect size (p>0.292) and within regenerated

bone and tooth (p>0.90) (Figure 3-7 a-b respectively). Quantitatively, percent of

cementum + cementoid tissue per defect size and between regenerated bone and teeth

demonstrated higher mean values for Tp group when compared to H and R (p=0.026)

and significantly higher values between Ts and H groups (p=0.44), respectively (Figure

3-8 a-b).

Mineralized cementum full regeneration (between teeth and regenerated) bone

estimated mean percentage for Ts group was significantly higher than E and S

(p<0.042), groups which failed to present any sample with full mineralized cementum

regeneration between tooth and bone. In fact, group E was significantly lower relative to

H, R and Ts groups (p<0.046) (Figure 3-12: a, b). The percentage of samples, which

presented mineralized cementum + cementoid covering 100% of the root surface within

55

bone walls for group E was significantly higher than S (p=0.022), with all other groups

being statistically homogeneous Figure 3-13: a, b).

PDL histologic micrographs depicting no PDL regeneration, full PDL

regeneration, non-functional PDL regeneration, and functional PDL regeneration are

presented in Figure 3-9. Full PDL regeneration only occurred when mineralized

cementum and/or cementoid fully covered instrumented dentin. Quantitatively, no full

PDL and functional PDL regeneration significant differences between groups was

detected (p>0.10) per defect size and between tooth and regenerated bone (Figures

310a and b). An identical trend was observed for functional PDL per defect size and

between tooth and regenerated bone, where groups were statistically homogenous

(Figures 3-11a and b). Nevertheless, qualitative analysis demonstrated higher

percentage of full PDL regeneration between tooth and regenerated bone for Ts and E

groups compared to S and H. It is noteworthy to mention that the significance value for

Ts relative to S exceeds one order of magnitude relative to E versus S at p<0.001 and

p=0.022, respectively. When the percentage of full functional PDL regeneration is

considered, the Ts, H, and R presented significantly higher values compared to E and S

(p=0.046 between E and S relative to H and R; p=0.42 between Ts relative to E and S)

(Figure 3-14).

56

Figure 3-1. Percent bone formation within defect quantified through CT reconstructions. (a) 2-, (b) 4-, (c) 6- and (d) 8-weeks in vivo. (e) Percent bone formation within defect obtained using microCT and (f) 2D histometric quantification. *Identical letters indicate statistically homogeneous groups.

57

Figure 3-2. Percent bone height maintenance at the (a) center of the defect and (b) adjacent to tooth.

58

Figure 3-3. Histologic sections. (a) E, (b) H, (c) R, (d) S, (e) Tp and (f) Ts groups after

8-weeks in vivo.

Figure 3-4. Histologic high magnification micrographs depicting PDL formation of (a) E,

(b) H, (c) R, (d) S, (e) Tp and (f) Ts groups after 8-weeks in vivo. NFcPDL: non-functional PDL; FcPDL: Functional PDL

59

Figure 3-5. Histologic images of the areas of interest observed in the study. (a) A descriptive histologic section delimiting defect margins (marked in black). (b) Typical anatomic features of hard (alveolar bone, cementum, and root dentin), and soft tissues (periodontal ligament). (c) Morphological features of regenerated mineralized cementum, PDL and new bone.

Figure 3-6. Histomorphologic features utilized for cementum regeneration quantification. (a) No Cementum (NC), (b) Cementum + Cementoid tissue (CT) and (c) Mineralized Cementum (MC).

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Figure 3-7. Mineralized Cementum a) Per defect size b) within bone and tooth walls

Figure 3-8. Cementum and Cementoid a) Per defect size b) within bone and tooth walls

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Figure 3-9. Histomorphologic features assumed to classify PDL regeneration. (a) Non-full PDL (NFPDL), (b) Full PDL (FPDL), (c) Non-functional (NFcPDL) - fiber insertion into cementoid, and (d) functional PDL (FcPDL) - fiber insertion in bone and mineralized cementum.

Figure 3-10. PDL Regeneration a) Per defect size b) within bone and tooth walls

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Figure 3-11. Functional PDL. a) per defect size b) within bone and tooth walls

Figure 3-12. Cementum regeneration and bone percentage. A) Mineralized cementum full regeneration B) Bone estimated mean percentage

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Figure 3-13. Cementum + cementoid regeneration and mean percentages. A) Mineralized cementum + cementoid regeneration B) estimated mean percentage

Figure 3-14. Periodontal ligament regeneration. a) Full PDL % b) % Full Functional PDL

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CHAPTER 4 DISCUSSION

This study tested the hypothesis that BDNF combinations regenerates the

periodontal tissue defect and demonstrates superior histologic regeneration when

compared to Emdogain and no treatment. The term ‘regeneration’ or ‘regenerative’ was

used in this study to point out that the intention of the study was to repair, as much as

possible, the periodontal tissue to its original structure and morphology. Although a

chronic periodontal disease model was not utilized, a surgically created one-wall critical

size defect was employed in the present study creating an extremely challenging

situation to regenerate.

The worst and final outcome of periodontally diseased dentition left untreated is

tooth loss.4,5 For this reason the main goal in periodontal treatment is to contain the

destruction of the periodontium. Due to the extensive research in the field, the

treatment of periodontal disease has advanced in such a way that the prevention of the

recurrence of the disease is of equal importance. 6, 7, 12, 13,39 There has been a paradigm

shift in the field from halting the progression to regenerating the once lost tissue by

using surgical techniques such as GTR with or without the combination of growth

factors. 25,26

Typically, the shallower and wider the defect, the less amount of regeneration

expected.51 This study addressed regeneration in a critical size one wall defect, a

highly challenging regenerative procedure to obtain successful outcomes. An ideal

periodontal defect for regeneration is a deep, narrow intrabony defect as described by

Tonetti, Cortellini, et al. in 1993. The biological explanation is that the greater the

number of walls remaining, the faster the source of osteoprogenitor cells reach the site

65

of interest. This is due to the generous increase in blood supply to the site. Blood

supply is a key element to regeneration as it provides nutrients and transports the

necessary cells to the site of interest. Also, mechanically with the presence of more

walls, the material added (graft, growth factor, i.e., scaffold.) is better contained, and if a

membrane is utilized, will adapt over the site instead of collapsing over it. Since in this

study we used a large critical size defect only partial regeneration was observed. This

demonstrates that if some regeneration occurs in critical size defects this size, this

could translate in potential efficacious results in a human chronic periodontitis model.

To the authors knowledge no other study has proven successful periodontal

regeneration with the use of BDNF in one wall bone defects. On the other hand,

periodontal regeneration with the implementation of BDNF growth factor and a scaffold

has been proven to be successful in Class II furcation defects.104 In another extremely

challenging clinical situation, Takeda et al. reported in in vivo canine class III furcation

defect studies that the use of BDNF in combination with atelocollagen sponge, or with

synthesized high molecular weight hyaluronic acid, seemed to regenerate the

periodontal tissue in a dose dependent manner. 101, 107 This study suggested the

potential successful outcomes with the use of BDNF growth factor to induces

regeneration in complex situations such as class III furcations. Thus, it was of great

interest to observe histologically in detail the effect of BDNF in combination in one-wall

defects, and to correlate to clinical reality.

Both histologic and histomorphometric outcomes of the current study presented

partial bone regeneration for all groups. Group Ts proved to be the defect with the

higher mean values of new bone volume formation and chiefly comprised full PDL

66

regeneration and mineralized cementum/functional PDL regeneration. While in most

instances no significant differences were detected in linear measurements (quantitative

analyses) due to no or partial tissue regeneration, when one considers full regeneration

success of functional PDL at 8 weeks in vivo along with higher full PDL formation, and

significantly higher bone regeneration within defect volume, strongly suggest that

regeneration takes place for this group in higher overall amounts and faster (supported

by significantly higher mineralized cementum formation and full conversion of 100% of

these into functional PDL). The H and R groups were significantly better than E and

comparable to Ts when mineralized cementum and functional PDL are considered.

However, a strong indicator that these collagens and BDNF combinations may take

longer to heal relative to Ts arises from the low values of PDL formation with insertion in

both mineralized cementum and cementoid; these values are not significantly different

than the S group.

Group R showed values are not significantly different from S group, new bone

and chiefly comprised non-full PDL and functional PDL regeneration. This group

presented significantly more mineralized cementum and cementoid coverage and full

functional PDL regeneration (significantly more than E and S – potential effect of

BDNF). All of the samples that presented full mineralized cementum regeneration

resulted in full functional PDL regeneration.

Group Tp showed comparable new bone within defect and essentially involved

no PDL and no full functional PDL regeneration There was also a non-significant effect

of BDNF and collagen type relative to E and S. The comparator product, Emdogain

group, presented with significantly less bone volume than H, S, Tp, and Ts and primarily

67

partial/no functional PDL regeneration along with full defect perimeter. While cementoid

allows for fiber insertion and PDL formation/organization along with mineralized

cementum, no sample presented 100% mineralized cementum leading to functional

PDL formation. It is not certain that such cementoid will mineralize and generate

functional PDL as time elapses. At 8 weeks, none of the defects from the E group

presented full functional PDL formation between the tooth structure and regenerated

bone. Regarding this matters, it is important to understand that the formation of PDL

fibers was not potentially encountered in some groups due to the lack of proper

occlusion in the dogs. The dogs were placed in a soft diet thus PDL formation and

proper fiber alignment could’ve have been hindered by this.

While the other groups presented partial or full bone regeneration, histologic

observations evidenced that the multiple periodontal tissue components did not

completely regenerate. This may be due to the large extent of the one-wall defect and

its poor regenerative potential. Based on the histomorphometric results, it is indicative

that full cementum coverage (cellular or acellular) is one of the essential factors for

periodontal ligament regeneration. This is supported by Group Ts having the highest

percentage of complete periodontal ligament regeneration.

A future recommendation for this study is to standardize the scaffold material,

and to study different defect types (i.e., 2- wall, furcation defects), along with a different

carrier material. Additionally, there should be an attempt to maximize bone formation

and ridge height maintenance by using particulate material loaded with the experimental

products, or reinforced membranes. A potential but controversial limitation to this study

is the use of a surgically created defect versus utilizing a model where controlled

68

chronic periodontitis is induced. Although a controlled chronic periodontitis model in

dogs would simulate a better human clinical representation, this is highly difficult to

archive. For this reason a critical size defect would be the most illustrative for

regeneration in a challenging situation. Another limitation encountered is the CT and

micro CT evaluation should include shorter and longer time points. Also include a larger

sample for future studies.

In summary, within the limitations of this study, it was demonstrated that Brain

Derived Neurotrophic Factor, in combination with a collagen sponge carrier is an upand-

coming regeneration product that induced higher degrees of complete periodontal tissue

healing in comparison to the comparator product, supporting the postulated hypothesis.

It is strongly suggested that the application of BDNF and collagen carrier combination

being the most desirable for periodontal regeneration of challenging defects.

69

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BIOGRAPHICAL SKETCH

Camille Medina-Cintron was born in San Juan, Puerto Rico. She completed her

undergraduate degree with Bachelor of Science in biology and dental degree at the

University of Puerto Rico and graduated on May 2014 with Doctor of Dental Medicine.

She then completed a one year General Practice Residency at the VA hospital in San

Juan. She received her master’s certificate in science from the University of Florida in

the spring 2018 and anticipates graduating in May 2018 with a certificate in

periodontics. Following graduation, she plans to practice clinical periodontology in

Tampa, Florida.