Dr. Nida Sultana Thesis

PANDIT DEENDAYAL UPADHYAY MEMORIAL HEALTH
SCIENCES & AYUSH UNIVERSITY OF CHHATTISGARH

RAIPUR [C.G.]
“A COMPARATIVE EVALUATION OF THE EFFICACY OF

AMNION MEMBRANE (AM) ALLOGRAFT BARRIER WITH
DEMINERALISED FREEZE-DRIED BONE ALLOGRAFT
(DFDBA) AND DFDBA ALONE IN TREATMENT OF
PERIODONTAL INTRABONY DEFECT IN CHRONIC
PERIODONTITIS PATIENTS: A CLINICO-
RADIOGRAPHICAL STUDY.”
By
Dr. NIDA SULTANA
Dissertation submitted to Pandit Deendayal Upadhyay Memorial
Health Sciences & Ayush University of Chhattisgarh, Raipur,
Chhattisgarh, in the partial fulfillment of the requirements for the
degree of
MASTER OF DENTAL SURGERY
In the speciality of
PERIODONTOLOGY
Under the guidance of

Dr. KARTHIK KRISHNA M
PROFESSOR & HOD
DEPARTMENT OF PERIODONTOLOGY
RUNGTA COLLEGE OF DENTAL SCIENCES AND
RESEARCH, BHILAI-490024
CHHATTISGARH.
Batch 2018-2021
Acknowledgement
ACKNOWLEDGEMENT
Writing this thesis has been fascinating and extremely rewarding. As a
Post Graduate Student, during my thesis tenure people were instrumental directly
or indirectly in shaping up my academic career. It was hardly possible for me to
thrive in my thesis work without precious support of these personalities. Here is
the small tribute to all those people.
To commence with, I bow my head in deep reverence to the ALMIGHTY
ALLAH the savior, for giving me energy, perseverance and the will, to finish this
work successfully.
At this moment of accomplishment, I am greatly indebted to my guide
Dr. KARTHIK KRISHNA M Professor and Head of Department of Periodontology at
Rungta College of Dental Sciences and Research for being my support pillar in this
pathway full of hurdles. Whenever I digressed, his amazing gift of explaining
complicated things in a simplified manner helped me in regaining my focus. His
vast knowledge and attention to detail, hard work is an example I hope to match
IV
Acknowledgement
someday. His unprecedented calm and patient persona, an unfailing caring and
understanding demeanor made each endeavor easier. His own zeal for perfection,
passion, unflinching courage and conviction has always inspired me to do more. He
is one teacher who truly made a difference in my life. No matter what I do to
show my gratitude towards him, it always will be infinitely small and thereby I
owe him, my sincere thanks.
I humbly acknowledge Mr. SANJAY RUNGTA, Chairman, Rungta College of
Dental Sciences and Research and Dean Dr. SUDHIR PAWAR for providing an
efficient and able platform for me to gain knowledge through this effective and
highly advanced research institute and allowed me to do this research.
My earnest thanks to Dr. V GOPINATH and Dr. PADMA R Professor &
former H.O.D in Rungta College of Dental Sciences and Research, for introducing
me to the world of research. It was only due to their valuable guidance, cheerful
enthusiasm, and ever friendly nature that gave me boosts to start my work.
I would like to express my heartfelt gratitude and deep appreciation to
Dr. AENA JAIN PUNDIR Professor in the Department of Periodontology at Rungta
College of Dental Sciences and Research. Her personified excellence, not only
through deep insight into the field of periodontology, but also through the unique
V
Acknowledgement
art, to exhume from her students the strings of an inquisitive mind and dedication
to work which made me realize the worth of discovering my own capabilities.
I am grateful to my prized staff Dr. SONIKA BODHI and Dr. SHRUTI
BHATNAGAR Senior Lecturers in the Department of Periodontology at Rungta
College of Dental Sciences and Research who had always been there for me for
unravelling the hitches of my course, giving constant advices to tackle glitches and
also for their constant guidance.
I also express my deep thankfulness to Dr. BALASUBRAMANYAM VASANT
for his meticulous statistical data analysis, without which this dissertation would
have been meaningless and incomplete.
I am extremely thankful to Tissue bank of TATA Memorial Centre, Advanced
Centre for Treatment, Research & Education in Cancer, Navi Mumbai. for providing
me the Amniotic Membrane and Bone Graft used in the study.
I extend my heartfelt thanks to my revered seniors Dr. TEH CHAI LIU and
Dr. NIKITA AGRAWAL for constant support and guidance, who have always been
there for me for unraveling the hitches of my course, giving constant advices to
tackle glitches and also for their constant guidance, infinite patience and
encouragement in shaping this thesis.
VI
Acknowledgement
I owe thanks to a very special person, my husband Dr. SHAHZAD AHMAD
for his continued and unfailing love, and understanding during my pursuit of post-
graduation that made the completion of thesis possible. who was always around
at times I thought that it is impossible to continue, he helped me to keep things
in perspective. I greatly value his contribution and deeply appreciate his belief in
me. Words would never say how grateful I am to him. I consider myself the
luckiest in the world to have such a caring husband standing beside me with his
love and unconditional support.
I am bound to express my thanks to my colleagues Dr. SAMARPITA NANDA
and Dr. SMRITI GUPTA for constant encouragement, motivation, unconditional
support and positive appreciation throughout my post-graduation period. Even in
those very dreaded days, you both made my life easier. Thanks for supporting me
wholeheartedly, I couldn’t appreciate it enough.
It’s my fortune to gratefully acknowledge the support of my juniors
Dr. SABREENA WILLIAMSON, Dr. KANCHAN AGRAWAL and Dr. LAWANYA
CHANDRAKAR for helping and enabling me comprehend and overcome the
hardships and hurdles throughout the course of my post-graduation.
VII
Acknowledgement
I am also thankful to my sub juniors Dr. ABHISHEK HIRWANI, Dr. SYEDA
NAZIYA HASAN, Dr. ASHIDA SARA BLESSON, Dr. SASWATI MOHANTY and
Dr. KRITIKA KEJRIWAL for their valuable help and support in all possible aspects.
I wish to acknowledge the help and cooperation from our non-teaching
staff Mrs. JAYA DAS and Mrs. KAMLAWATI DEVI.
No words could ever express the love and appreciation that I hold for my
loving PARENTS AND FAMILY, for their unconditional love, constant prayers,
support and sacrifice that they have made and also for instilling in me those
values and principles in my life.
I would like to pay high regards and deep sense of gratitude to my parents
Mr. JAMAL KHAN, Mrs. NAJMA SULTANA, my in Laws Mr. SHAHABUDDIN and
Mrs. SHAMIM BANO, for their sincere encouragement and inspiration throughout
my work and lifting me uphill in this phase of life. I hope I have made them
proud and I owe everything to them.
I extend my sincere thanks to my brother NAHEED KHAN, my sister
SEDRA SULTANA, my brother in laws MINHAJ AHMAD and Dr. MOHD SHAHID
and my sister in laws ALFIYA BANO and SANIYA NASEEM for their kind co-
VIII
Acknowledgement
operation and support. Their faith in me gave me the strength to carry out the
work successfully.
Narrow border of language could never express my respect and gratitude to
all the patients who co-operated with me, they deserve maximum credit for the
success of this dissertation.
Besides this, I want to thank all the people that have knowingly and
unknowingly helped me in the successful completion of my dissertation.
Dr. NIDA SULTANA
IX
List of abbreviations
LIST OF ABBREVIATIONS
ACRONYMS FULL DESCRIPTION
ABM Allogenic Bone Matrix
ACM Amnion Chorion Membrane
AIAM Antibiotic Impregnated Amniotic Membrane
AM Amnion Membrane
ANOVA Analysis of Variance
BMP Bone Morphogenic Protein
BOP Bleeding on Probing
BP Bard Parker
CAL Clinical Attachment Level
CBCT Cone Beam Computed Tomography
CM Chorion Membrane
CPS Calcium Phosphosilicate
DBM Demineralized Bone Matrix
X
DFDBA Demineralized Freeze-Dried Bone Allograft
DMSD Digital Media Sustainable Development
ECM Extracellular Matrix
EGF Epidermal Growth Factor
EMD Enamel Matrix Derivatives
FDBA Freeze-Dried Bone Allograft
GI Gingival Index
GR Gingival Recession
GTR Guided Tissue Regeneration
HA Hydroxyapatite
HCL Hydrochloride
HIV Human Immunodeficiency Virus
HLA Human Leukocyte Antigen
HS Highly Significant
HSV Herpes Simplex Virus
XI
IL Interleukin
KGF Keratocyte Growth Factor
mm Millimeter
NCP Non-Collagenous Proteins
NS Non-Significant
OFD Open Flap Debridement
OPG Orthopantomogram
PDL Periodontal Ligament
PI Plaque Index
PPD Probing Pocket Depth
PRF Platelet Rich Fibrin
PRP Platelet-Rich Plasma
PTFE Polytetrafluoroethylene
RAL Relative Attachment Level
RVG Radiovisiography
XII
S Significant
TGF Transforming Growth Factor
TIMP Tissue Inhibitors of Metalloproteinase
UNC University of North Carolina
XIII
Table of contents
TABLE OF CONTENTS
S.NO. PARTICULARS PAGE NO.
1. INTRODUCTION 1-4
2. AIM & OBJECTIVES 5
3. REVIEW OF LITERATURE 6-52
4. MATERIALS AND METHOD 53-89
5. OBSERVATION & RESULTS 90-118
6. DISCUSSION 119-131
7. SUMMARY & CONCLUSION 132-133
8. BIBLIOGRAPHY 134-153
9. ANNEXURES 154-164
XIV
List of tables
LIST OF TABLES
Sr. No. TITLE PAGE.NO.
Table 1 Bone replacement grafts 18
Table 2 Scoring criteria for Plaque Index 57
Table 3 Suggested Nominal Scale for Patient Evaluation 58
for Plaque Index
Table 4 Scoring criteria for Gingival Index 58
Table 5 Suggested Nominal Scale for Patient Evaluation 59
for Gingival Index
Table 6 Intragroup comparison of mean Plaque Index at 91
Baseline, 90th day and 180th day in group I
Table 7 Intragroup comparison of mean Plaque Index in 91
group I at different time interval
Table 8 Intragroup comparison of mean Plaque Index at 92
Baseline, 90th day and 180th day in group II
Table 9 Intragroup comparison of mean Plaque Index in 92
group II at different time interval
XV
List of tables
Table 10 Intergroup comparison of mean Plaque Index 93
between various time intervals
Table 11 Intragroup comparison of mean Gingival Index at 95
Baseline, 90th day and 180th day in group I
Table 12 Intragroup comparison of mean Gingival Index in 95
group I at different time interval
Table 13 Intragroup comparison of mean Gingival Index at 96
Baseline, 90th day and 180th day in group II
Table 14 Intragroup comparison of mean Gingival Index in 96
group II at different time interval
Table 15 Intergroup comparison of mean Gingival Index 97
between various time intervals
Table 16 Intragroup comparison of mean Probing Pocket 99
Depth at Baseline, 90th day & 180th day in group I
Depth in group I at different time interval
Depth at Baseline, 90th day & 180th day in group II
XVI
List of tables
Depth in group II at different time interval
Table 20 Intergroup comparison of mean Probing Pocket 101
Depth between various time intervals
Table 21 Intragroup comparison of mean Relative 103
Attachment Level at Baseline, 90th day and 180th
day in group I
Attachment Level in group I at different time
interval
Attachment Level at Baseline, 90th day and 180th
day in group II
Attachment Level in group II at different time
intervals
Table 25 Intergroup comparison of mean Relative 105
Attachment Level between various time intervals
XVII
List of tables
Table 26 Intragroup comparison of mean Gingival 107
Recession at Baseline, 90th day and 180th day in
group I
Table 27 Intragroup comparison of mean Gingival 107
Recession at Baseline, 90th day and 180th day in
group II
Table 28 Intergroup comparison of mean Gingival 108
Recession between various time intervals
Table 29 Intragroup comparison of mean Radiographic 110
Defect Depth at Baseline and 180th day in group I
Table 30 Intragroup comparison of mean Radiographic 110
Defect Depth at Baseline and 180th day in group I
Table 31 Intergroup comparison of mean Radiographic 111
Defect Depth between various time interval
Table 32 Intergroup comparison of mean Amount of Defect 113
Fill at 180th day
Table 33 Intergroup comparison of Percentage of Defect 114
Fill at 180th day
XVIII
List of figures
LIST OF FIGURES
S. No. TITLE PAGE.NO.
Fig 1 Classification According to the number of osseous 7
walls present
Fig 2 Infrabony defects 8
Fig 3 Predictability of treatment outcome based on defect 10
anatomy
Fig 4 Sources of regenerating cells in the healing stages of 12
a periodontal pocket
Fig 5 New Attachment 14
Fig 6 Epithelial adaptation after periodontal treatment 15
Fig 7 Layers of Placenta 26
Fig 8 Cross section of the human amniotic membrane 26

indicating the biochemical characteristics of the
section
Fig 9 Inhibition of Fibrosis by Amnion membrane 27
Fig 10 Defect landmarks 62
Fig 11 Defect landmarks in the radiograph 62
XIX
List of figures
Fig 12 Diagnostic cast with acrylic stent 74
Fig 13 Millimeter grid mount 74
Fig 14 Radiograph with millimeter grid mount 75
Fig 15 Surgical armamentarium 75
Fig 16 Demineralized freeze-dried bone allograft 76
Fig 17 Amnion membrane (AM) allograft 76
CLINICAL PHOTOGRAPHS SHOWING THE PROCEDURES IN GROUP I
(DFDBA)
Fig 18 RAL measured using UNC -15 probe preoperatively 77
Fig 19 PPD measured using UNC -15 probe preoperatively 77
Fig 20 Incision placement 78
Fig 21 Osseous defect between right mandibular first molar 78
and second molar after debridement
Fig 22 DFDBA being carried to the defect 79
Fig 23 DFDBA placement in the defect 79
Fig 24 Flap closure with simple interrupted suture 80
XX
List of figures
Fig 25 Periodontal dressing given 80
Fig 26 RAL measured using UNC -15 probe postoperatively 81
Fig 27 PPD measured using UNC -15 probe postoperatively 81
Fig 28 RVG at baseline 82
Fig 29 RVG at 180th day months 82
CLINICAL PHOTOGRAPHS SHOWING THE PROCEDURES IN GROUP
II (DFDBA with AM)
Fig 30 RAL measured using UNC -15 probe preoperatively 83
Fig 31 PPD measured using UNC -15 probe preoperatively 83
Fig 32 Incision Being Placed 84
Fig 33 After debridement 84
Fig 34 Osseous defect between right mandibular second 85
premolar and first molar after debridement
Fig 35 DFDBA and Trimmed AM with template 85
Fig 36 Defect grafted with DFDBA 86
Fig 37 AM placed over the grafted defect 86
XXI
List of figures
Fig 38 Flap closure with simple interrupted suture 87
Fig 39 Periodontal dressing given 87
Fig 40 RAL measured using UNC -15 probe postoperatively 88
Fig 41 PPD measured using UNC -15 probe postoperatively 88
Fig 42 RVG at baseline 89
Fig 43 RVG at 180th day 89
XXII
List of graphs
LIST OF GRAPHS
Graph 1 Comparison of mean Plaque Score Index between 115
group I and group II at baseline, 90th day and
180th day
Graph 2 Comparison of mean Gingival Score Index 115
between group I and group II at baseline, 90th day
and 180th day
Graph 3 Comparison of mean Probing Pocket Depth 116
and 180th day
Graph 4 Comparison of mean Relative Attachment Level 116
and 180th day
Graph 5 Comparison of mean Gingival Recession between 117
group I and group II at baseline, 90th day and
180th day
Graph 6 Comparison of mean Radiographic Defect Depth 117
between group I and group II at baseline and
XXIII
List of graphs
180th day
Graph 7 Comparison of mean Amount of Defect Fill 118
between group I and group II at 180th day
XXIV
List of annexures
LIST OF ANNEXURES
1 Institutional ethics committee – Certificate of ethics 154
approval
2 Scientific research committee certificate 155
3 Patient’s consent form 156
4 Case history Proforma 157-161
5 Master Table 162-164
XXV
Abstract
ABSTRACT
Background: Periodontal regeneration in intrabony defects is an arduous task to
achieve, regardless of all advancement. Several treatment modalities have been
applied over the years to reconstruct intrabony periodontal defects surgically.
Perhaps the most obvious shift has been from resective periodontal surgical procedures
to techniques and methods aimed at regeneration and reconstruction of the lost
periodontium.
Objectives: To compare the efficacy of Amnion Membrane (AM) allograft barrier with
DFDBA and DFDBA alone in treatment of periodontal intrabony defect in chronic
periodontitis patients.
Methods: A total of 40 sites (20 in each group) with clinical and radiographical
evidence of intrabony defects were selected from the Out-Patient Department, Rungta
College of Dental Sciences and Research, Bhilai, Chhattisgarh. The selected sites were
randomly divided into group I (Open flap debridement with DFDBA) and group II
(Open flap debridement with DFDBA+ AM). The clinical parameter evaluated were
Plaque index (PI), Gingival index (GI), Probing pocket depth (PPD), and Relative
attachment level (RAL), at baseline, 90th day and 180th day postoperatively.
Radiographic parameters recorded vertical osseous defect depth, amount of defect fill
and percentage of defect fill at baseline and 180th day. All clinical and radiographic data
were subjected to statistical analysis using appropriate tests for within group
comparison and intergroup comparison.
Results: The clinical and radiographical parameters showed improvement throughout
the study period. The plaque and gingival index showed significant reduction from
baseline (Group I: 2.07±0.18, Group II: 2.05±0.25 and Group I: 2.14±0.24, Group II:
XXVI
Abstract
2.28±0.21 respectively) to 180th day (Group I: 0.57±0.10, Group II: 0.61±0.13 and
Group I: 0.69±0.12, Group II: 0.66±0.15 respectively). Both the group showed
reduction in mean PPD and RAL which was statistically significant when comparing
baseline (Group I: 6.95±0.94, Group II: 7.30±0.92 and Group I: 12.05±1.39, Group II:
12.95±1.93 respectively) to 180th day (Group I: 4.10±0.78, Group II: 3.45±0.60 and
Group I: 9.30±1.86, Group II: 8.04±2.05 respectively). Group II showed greater
reduction in PPD (p= 0.005) and improvement in RAL (p= 0.048) than group I. There
was significant reduction in defect depth (Group I: 2.11± 0.57 and Group II: 1.85±0.55)
and increase in percentage of defect fill (p= 0.04) in both groups.
Conclusion: The results of this investigation indicated that AM as a novel barrier
membrane, in conjunction with DFDBA, is quite predictable and comparable to
DFDBA alone when used in intrabony defects in bringing improvements in clinical
parameters.
Keywords: Guided tissue regeneration; intrabony defect; demineralized freeze-dried
bone allograft; amnion membrane.
XXVII
INTRODUCTION
Introduction
INTRODUCTION
Periodontitis is a chronic infectious disease of the supporting tissues of the
teeth. It results from pathogenic bacterial infection, which produces factors that
destroy collagenous support of the tooth, as well as loss of alveolar bone. 1 Loss of
alveolar bone support is one of the characteristics signs of destructive periodontal
disease and is generally considered to represent the anatomical sequela to the apical
spread of periodontitis.2
The main goals of periodontal treatment are the elimination of infection and
the resolution of chronic inflammation in order to arrest disease progression and
prevent its recurrence. Persistent probing depths following treatment are often related
to the presence of intrabony (angular) periodontal defects, thus worsening the long-
term prognosis for teeth. The rationale behind the treatment of intrabony defects is
therefore to reduce residual probing depths to improve tooth prognosis. During the
last three decades, various treatment approaches involving nonsurgical techniques,
have been employed for the treatment of intrabony defects and have achieved variable
success.3
The ideal therapeutic goal of periodontal therapy is regeneration. Periodontal
regeneration is defined as complete replacement of lost periodontal structure
including formation of new cementum with inserting periodontal ligament, bone and
gingiva.4 Several treatment procedures including bone grafts,5 guided tissue
regeneration,6 bioactive agents7 or combined approaches8 have been suggested for
regenerative periodontal therapy.
1
Introduction
Although bone tissue exhibits a large regeneration potential and may restore
its original structure and function completely, bony defects may often fail to heal with
bone tissue. In order to facilitate and/or promote healing, bone grafting materials can
be placed into bony defects. It is generally accepted that the biologic mechanisms
forming the basis for bone grafting include three basic processes: osteogenesis,
osteoconduction, and osteoinduction.9
Combination regenerative approaches like open flap debridement (OFD) with
bone graft,10 guided tissue regeneration,11 growth factors,12 enamel matrix derivative,8
stem cells13 have been quite successful for treating the more challenging intrabony
lesions, particularly as the size and complexity of the lesion increases. 14 Typically
these efforts have included a bone replacement graft that has been covered by an
exclusionary barrier to achieve guided tissue regeneration (GTR).15
GTR has an advantage of permitting or encouraging wound repopulation by
cells derived from tissues with known or suspected regenerative potential. In
periodontal regeneration with GTR it is noted that the GTR and bone substitutes
demonstrate better results as compared with GTR alone. 6 Demineralized freeze-dried
bone allograft (DFDBA), is an allograft that possess osteoconductive and
osteoinductive properties and eliminates the need for a second surgical site. DFDBA
has shown evidence of greater bone formation in healing of non-molar extraction
socket.16 DFDBA has the potential for osteoinduction with more expression of bone
morphogenetic protein, and hence indicated for periodontal regeneration. 17-20
Tissue engineering (TE) is defined as the development of biological
substitutes for the purpose of restoring, maintaining or improving tissue function and
requires the application of principles and methods from engineering and life
2
Introduction
sciences.21 Stem cells represent an excellent source for use in TE. Recently Amniotic
Membrane (AM) has been investigated as a possible source of stem/progenitor cells
for therapeutic application and has a beneficial role in tissue engineering.
The placental based amniotic membranes have been used for tissue
engineering in the reconstruction of ocular surface, intractable epithelial defects,
management of Steven Johnsons syndrome, chemical burns, nerve regeneration,
feeder layer for stem cells, skin reconstruction, endothelial cell cultivation,21 local
drug delivery, as a graft material after vestibuloplasty,22 barrier membrane in
treatment of periodontal osseous defects,23 and as a guided tissue regeneration
membrane in the management of gingival recessions. AM have inherent biological
properties that actively promote wound healing in lieu of simply providing an occlusal
barrier for selective cell population and also promotes bone induction.24,25
The AM represents the innermost layer of the placenta, composed of single
epithelial layer, thick basement membrane and avascular stroma. AM has anti-
inflammatory, anti-microbial, anti-scarring, low immunogenicity and reasonable
mechanical property.21 The amniotic membrane is considered an important potential
source for scaffolding material, as it creates an almost a native scaffold for self-
seeding in tissue engineering as the epithelium might retain reservoir of stem cells. 21
The above-mentioned facts about AM and DFDBA may positively influence
the treatment of a periodontal intrabony defect by enhancing wound healing,
promoting periodontal regeneration increased bone formation as compared to sites
treated with conventional open flap debridement and bone substitutes. However, the
scientific literature has limited information evaluating the benefits of AM in
periodontal regeneration.
3
Introduction
Hence in order to compare and evaluate this hypothesis, the present
randomized control trial will be aimed at clinically and radiographically evaluating
(bone fill) the efficacy of AM in combination with DFDBA in the treatment of
periodontal intrabony defects of chronic periodontitis patients.
4
AIM &
OBJECTIVES
Aim and objectives
AIM AND OBJECTIVES
AIM
The aim of this study was to evaluate clinically and radiographically the efficacy
of Amnion Membrane (AM) allograft barrier with DFDBA and DFDBA alone in
treatment of periodontal intrabony defect in chronic periodontitis patients.
OBJECTIVES
The present study was undertaken with the following objectives:
1. To compare the efficacy of Amnion Membrane (AM) allograft barrier and
Demineralized Freeze-Dried Bone Allograft (DFDBA) in the reduction of
probing pocket depth, gain in the clinical attachment level and reduction in
gingival recession.
2. To assess radiographically the effect of Amnion Membrane (AM) allograft
barrier and Demineralized Freeze-Dried Bone Allograft (DFDBA), measure
the defect depth and to calculate the percentage of defect fill.
5
REVIEW OF
LITERATURE
Review of literature
REVIEW OF LITERATURE
Chronic periodontitis is the most prevalent form of periodontitis, and it
generally shows the characteristics of a slowly progressing inflammatory disease.
Periodontitis belongs to the group of complex inflammatory diseases in humans. 26
The classic definition described chronic periodontitis as “an infectious disease
resulting in inflammation within the supporting tissues of the teeth, progressive
attachment loss, and bone loss.”27 The clinical feature that distinguishes periodontitis
from gingivitis is the presence of clinically detectable attachment loss as a result of
inflammatory destruction of the periodontal ligament and alveolar bone. This loss is
often accompanied by periodontal pocket formation and changes in the density and
height of the subjacent alveolar bone. 26
DEFINITIONS
Regeneration is defined as the biologic process by which the architecture and
function of lost tissues are completely restored. With periodontal regeneration this
would mean regeneration of the lost supporting tissues of the tooth including new
alveolar bone, a new periodontal ligament and gingival structures. New connective
tissue attachment is defined as the reunion of connective tissue to a root surface that
has previously been pathologically exposed. Reattachment means the reunion of
connective tissue to a root surface exposed by incision or injury. Bone fill is defined
as clinical restoration of bone tissue in a treated periodontal defect. Bone fill does not
address the presence or absence of histologic evidence of new connective tissue
attachment or the formation of a regenerated periodontal ligament. 28
6
OSSEOUS DEFECTS:
For proper diagnosis and better treatment outcome of regenerative procedure
for periodontal osseous defects, their morphology and classification is important.
Vertical or angular defects occur in an oblique direction, leaving a hollowed-out
trough in the bone alongside the root. The base of the defect is located apical to
alveolar crest and it forms when junctional epithelium is apical to alveolar crest. 34 An
intrabony defect is defined as a ‘periodontal defect within the bone surrounded by
one, two or three bony walls or a combination of these’. 29 Periodontal disease alters
the morphologic features of the bone in addition to reducing bone height and result in
different types of bone deformities.30
Classification According to the number of osseous walls present (Figure 1)
(Goldman & Cohen 1958):31
OSSEOUS DEFECTS
SUPRABONY INFRA BONY

DEFECT DEFECT
ONE WALL DEFECT
TWO WALL DEFECT
THREE WALL DEFECT
COMBINED DEFECT
7
ONE WALL DEFECT TWO WALL DEFECT
THREE WALL DEFECT INTERPROXIMAL CRATER
Figure 2- Infrabony defects
8
Infrabony defects are usually classified according to their morphology i.e. the
number of walls present and their topographic extension around the tooth: 31,32
1) one-wall defects: defects limited by one osseous wall and the tooth surface;
2) two-wall defects: defects limited by two osseous walls and the tooth surface; and
3) three-wall defects: (originally called intrabony defect) defects limited by three
osseous walls and the tooth surface.33
Combined osseous defect:
The number of bony walls in the apical portion of the defect may be greater
than in its coronal portion i.e. three wall defect in most apical portion of the defect
and two/one wall defect in more superficial portion.34
Grading of angular defects are as follow:35
1) Shallow and narrow.
2) Shallow and wide.
3) Deep and narrow.
4) Deep and wide.
The morphology of the osseous defect is a critical factor in determining the
outcome of the regenerative procedures. The deeper the defect, greater is the amount
of clinical improvement and the wider the defect the lower the clinical attachment and
bone gain.36
9
Regeneration is better with 2 or 3 wall deep, narrow intrabony defects and
deep intraosseous craters.37 Defects presenting with a radiographic angle of 25% or
less, an intrabony component deeper than 3 mm and gingival tissues at least 1 mm
thick have the greatest chances to result in consistent amounts of clinical attachment
and bone gains, irrespective of the number of residual bony walls. The thickness of
the gingival tissues, if unfavorable, can be improved with mucogingival surgery. 38
Figure 3: Predictability of treatment outcome based on defect anatomy: 38,39,40
DEFECT ANATOMY
Gingival Thickness (≥1mm)
1-,2-,3- Wall Defect
Wide (≥37 degrees) Narrow (≤25 degrees)
Shallow (≤ 3 mm) Deep (>3 mm) Shallow (≤ 3 mm) Deep (>3 mm)
Increasing Predictability
Periodontal regeneration is defined as “the restoration of lost periodontium
or supporting tissues and includes formation of new alveolar bone, new cementum,
and new periodontal ligament.”
The ultimate goal of periodontal regeneration therapy is reconstitution of lost
periodontium. On cellular level, periodontal regeneration is a complex process
requiring coordinated proliferation of progenitor stem cells remain in PDL and
10
perivascular region of alveolar bone after tooth development and their synchronized
differentiation, and maturation to form new alveolar bone, PDL and cementum in a
sequence that these three individual tissues are integrated to function as a new
periodontal supporting apparatus.40
Regeneration of the periodontium is a continuous physiologic process. Under
normal conditions, new cells and tissues are constantly being formed to replace those
that mature and die; this is termed wear and tear repair.41
It is manifested by:
1. Mitotic activity in the epithelium of the gingiva and the connective tissue
of the periodontal ligament,
2. The formation of new bone, and
3. The continuous deposition of cementum.
Regeneration is occurring even during destructive periodontal disease as a part
of healing. Most gingival and periodontal diseases are chronic inflammatory
processes and as such are healing lesions. However, bacteria and bacterial products
that perpetuate the disease process, along with the resulting inflammatory exudate, are
injurious to the regenerating cells and tissues, thus preventing completion of the
healing process. By removing bacterial plaque and creating the conditions to prevent
its new formation, periodontal treatment removes the obstacles to regeneration and
enables the patient to benefit from the inherent regenerative capacity of the tissues. A
brief “spurt” in regenerative activity occurs immediately after periodontal treatment,
but no local treatment procedures promote or accelerate regeneration.
11
Repair
Repair simply restores the continuity of the diseased marginal gingiva and
reestablishes a normal gingival sulcus at the same level on the root as the base of the
preexisting periodontal pocket. This process, called “healing by scar” arrests bone
destruction but does not result in gain of gingival attachment or bone height. 42 This
return of the destroyed periodontium to health involves regeneration and mobilization
of epithelial and connective tissue cells into the damaged area and increased local
mitotic divisions to provide sufficient numbers of cells.
Figure 4- Sources of regenerating cells in the healing stages of

a periodontal pocket. Left, intrabony pocket. Right, after therapy the
clot formed is invaded by cells from, A, the marginal epithelium; B,
the gingival connective tissue; C, the bone marrow; and, D, the
periodontal ligament.
12
For the diseased gingiva and attachment apparatus to regain (totally or
partially) their level on the root, therapy must include special materials and
techniques. If these are not used or are not successful, tissues undergo repair only,
which involves regeneration of tissue to remodel the attachment apparatus but does
not include regaining attachment level or new bone height. For this reason, we prefer
to use the term reconstruction of the periodontium to refer to the crucial therapeutic
techniques that seek to rebuild the periodontium and result in a significant gain of
attachment and bone height.
New Attachment
New attachment is the embedding of new periodontal ligament fibers into new
cementum and the attachment of the gingival epithelium to a tooth surface previously
denuded by disease. The critical phrase in this definition is “tooth surface previously
denuded by disease”. The attachment of the gingiva or the periodontal ligament to
areas of the tooth from which they have been removed in the course of treatment (or
during preparation of teeth for restorations) represents simple healing or reattachment
of the periodontium, not new attachment. The term reattachment refers to repair in
areas of the root not previously exposed to the pocket such as after surgical
detachment of the tissues or following traumatic tears in the cementum, tooth
fractures, or the treatment of periapical lesions.
13
Figure 5- New Attachment- Enamel surface (A). Area of cementum

denuded by pocket formation (B). Area of cementum covered by junctional
epithelium (C). Area of cementum apical to junctional epithelium (D). The
term new attachment refers to a new junctional epithelium and attached
connective tissue fibers formed on area B.
Epithelial adaptation differs from new attachment in that it is the close
apposition of the gingival epithelium to the tooth surface, with no gain in height of
gingival fiber attachment. The pocket is not completely obliterated, although it may
not permit passage of a probe.43,44
The term periodontal reconstruction refers to the process of regeneration of
cells and fibers and remodeling of the lost periodontal structures that results in-
1. Gain of attachment level,
2. Formation of new periodontal ligament fibers, and
3. A level of alveolar bone significantly coronal to that present before treatment.
14
Figure 6- Epithelial adaptation after periodontal treatment. A,

Periodontal pocket. B, After treatment. The pocket epithelium
is closely adapted to but not attached to the root.
A technique to attain these ideal results has been a constant but elusive goal of
periodontal therapy for centuries.45 Since the 1970s, renewed laboratory and clinical
research efforts have resulted in new concepts and techniques that have moved us
much closer to attaining this ideal result of therapy. Melcher pointed out that the
regeneration of the periodontal ligament is the key to periodontal reconstruction
because it “provides continuity between the alveolar bone and the cementum and also
because it contains cells that can synthesize and remodel the three connective tissues
of the alveolar part of the periodontium.” 46
During the healing stages of a periodontal pocket, the area is invaded by cells
from four different sources: oral epithelium, gingival connective tissue, bone, and
periodontal ligament.
15
The final outcome of periodontal pocket healing depends on the sequence of
events during the healing stages.46 If the epithelium proliferates along the tooth
surface before the other tissues reach the area, the result will be a long junctional
epithelium. If the cells from the gingival connective tissue are the first to populate the
area, the result will be fibers parallel to the tooth surface and remodeling of the
alveolar bone with no attachment to the cementum. If bone cells arrive first, root
resorption and ankylosis may occur. Finally, only when cells from the periodontal
ligament proliferate coronally is there new formation of cementum and periodontal
ligament.46
BONE GRAFTS AND BONE SUBSTITUTES
Bone replacement grafts are widely used to promote bone formation and
periodontal regeneration. The use of bone grafts for reconstructing osseous defects
produced by periodontal disease dates back to Hegedus in 192347 and was reviewed
by Nabers & O’Leary in 1965.48
Osteogenesis occurs when viable osteoblasts and precursor osteoblasts are
transplanted with the grafting material into the defects, where they may establish
centers of bone formation. Autogenous iliac bone and marrow grafts are examples of
transplants with osteogenic properties.
Osteoconduction occurs when non-vital implant material serves as a scaffold
for the ingrowth of precursor osteoblasts into the defect. Autogenous cortical bone or
banked bone allografts may be examples of grafting materials with osteoconductive
properties.
16
Osteoinduction involves new bone formation by the differentiation of local
uncommitted connective tissue cells into bone-forming cells under the influence of
one or more inducing agents.
Bone grafting material function, in part as structural scaffolds and matrices for
attachment and proliferation of anchorage-dependent osteoblasts.49
Objectives of bone grafting: (Schallhorn, 1977) 50
1. Pocket reduction/ elimination
2. Gain in clinical attachment
3. Restoration of host alveolar bone
4. Regeneration of new bone, cementum and periodontal ligament as
determined by histologic analysis.
5. To establish a healthy maintainable environment
Characteristics of ideal bone graft materials: (Boyne, 1973)51
1. Should be readily available and not requie surgical intervention at a second
donor site.
2. Should provide rapid osteogenesis.
3. Should not elicit immunological responses.
4. Should enhance revascularization.
5. Should be highly osteoinductive.
6. Should provide osteoconduction.
17
7. Should provide for the formation of new attachment in periodontal lesion.
8. Should not impede bone growth.
The use of bone grafting or replacement materials is based on the assumption that
these materials may facilitate formation of alveolar bone, periodontal ligament and
root cementum.47
Table 1. Classification of bone graft materials (Nasr et al 1999) 52
BONE REPLACEMENT GRAFTS
HUMAN BONE
Autogenous grafts (autografts)
Extraoral
Intraoral
Allogeneic grafts (allografts)
Fresh frozen bone
Freeze-dried bone allografts
Demineralized freeze-dried bone allografts
BONE SUBSTITUTES
Xenogeneic grafts (xenografts)
Bovine-derived hydroxyapatite
Coralline calcium carbonate
Alloplastic grafts (alloplasts)
Polymers
Bioceramics
Tricalcium phosphate
Hydroxyapatite
dense, nonporous, nonresorbable
18
porous, nonresorbable (xenograft)

resorbable hydroxyapatite derived at
low temperature
Bioactive glasses
Autogenous bone grafts: According to the “American Academy of Periodontology’s
Glossary of Periodontic Terms”; an autograft is defined as a tissue graft (bone)
transferred from one position to a new position in the same individual. Autogenous
bone is often referred to as the “gold standard” grafting material since it contains both
osteoprogenitor cells and scaffolding. They can be obtained either from intraoral sites
such as edentulous ridge, maxillary tuberosity, retromolar area, extraction sockets or
from extaoral sites such as iliac crest. 53
Allografts: The inability to obtain sufficient quantities of autogenous material
at the time of surgery led to the development of several types of bone allografts. The
allografts are obtained from other individuals of the same species but different
genotype. Two types of bone allografts in routine use are mineralized freeze-dried
bone allograft and demineralized freeze-dried bone allograft. They come in different
forms like particulate, gel and putty. They provide a source of type-I collagen, which
is the sole organic component of bone. 49
Xenografts: Xenografts are grafts shared between different species. Currently,
there are two available sources of xenografts used as bone replacement grafts in
periodontics: bovine bone and natural coral. Both sources, through different
processing techniques, provides products which are biocompatible and structurally
19
similar to human bone. They are osteoconductive with the advantage of being free of
risk of disease transmission and are readily available. 52
Alloplasts: An alloplasts is a biocompatible, inorganic synthetic bone grafting
material. Alloplastic materials can be used alone in some applications, or more
commonly in combination with freeze dried or autogenous bone. At times, these
materials are used simply as volume expanders, such as when there is insufficient
volume of autogenous bone. Other times, they are used as biocompatible space fillers,
such as to occlude the gap between a dental implant and an extraction socket wall.
Another common use of these materials is to ostensible provide a ready mineral
source for new bone formation. Used alone as a single graft material, these materials
can result clinically in improved bone density and can lead to more complete bone fill
of defects via osteoconduction. Alloplastic materials used recently to reconstruct
osseous periodontal defect include bioactive glasses, bioceramics, collagen and
polymers.49
DEMINERALIZED FREEZE-DRIED BONE ALLOGRAFT
(DFDBA) BONE GRANULES
Allografts are graft transferred between genetically dissimilar members of the
same species. Urist and Strates (1965) stated that demineralizing and freeze drying
the bone greatly enhances the osteogenic potential. HCL demineralization exposes the
bone morphogenic proteins that are composed of acidic polypeptides. The bone
morphogenic proteins are located in the bone matrix and therefore are abundant in
cortical bone where bone matrix is more abundant.54
20
DFDBA was first used in dentistry and medicine in 1965 and in 1975 for the
first time it was used for the treatment of periodontal defects in humans. 55 DFDBA
provides osteoconductive surface and in addition it also acts as a source of
osteoinductive factors. So, it elicits mesenchymal cell migration, attachment and
osteogenesis when implanted in well-vascularized bone; it induces endochondral bone
formation when implanted in tissues that would otherwise not form bone. DFDBA
contains bone morphogenic proteins (BMPs) such as BMP 2, 4, and 7 which help
stimulate osteoinduction.56
Thus, commercially prepared, allograft-retained proteins have the capacity to
influence cell behavior in vivo. BMPs produce multiple effects on bone by: acting as
mitogens on undifferentiated mesenchymal cells and osteoblast precursors, inducing
the expression of the osteoblast phenotype bone cells, and acting as chemo attractants
for mesenchymal cells and monocytes as well as binding to extracellular matrix type
IV collagen.57
Advantages of allografts include:58
 Availability in adequate quantities
 Predictable results
 The elimination of an additional donor site surgery
Disadvantages of allografts include:58
 Host incompatibility
 Potentially contaminated specimens resulting in recipient site infections
 Potential transmission of disease from donor to recipient of the allograft and
impractical or biologically ineffective usefulness
21
Processing Sequence:59
1. Cortical bone is obtained in a sterile manner within 12 hours of death of the
donor. It is preferred over cancellous bone as it is less antigenic and contains
more bone inducing proteins.
2. Then the bone is fragmented into 0.5 to 1mm particles and decalcified in 0.6 N
HCL and immersed in 100% ethyl alcohol for 1 hour to remove fat and to
inactivate virus.
3. After which, bone is frozen at -80֯C for 1-2 week to interrupt the degradation
process, further decreasing the risk of disease transfer. Freeze drying removes
more than 95% of water content from bone.
4. The bone is grounded to a particle size of 250-800μm. This particle size range
is shown to promote osteogenesis, whereas a particle size smaller than 125μm
induce a macrophage response.
5. This graft material is again immersed in ethyl alcohol and washed repeatedly
to remove chemicals used in processing.
6. Then it is decalcified with 0.6 N HCL which removes Ca ++ leaving the bone
matrix and exposing the bone inductive proteins.
7. Then washed with Na phosphate buffer to remove the residual acid and stored.
Healing following the use of DFDBA follows a highly regulated cascade of
events, ultimately resulting in cellular migration, differentiation and synthesis of
bone. Although the precise origin of these progenitor cells remains unknown, it is
clear that they have the capacity to migrate and differentiate into synthetically
specialized cell types in response to signals, such as bone morphogenic proteins
present within DFDBA. Although, poorly understood, similar signaling mechanisms
22
are likely responsible for cellular recruitment, differentiation and synthesis of
cementum and periodontal ligaments. 60
When DFDBA is used in particulate form, particle size also appears to be an
important variable in the success of DFDBA as a bone inductive material. Particles in
the range of 125 to 1000 μm possess a higher osteogenic potential than do particles
below125 microns.61 Optimal particle size appears to be between 100 to 300 μm. This
may be due to a combined effect of surface area and packing density. Very small
DFDBA particles elicit a macrophage response and are rapidly resorbed with little or
no new bone formation. Tissue banks providing DFDBA for dental use usually have
this graft material in various particle sizes, and the range from 250 to 750 μm is the
most frequently available.67
Factors related to particle size that cause differences in healing include
interparticle spacing, surface area and exfoliation of osseous graft particles. 62
AMNION MEMBRANE
Amniotic Membrane (AM) is a thin patch of multi-layered human collagen and
fibronectin derived from the inner lining of the human placenta. It is composed of
3 layers: an epithelial monolayer, a thick basement membrane and underlying
stroma. Normal amniotic membrane is 0.02-0.5 mm thick, which is equivalent to 6-
8 layers of cells. It is tough and lacks blood vessels, lymphatic system and nerves.
The amnion withstands tensile forces better than the chorionic membrane because
of its rich collagen content.65
23
EMBRYOLOGY
Following implantation of the blastocyst in the endometrium, its wall becomes
the chorion, the outer layer of membranes surrounding the fetus. The innermost
trophoblastic cells, the so called amniogenic cells, form the amniotic membrane
(amnion) which is visible by day 10 post-conception. 65 The amniotic sac fills
with fluid, expands gradually and adheres to the inner surface of the chorion; the
chorionic cavity disappears but the two layers do not fuse histologically and
remain separable. The amnion is thick and hard but a flexible sheet of tissue at
full term. 65
HISTOLOGY AND PHYSIOLOGY
The Amnion and Chorion are multiple layers of collagen and are hydrophilic
(absorbs liquid). The amnion is presumably derived from embryonic ectoderm.
Human amniotic membrane is totally free of smooth muscle cells, nerve fibers
and lymphatics or blood vessels. The tractional resistance of the amniotic
membrane is related mainly to the condensed layer of interstitial collagens type
I and II.
Collagen type I is the main interstitial collagen in tissues with high
tractional resistance such as bone and tendon, but in other tissues, collagen III is
known as the main factor of integrity and firmness. 65 Elastin exists in small
amounts in the amniotic membrane and its elasticity is mainly due to collagen
type III. Owing to the presence of interstitial collagens, one of the important
properties of amniotic membrane is its resistance to proteolytic factors. 66 The
24
amniotic membrane is not just a simple avascular membrane. It has multiple
metabolic functions such as transport of water and soluble materials and
production of bioactive factors including vasoactive peptides, growth factors,
and cytokines. 66
PROPERTIES
Since 1910, hundreds of studies have found many characteristics of AM which
makes it a suitable substrate for use in surgery:
Promotion of Epithelialization
Amniotic membrane serves as a basement membrane which facilitates epithelial
cell migration, reinforces adhesion of basal epithelial cells, promotes epithelial
differentiation and prevents epithelial apoptosis. 64,67,68 It produces various
growth factors such as platelet-derived growth factor, vascular endothelial
growth factor, angiogenin, transforming growth factor beta 2 (TGF-b2),
epidermal growth factor (EGF), keratocyte growth factor (KGF) which can
stimulate epithelialization. 64 The epithelial cells of the membrane produce brain
natriuretic peptide and corticotrophin releasing hormone which have roles in
increasing cellular proliferation and calcium metabolism. 66 Amniotic membrane
can accelerate epithelial healing through several mechanisms of action
mentioned above. It also has good permeability providing sufficient
oxygenation for epithelial cells which is in contrast to many synthetic materials.
25
Figure 7- Layers of Placenta
Figure 8- Cross section of the human amniotic membrane indicating the
biochemical characteristics of the section.
26
Inhibition of Fibrosis
Fibroblasts are naturally responsible for scar formation during wound healing and are
activated by transforming growth factor β (TGF- β). Amniotic membrane inhibits
expression of TGF-β receptors in fibroblasts resulting in less fibrosis.64
Figure 9- Inhibition of Fibrosis by Amnion membrane
Inhibition of Inflammation and Angiogenesis
The exact mechanism of the anti-inflammatory properties of amniotic membrane
is not clear. It is postulated that it serves as a barrier, decreasing influx of
inflammatory cells to the affected area and consequently reducing inflammatory
mediators. When applied as a patch in vivo, amniotic membrane entraps T
lymphocytes. 64 Additionally, other anti-inflammatory factors such as tissue
27
inhibitors of metalloproteinase (TIMPs-1,2,3,4), interleukin-10 (IL-10), and IL-1
receptor antagonists as well as endostatin which inhibits endothelial cell
proliferation, angiogenesis, and tumor growth have been isolated in human
amniotic membrane. The presence of proteinase inhibitors may facilitate wound
healing. Thrombospondin-1, an antiangiogenic factor, is secreted by the
epithelium of amniotic membrane. IL-1α and IL-1β, two very potent
proinflammatory mediators, are suppressed by the stromal matrix of the amniotic
membrane. 64
Lack of Immunogenicity
Studies showed that both amniotic epithelial and mesenchymal cells and fibroblasts
express all HLA class I molecules including class Ia (HLA-A, B, C, DR) and class
Ib (HLA-G, E) antigens. However, HLA class II antigens are not expressed by
amnion epithelial cells. INFγ and other immunologic factors have been
recognized in the amniotic membrane. It seems that in the presence of viable
epithelial cells, amniotic membrane may induce immunologic reactions. One study
revealed that transplantation of fresh amniotic membrane is associated with a mild
inflammatory reaction probably due to expression of HLA-I antigens by viable
epithelial cells.
However cryopreserved amniotic membrane does not lead to immunologic
rejection. The main reason is loss of epithelial cells due to cryopreservation.
Human amniotic membrane has the ability to suppress T lymphocytes in
28
allografted limbus cells. This implies immunosuppressive properties which can
increase the success rate of grafting. 64
Antimicrobial and Antiviral Properties
Amniotic membrane may decrease the risk of infection due to antimicrobial
properties. Amniotic membrane has cystatin E, the analogue of cysteine proteinase
inhibitor, which exhibits antiviral properties.64 Amniotic membrane may function as
a barrier against bacterial infiltration by adhesion to the wound surface, prevents
dead space formation and serous discharge accumulation thereby reducing bacterial
load. In clean surgical wounds, the hemostatic property of collagen fibers in
amniotic basement membrane prevents hematoma formation reducing microbial
accumulation and thereby the risk of infection.
Furthermore, formation of fibrin filaments during wound healing results in
adhesion of the wound bed to amniotic membrane collagens leading to bacterial
entrapment and stimulation of phagocyte migration. There is a report that amniotic
membrane may decrease bacterial proliferation even in contaminated wounds. 70
Other Properties
Amniotic membrane acts as a biologic dressing resulting in significant pain relief
owing to adhesion to the wound surface and coverage of dermal nerve endings. It
also prevents wound surface drying, which accelerates wound healing.64
29
AMNIOTIC MEMBRANE PREPARATION
Amniotic membrane may be obtained under sterile conditions through elective
cesarean section after a full -term pregnancy. The donor is initially evaluated for
the possibility of blood borne infections by taking a careful history of high-risk
sexual behavior, intravenous drug abuse, blood transfusion or malignant disease
and by performing physical examination specifically for tattoos and needle marks.
After obtaining written consent, serum samples of the potential donor are tested for
anti-HIV-1 and 2 antibodies, hepatitis B surface antigen, hepatitis B core antigen,
anti-hepatitis C virus antibody, and rapid plasma reagin test for syphilis. The
serologic tests are repeated 6 months later in seronegative subjects to recognize
infections in the window period of the previous tests. Until confirmation of the
seronegative state of the donor, the amniotic membrane is preserved at -80ºC.
Absolute aseptic techniques should be applied at all stages.64
There are several methods of tissue preparation and preservation which are
described hereunder.
Heat-dried Amniotic Membrane
In this method, the tissues are dried overnight in an oven at 40±2°C. It is then
sterilized using gamma irradiation. In this method, the membrane loses many of its
biologic properties due to the high temperature employed and serves as a biologic
dressing which is often used for management of burns.70
30
Air-dried Amniotic Membrane
After separating and washing, the amniotic membrane is flattened under a lamellar
flow hood and exposed to the air overnight to get dried. Packing and sterilization
using gamma irradiation is then performed. Although high temperature is not
applied in this method, some properties of the amnion are lost or altered
remarkably due to dehydration. This type of prepared amniotic membrane is also
often used for wound dressing.71
Lyophilized (Freeze-dried) Amniotic Membrane
In this method, placental membrane is cut into pieces and rapidly frozen at -50ºC
to -80ºC. Thereafter it is dried under high vacuum using a freeze drier device.
Tissue water is extracted through sublimation reaching a final water content of 5-
10%. At the end, packing and sterilization using gamma irradiation is performed.
This type of preparation induces minimal changes in amniotic membrane properties
and the product can be stored at room temperature. The technique is complex and
more expensive than the previous two methods. This preparation is mainly used
for management of wounds. 72
Preservation in Cold Glycerol
Glycerol has been used as a cryoprotective agent for a long time. Due to its high
osmotic pressure it extracts interstitial water from the amniotic membrane. In this
method, 80% glycerol is used for drying the amniotic membrane which can
31
thereafter be preserved at 4ºC for a long time, although it loses some of its biologic
properties. This type of preserved amnion is used for dressing burn wounds. 63,73
Cryopreserved Amniotic Membrane
Under sterile conditions, the placenta is washed free of blood clots with isotonic
solutions. Next, the amniotic membrane is separated from the chorion and washed
again with isotonic solutions containing antibiotics. The amniotic membrane is
then flattened onto a nitrocellulose paper & then cut into desired sizes and placed
in a solution containing glycerol or DMSD (digital media sustainable development)
as a cryoprotective agent. Due to the toxicity of DMSD, glycerol may be
preferable.59 The tissue can next be preserved by two methods:
 The vial containing the amniotic membrane and the solution is frozen at -
80ºC.
 The tissue is packed within two layers of polycarbonate and aluminum foil
and then frozen at -80ºC.
This type of preserved amniotic membrane maintains maximum biologic
properties compared to other methods. It seems that the viability of amniotic epithelial
cells has little effect on the biologic properties of the membrane; these properties are
correlated on the integrity of its matrix.65
32
Antibiotic Impregnated Amniotic Membrane (AIAM)
After separation, the amniotic membrane is placed in an antibiotic solution composed
of 5 types of wide-spectrum antibiotics and an antifungal agent overnight and then
frozen at -80°C. The resultant amniotic membrane is suitable for management of
infected wounds by providing an appropriate concentration of antibiotics to the
wound surface.74
APPLICATIONS OF AMNION MEMBRANE
Human amnion has a long history of clinical applications. It was reported for
the first time as a biological dressing to heal skin wounds a century ago. In the
management of open wounds, the major goal is to obtain a clean and closed wound in
the shortest time possible, thereby preventing fluid, heat, and nutrient loss as well as
wound infection, pain, and decreased mobility.
Amniotic membranes are efficiently used as allografts for treating skin burns;
open and nonhealing ulcers; pressure sores; and surgical, infected, and traumatic
wounds. An alternative treatment to manage wounds in the oral cavity, such as the
tongue, buccal mucosa, vestibule, palatal mucosa, and floor of the mouth; in the
reconstruction of the oral cavity, bladder, and vagina; tympanoplasty; arthroplasty,
and so forth. Its adhesive and tight contact with the injured surface promotes
hemostasis and good pain relief due to exposition of nerve fibers. Good
biocompatibility and mechanical properties such as permeability, stability, elasticity,
flexibility, plasticity, and resorbability also make it a promising scaffolding material
33
in tissue engineering as in cell adhesion, and the potential for delivery of
biomodulatory agents such as growth factors and genetic materials.
Anti-inflammatory and anti-scarring property of amniotic membrane have
shown decreased necrosis and rapid healing of ulcers with herpes simplex virus
(HSV), varicella zoster virus infected tissues, erythema multiforme major (Stevens–
Johnson syndrome), and cervical necrotizing fasciitis. AM has been tried in the
reconstruction of temporomandibular joint ankylosis because it prevents fibrosis and
re-ankylosis when used as an interpositional material. Amniotic membrane is even
used as a carrier for local delivery of various drugs such as antibiotic netilmycin and
antiviral drugs such as acyclovir and trifluridine. Amnion has been tried as a graft
material after vestibuloplasty where it prevents secondary contraction after surgery
and maintains postoperative vestibular depth.
A novel allograft composed of amnion tissue has recently been introduced for
periodontal plastic surgery. Collected data and subjective observation by the authors
indicate that the use of processed dehydrated allograft amnion provides good results
in terms of root coverage, increased tissue thickness, and increased attached gingival
tissue. Processed dehydrated allograft amnion demonstrated excellent esthetic results
in terms of texture and color match without postoperative discomfort and adverse
reactions. The allograft has been reported to treat Grade II furcation defects with
DFDBA and xenograft by guided tissue regeneration.
34
REVIEW OF LITERATURE
Studies on Amnion membrane
Animal Studies:
Adachi K et al (2014)75 conducted an analysis of periodontal ligament (PDL)
cells cultivated on AM to determine the distribution of factors responsible for
maintaining the characteristics of PDL. Amniotic membrane was obtained from
women undergoing cesarean sections, whereas PDL tissue was obtained from human
maxillary third molars. The harvested PDL cells were maintained in explant culture
for three or four passages, following which they were cultured on AM. After 3 weeks
of culture, the PDL cells had grown well on AM. Immunofluorescence showed that
these cells were capable of proliferating and potentially maintaining their PDL-like
properties. In addition, strong cell-cell adhesion structures, namely desmosomes and
tight junctions, were shown to be present between cells. Electron microscopy images
showed that the cultured PDL cells had differentiated and proliferated on AM with
lateral conjugation and adhesion to AM. We conclude that AM may represent a
suitable substrate for culturing PDL cells and that PDL cells cultured on AM show
sheet formation.
Honjo K et al (2014)76 conducted a study in which mesenchymal stem cells
are transplanted for periodontal tissue regeneration, and the PDL is regenerated using
a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells,
growth factors, and AM. Dental pulp-derived cells can be easily obtained from
extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial
infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells
were cultured on AM as a culture substrate for immunohistochemical examination.
35
Wisdom teeth extracted from three adults were cut along the cement-enamel border.
DP tissue was collected, minced, and primarily cultured. After three or four passage
cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-
E) and immunofluorescence staining. DP-derived cells cultured on AM formed a
layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44,
105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. DP-
derived cells proliferated on AM, while retaining the properties of DP, which allowed
the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained
MSC, which suggests its potential application in periodontal tissue regeneration.
Iwasaki et al (2014)77 conducted a study to investigate the regenerative
potential of PDLSC-amnion in a rat periodontal defect model. Cultured PDLSCs were
transferred onto amniotic membranes using a glass substrate treated with polyethylene
glycol and photolithography. The properties of PDLSCs were investigated by flow
cytometry and in vitro differentiation. PDLSC-amnion was transplanted into
surgically created periodontal defects in rat maxillary molars. Periodontal
regeneration was evaluated by microcomputed tomography (micro-CT) and
histological analysis. PDLSCs showed mesenchymal stem cell-like characteristics
such as cell surface marker expression (CD90, CD44, CD73, CD105, CD146, and
STRO-1) and trilineage differentiation ability (i.e., into osteoblasts, adipocytes, and
chondrocytes). PDLSC-amnion exhibited a single layer of PDLSCs on the amniotic
membrane and stability of the sheet even with movement and deformation caused by
surgical instruments. We observed that the PDLSC-amnion enhanced periodontal
tissue regeneration as determined by micro-CT and histology by 4 weeks after
36
transplantation. These data suggest that PDLSC-amnion has therapeutic potential as a
novel cell-based regenerative periodontal therapy.
Human Studies:
Kiany F et al (2015)78 conducted a study to evaluate and compare the efficacy
of AM with deproteinized bovine bone mineral (BBM) and a collagen membrane
(CM) with BBM in GTR for the treatment of intrabony periodontal defects. Ten
chronic periodontitis patients with bilateral intrabony defects with radiographic
evidence of intrabony component ≥ 4 mm and probing pocket depths (PPDs) ≥ 6 mm
were randomly divided into two groups. The test group was treated with AM+BBM,
and the control group was managed with CM+BBM. Periodontal clinical parameters
were recorded at baseline and at 6 months after treatment. PPD, clinical attachment
level (CAL), and probing bone (PB) showed significant improvements after 6 months
in both the test and control groups. Gingival recession showed a significant increase
in the control group but not in the test group. The changes in mean PPD, PB, and
CAL preoperatively and postoperatively between the groups were not significant.
There was no significant relationship between the depth of the baseline bony defect
and CAL gain. Both AM and CM in conjunction with BBM provided improvement of
clinical periodontal parameters. AM did not induce significant gingival recession and
is suggested as a new barrier membrane in GTR treatment.
Temraz A et al (2019)79 compared the clinical and radiographic outcomes of
amnion chorion membrane (ACM) with demineralized bone matrix (DBM) in a putty
form in management of periodontal intrabony defects. Twenty-two participants with
severe chronic periodontitis and intrabony defects were randomly assigned in two
37
equal parallel groups. Each group was treated with open flap debridement (OFD) and
ACM or OFD and DBM putty. Plaque index, gingival index, PD, CAL and
radiographic measurement of bone defect area (BDA) were recorded at baseline, 3
and 6 months postoperatively. Both ACM and DBM putty demonstrated significant
improvement in all clinical and radiographic outcomes at 6 months compared to
baseline values. However, no significant difference was observed between the two
treatment modalities when compared at different time intervals. Six months
postoperatively, ACM showed PD reduction and CAL gain, while DBM putty
revealed PD reduction and CAL gain. Radiographic assessment showed that mean
baseline BDA for ACM group, which significantly reduced after 6 months. Mean
BDA mm2 in DBM putty group also significantly improved after 6 months, when
compared to baseline values. Both ACM barrier and DBM putty allograft provided
significant improvement in clinical and radiographic outcomes after 6 months, yet no
significant differences were noticed between them. This trial implied that both
biomaterials have a potential regenerative capacity in treating periodontal intrabony
defects.
STUDIES ON DFDBA
Animal studies:
Becker W et al (1995)80 performed a study to test the osteoinductive
properties of DFDBA randomly from four commercial bone banks. Twenty-five
milligrams of bone from each of the bank was implanted into the hindquarter muscles
of athymic mice. Two samples from each of the banks were compared with samples
from the other banks. A total of 16 implants were grafted into 8 mice. Two additional
38
mice served as controls. One mouse received an implantation of deactived human
cortical bone matrix (DBM). The other mouse received an implant of human bone
morphogenetic protein/non-collagenous proteins (hBMP/NCP) infused to surface
demineralized human cortical bone (positive control). At 21 days the mice were
killed, the hindquarters were photographed, and the tissues were prepared for
histologic evaluation. Of the 16 commercial DFDBA implants, 12 were available for
histologic evaluation. There was no radiographic evidence of bone formation for the
DFDBA implanted mice or the DBM implants. Small bone ossicles were scarcely
visible in the hindquarters of the mouse which received the hBMP/NCP infused bone.
The result of this exploratory study questions the continued use of commercially
available DFDBA for bone induction in periodontal defects and adjacent to dental
implants. One potential cause might be that bone induction proteins are not present in
sufficient quantity to produce detectable bone formation. Another possibility is that
the bone inductive components of DFDBA are present but in an inactive form.
Carnes L et al (1999)81 conducted a study to develop a rapid reliable method
for assessing the induction ability of DFDBA in vitro. They characterized the
response of 2T9 cells, an immature osteoprogenitor cell line derived from the
calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone
morphogenic protein (BMP-2) promoter to recombinant human BMP-2 by measuring
alkaline phosphates specific activity. They also tested the hypothesis that the radio
opacity of tissue following implantation of DFDBA in vivo correlated with the ability
of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by
age, were implanted subcutaneously in the thorax of 18 nude mice. Tissue was
harvested at 36 days postoperatively and examined histologically and biochemically
for calcium and phosphorous uptake. In conclusion the calcium and phosphorous
39
uptake by DFDBA implanted, tissue correlated with the age dependent decrease in
bone induction. These data showed that early x-rays might actually detect
remineralization and not new bone formation.
Human studies:
Libin BM et al (1975)82 presented three case reports of patients with severe
periodontal defects treated with a decalcified, lyophilized bone allograft. Two patients
received grafts of cancellous bone and one patient received a cortical bone graft. The
patients were observed up to 2 years following implantation. The results showed that
the implantation of decalcified, lyophilized bone allograft of both the cortical and
cancellous bone types resulted in new bone formation and a gain in attachment level
and no evidence of rejection of graft material for up to 2 years following implantation.
Pearson GE et al (1981)83 conducted a controlled pilot study to evaluate the
effectiveness of decalcified freeze-dried cancellous bone allograft material in the
treatment of intrabony periodontal defects in humans. Twenty-two defects were
selected for the study, out of which 16 defects were randomly selected for grafting
while the remaining 6 served as controls. Results showed a significant gain in
attachment with the allografting procedure but not with the control procedure, which
consisted of flap and curettage only.
Quintero et al (1982)84 evaluated the osteogenic potential of DFDBA in the
treatment of human periodontal osseous defects over a 6-month period. Cortical bone
obtained under sterile conditions from a human donor within 24 hours after death, was
decalcified, freeze dried and grounded to particle size of 250-500 microns. 27 osseous
defects with one, two and wide three wall morphology were treated. Clinical
40
measurements were made before surgery, at the time of surgery and re-entry. The
results showed a combined mean osseous regeneration for all defects was 2.4mm.
This represented a 65% mean bone fill of the original defect.
Werbitt M et al (1987)85 presented several case reports where DFDBA had
been used to treat advanced intrabony defect and where new bone formation had
occurred. A total of 20 defects were treated, and at the nine-month evaluation, the six
cases presented in this report had minimal probing depth and showed radiographic
evidence of substantial bone fill. The amount of repair ranged from 75% to 95% of
the original defect. Bone fill was achieved on both vital and non-vital teeth and in
some cases, there was a radiographic evidence of a lamina dura and a discernable
periodontal ligament space. Furthermore, the teeth that were initially mobile showed a
decrease in mobility.
Fucini SE et al (1993)86 conducted a study to compare the bony defect
resolution by using two different particle size of DFDBA. Cortical bone of final
particle sizes of 250μ to 500μ or 850μ to 1000μ were obtained. Paired interproximal
intrabony periodontal defects in 11 patients were grafted with DFDBA. Soft and hard
tissue measurements were made using an electronic constant force probe at the initial
and reentry surgeries at 6 months. Treated sites in 10 patients were reevaluated by
reentry approximately 6 months post operatively. The results showed no statistically
significant difference in bony fill between defects grafted with the different particle
sizes of DFDBA when used in humans.
Persson et al (2000)87 conducted a study to assess changes in intra-bony
defects after either osseous surgery or open flap debridement in combination with
grafting procedures with DFDBA. Pre- and post-surgical computer digitized images
of intra-oral radiographs from 60 patients who had received periodontal surgery to
41
manage intrabony defects were analyzed by linear measurements. A total of 36
patients were treated with osseous surgery and had received flap procedures with
DFDBA. Post-surgical radiographs were obtained. Results showed a minor mean
bone fill for osseous surgery sites and 0.5 mm for DFDBA sites.
Studies comparing DFDBA with other regenerative materials:
Bowen JA et al (1989)88 conducted a study to compare the healing potential
of the osteoinductive DFDBA with an osteoinductive porous hydroxyapatite (HA). A
total of six patients were selected for the study. Soft and hard tissue measurements
were taken at baseline and 6 months re-entry. There was no significant difference in
any of the soft tissue measurements when DFDBA and HA were compared. There
was a defect fill of 61% for DFDBA and 53% for HA which was not statistically
significant.
Rummelhart JM et al (1989)89 performed a comparative study to evaluate
freeze-dried bone allograft (FDBA) with demineralized freeze-dried bone allograft
(DFDBA) in intrabony defect. 22 defects in 9 patients were grafted with either
DFDBA or FDBA. Evaluations were based on standardized radiographs, presurgical
and post-surgical measurements using the cementoenamel junction as fixed reference
point and osseous measurements at the time of surgery. At reentry after 6 months a
mean osseous repair of 59% occurred with DFDBA and 66% with FDBA. Findings
revealed significant differences between the two materials in primarily intraosseous
defects when evaluated at a minimum 6 months post-surgery.
Francis et al (1995)90 evaluated an allogenic bone matrix (ABM) as a graft
material for the treatment of periodontal osseous defects. Paired osseous defects in 11
42
patients were randomized to receive ABM or DFDBA. Soft tissue and hard tissue
measurements were determined at baseline and 6 months reentry procedure.
Standardized radiographs were taken. Results of radiographic analysis suggested
similar density changes with each graft. These results demonstrated that both the
treatments were effective and that ABM may be a useful graft material in the
treatment of periodontal osseous defects.
Masters LB et al (1996)91 evaluated the use of DFDBA reconstituted with
50mg/ml tetracycline hydrochloride in the treatment of intrabony periodontal defects.
15 patients with three osseous defects each were selected for the study. Each site in
each subject was randomly assigned to one of the following groups: 1) DFDBA +
TCN, 2) DFDBA, 3) Debridement. Clinical measurements were taken on the day of
surgery, at 6months and 1 year. Radiographs were taken at baseline and 1 year and
were evaluated by CADIA. Osseous defect measurements were taken at baseline and
1-year reentry. Results showed statistically significant improvement in probing depth
and attachment level at 1 year. Although the grafted groups showed greater bone fill
and defect resolution there was no statistically significant difference in any of the
clinical parameters between the treatment groups. There is no significant benefit from
reconstituting the allograft with 50mg/ml of tetracycline hydrochloride.
Laurell L et al (1998)92 reviewed the studies presented during the last 20
years on the surgical treatment of intrabony defects. The treatments included open
flap debridement, open flap debridement with DFDBA, FDBA or autogenous bone
and GTR. The review included only those studies that presented baseline and final
data on probing depths, intrabony defect depth as measured during surgery, clinical
attachment level gain and or bone fill. Result of meta-analysis showed that open flap
debridement alone resulted in limited pocket reduction, clinical attachment level gain
43
averaged 1.5mm and bone fill 1.1mm. Open flap debridement plus bone graft resulted
in limited pocket reduction; clinical attachment level gain and bone fill averaged
2.1mm. Guided tissue regeneration resulted in significant pocket reduction, clinical
attachment level gain of 4.2mm and bone fill averaging 3.2mm.
Parashis et al (1998)93 performed a study to compare clinically and
radiographically the effectiveness of guided tissue regeneration, using a bioabsorbable
polylactic acid softened with citric acid ester barrier and commercially available
DFDBA in the treatment of 2 and 3 wall intrabony defects. 12 patients, each with one
treated defect comprised each group. Soft tissue measurements and hard tissue
measurements were taken and were comparable in both the groups at baseline. They
were repeated at 12 months. Results showed no statistically significant differences
between the 2 groups; only the exception was in the radiographic resolution of defect,
which was significantly greater in the GTR group.
Froum S et al (2002)94 compared healing extraction sockets 6 to 8 months
post implantation of a bioactive glass or DFDBA to an unfilled socket control. 30
sockets in 19 patients were randomly divided into 3 treatment groups. 10 sockets
received bioactive glass, 10 sockets DFDBA, and 10 sockets served as unfilled
controls, and histological cores of the treatment sites were obtained. Results
concluded that although the differences in percent vital bone were not statistically
significant among the 3-treatment group, bioactive glass material was observed to act
as an osteoconductive material and had a positive effect on socket healing at 6 to 8
months post extraction. Residual implant material was significantly higher in DFDBA
treated sockets versus bioactive glass treated sockets.
Reynolds MA et al (2003)95 systemically reviewed the efficacy of bone
replacement grafts in proving demonstratable clinical improvements in periodontal
44
osseous defects compared to surgical debridement alone. The therapeutic end points
examined included changes in bone level, clinical attachment level, probing depth,
gingival recession and crestal resorption with respect to the treatment if intrabony
defect, the results of meta-analysis supported the following conclusions; 1). Bone
grafts increased bone level, reduced crestal resorption, increased clinical attachment
level, and reduce probing depth compared to OFD procedures 2). Bone grafts in
combination with barrier membranes increased clinical attachment level and reduced
probing depth compared to graft alone 3). Histologically DFDBA supported the
formation of a new attachment apparatus in intrabony defects, whereas OFD resulted
in repair characterized by long junctional epithelium. The results of this systemic
review indicated that bone replacement grafts provided demonstrable clinical
improvement in periodontal osseous defects compared to surgical debridement alone.
Gurinsky BS et al (2004)96 conducted a study to evaluate the use of DFDBA
in combination with Enamel matrix derivative compared to enamel matrix derivative
alone in the treatment of human periodontal osseous defects. 40 patients with a total
of 67 sites were selected for the study; each subject received either enamel matrix
derivative alone, (34 sites) or enamel matrix derivative in combination with DFDBA
(33sites). Both soft tissue and hard tissue measurements were taken at baseline and
6months. Results showed significant improvements in soft tissue parameters for both
treatment groups as compared to preoperative measurements. There was no statistical
difference between the two groups.
Reidy et al (2004)97 conducted a study to establish whether there was a
significant difference in hard tissue fill of intrabony defects following treatment with
either calcium sulphate or ePTFE in combination with DFDBA. 19 patients with 38
defects were selected for the study. Soft tissue and hard tissue measurements were
45
recorded at baseline and 6 months. Defects were randomly treated with either a
combination graft of DFDBA with calcium sulphate covered by a calcium sulphate
barrier or with DFDBA and fitted with an ePTFE barrier. Results of this study
indicated that calcium sulphate, when used as a binder and barrier in combination
with DFDBA, supported significant clinical improvement in intrabony defects.
Calcium sulphate represented an alternative to non-resorbable ePTFE barrier in
combination with DFDBA for the treatment of intrabony defects.
Bender et al (2005)98 performed a study to determine the effectiveness of
DBX paste and putty compared to DFDBA in the treatment of human intraosseous
periodontal defects. Sixty systemically healthy individuals between the ages of 31 and
71 years with at least one intraosseous periodontal defect of ≥3 mm in depth and
radiographic evidence of at least 40% to 50% vertical bone loss were accrued.
Following initial non-surgical periodontal therapy, sites were randomly selected to
receive either DBX paste, DBX putty, or DFDBA (control). Baseline and 6-month
reentry soft and hard tissue parameter measurements were made. Results showed that
Probing depth reductions and attachment level gains were significantly improved in
all treatment groups. Bone fill was similar between all groups with DBX paste, putty,
and DFDBA control groups demonstrating 2.0 mm, 2.4 mm, and 2.2 mm,
respectively.
Ilgenli et al (2007)99 did a comparative study to evaluate the clinical and
radiographic outcomes of DFDBA/platelet-rich plasma (PRP) combination with PRP
alone for the treatment of infrabony defects and to examine the influence of
radiographic defect angle on the clinical and radiographic outcomes. Twenty-eight
infrabony defects were treated with DFDBA/PRP combination or PRP alone. Clinical
parameters and radiographic measurements were compared at baseline and 18
46
months. The results showed more favorable gains in both clinical and radiographic
parameters with DFDBA/PRP combination than PRP alone group A correlation
existed between defect angle, defect depth, and clinical/radiographic outcomes for the
defects treated with DFDBA/PRP. The narrow defects presented more favorable CAL
gain, probing pocket depth (PPD) reduction and defect resolution than wide defects in
the combination group.
Aspriello et al (2011)100 did a study to compare the use of enamel matrix
derivative (EMD) and DFDBA with DFDBA alone for the treatment of human
periodontal intrabony defects at 12 months post-surgery. Fifty-six intrabony osseous
defects in 56 periodontitis patients were randomly assigned to the test group (DFDBA
+ EMD) or the control group (DFDBA) for periodontal treatment. Clinical and
radiographic measurements were made at the baseline and after 12 months. Compared
to baseline, the 12-month results indicated that both treatment modalities resulted in
significant changes in all clinical parameters (gingival index, bleeding on probing,
PD, CAL, gingival recession and radiographic parameters (hard tissue fill (HTF) and
bone depth reduction). Furthermore, statistically significant differences were found in
the test group compared to the control group in PD reduction, CAL gain, and HTF.
Agarwal et al (2013)101 performed a cohort study to evaluate the regenerative
outcomes of bone graft with or without local doxycycline in non-contained infrabony
periodontal defects. 16 one or two wall intrabony defects, in 11 patients suffering
from moderate to severe chronic periodontitis, aged 35-60 years, were randomly
divided for bone graft, alone (control) and with doxycycline (test) for the study. At
baseline, after 3 months and after 6 months of post-operative period, PPD, CAL,
radiological bone fill (RBF) and alveolar height reduction were recorded. The results
47
showed no added benefits of local doxycycline, as compared with bone graft alone,
for regeneration of non-contained human periodontal infrabony defects.
Bansal et al (2013)102 did a study to clinically evaluate and compare the
efficacy of autologous PRF combined with DFDBA to DFDBA alone in the treatment
of periodontal intrabony defects. 10 patients having two almost identical intrabony
defects with clinical probing depth of at least 6 mm were selected for the study.
Selected sites were randomly divided into two groups. In Group I, mucoperiosteal flap
elevation followed by the placement of DFDBA was done. In Group II,
mucoperiosteal flap elevation followed by the placement of homogeneous mixture of
PRF with DFDBA was done. Clinical and radiographic parameters were recorded at
baseline and at 6 months post‑operatively. Results showed both treatment groups had
a significant probing pocket depth reduction, clinical attachment gain, defect fill, and
defect resolution 6 months after surgery compared to baseline. However, there was a
significantly greater probing pocket depth reduction and clinical attachment gain
when PRF was added to DFDBA.
Katuri et al (2013)103 evaluated the efficacy of DFDBA and bioactive glass
clinically and radiographically in periodontal intrabony defects for a period of 12
months. Ten systemically healthy patients diagnosed with chronic periodontitis, with
radiographic evidence of at least a pair of contralateral vertical osseous defects were
included in this study. Defect on one side was treated with DFDBA and the other side
with bioactive glass. Clinical and radiographic measurements were made at baseline 6
month and 12 months after the surgery. Compared to baseline, the 12-month results
indicated that both treatment modalities resulted in significant changes in all clinical
parameters (gingival index, probing depth, CAL and radiographic parameters (bone
48
fill), however, sites treated with DFDBA exhibited statistically significantly more
changes compared to the bioactive glass in probing depth reduction and CAL gain.
Kher et al (2013)104 conducted a study to compare the effectiveness of GTR
by using a collagen membrane barrier with or without DFDBA in the treatment of
periodontal infrabony defects characterized by unfavorable architecture. Sixteen
systemically healthy patients with 20 periodontal infrabony defects were selected for
the study. Baseline measurements included plaque index, papillary bleeding index,
PPD, gingival recession, clinical attachment level and radiographic defect depth
(DD). At the time of surgery, the defects were randomly assigned to either the test
group (collagen membrane plus DFDBA) or the control group (collagen membrane
only). At the 6-month examination, result showed PPD reduction was significantly
greater in the test group compared with the GTR group. Radiographic DD reduction
was greater in the test group compared with the GTR group.
Khosropanah H et al (2015)105 conducted a study on 12 patients with two
comparable bilateral intrabony defects. Each pair of defects was randomly treated
with DFDBA+PRP (test) or DFDBA alone (control). CAL, intrabony defect depth
(IDD), distance from the stent to the alveolar crest and PD as well as radiographic
parameters including the radiographic defect depth, width and angulation were
measured at baseline and six months post-operatively. This study showed that both
treatments resulted in significant PD reduction, CAL gain and IDD reduction. Also,
PRP failed to enhance the results obtained by DFDBA.
Agrawal A et al (2015)106 conducted a study to determine the additive effects
of PRF with a DFDBA in the treatment of human intrabony periodontal defects. Sixty
interproximal infrabony defects in 30 healthy, non-smoker patients diagnosed with
chronic periodontitis were randomly assigned to PRF/DFDBA group or the
49
DFDBA/saline. Clinical [PD, CAL and gingival recession (REC)] and radiographic
(bone fill, defect resolution and alveolar crest resorption) measurements were made at
baseline and at a 12-month evaluation. Compared with baseline, 12-month results
indicated that both treatment modalities resulted in significant changes in all clinical
and radiographic parameters. However, the PRP/DFDBA group exhibited statistically
significantly greater changes compared with the DFDBA/saline group in PD, CAL,
REC, bone fill and defect resolution. Observations indicate that a combination of
PRF and DFDBA is more effective than DFDBA with saline for the treatment of
infrabony periodontal defects.
Chadwick J K et al (2016)107 conducted a study to evaluate changes in
clinical attachment level and bone fill of periodontal intrabony defects treated with
DFDBA compared to platelet-rich fibrin (PRF) in humans. Thirty-six patients
completed the study protocol. Each patient contributed a single intrabony defect,
which was randomized to receive either DFDBA or PRF. Clinical and standardized
radiographic data were collected at baseline and 6 months after treatment. Primary
outcomes measures included radiographic bone fill as measured from the CEJ to base
of bony defect, and change in CAL. Both treatment groups had significant gains in
CAL as well as bone fill, with no significant differences in outcomes between groups.
Treatment of intrabony defects with either DFDBA or PRF resulted in a significant
gain in CAL as well as bone fill after 6 months of healing, with no significant
difference between materials.
Agrawal P et al (2016)108 evaluated the effectiveness of PRP alone in
infrabony defects. Thirty infrabony defects were treated with either autologous PRP
with OFD or autologous PRP + DFDBA with OFD or OFD alone. Clinical parameters
recorded were gingival index, plaque index, PD, CAL, and REC. Radiographic
50
parameters included defect depth reduction, defect resolution, and crestal bone level.
All the parameters were recorded at baseline and 12 months postoperatively. Mean
PD reduction and CAL gain were greater in PRP + DFDBA and PRP groups than the
control group. Within the limits of the study, all the three groups showed significant
improvement in clinical parameters from baseline to postoperative 12 months. The
amount of defect depth reduction and defect resolution treated with PRP alone group
were significantly <PRP + DFDBA. The results pertaining to these parameters were
significantly better than the control group.
Kothiwale S et al (2019)109 conducted a study to evaluate and compare
clinically and radiographically freeze-dried bone and demineralized freeze-dried bone
block allografts with chorion membrane in intra-bony defects at 12 months post-
surgery. Eighteen intra-bony defects (9 intra patient pairs) in 9 patients with chronic
periodontitis were randomly assigned to group 1 (FDBA + Chorion membrane) and
group 2 (DFDBA + Chorion membrane) for periodontal therapy. Clinical and
radiographic (RVG) measurements were made at base line and 12 months. Data
obtained was subjected to statistical analysis. At 12 months on intra-group
comparison both the groups showed statistically significant improvement in the
plaque and gingival indices with reduction in the mobility, probing pocket depth and
gain in clinical attachment. Radiographs showed significant bone fill and increased
bone density However, group 1 (FDBA) showed increase in the bone density which
was statistically significant. The use of the FDBA and DFDBA block allografts
showed significant improvement in the periodontal prognosis of teeth with intra-bony
defects.
51
Atchuta A et al (2020)110 evaluated the efficacy of PRF, DFDBA, and their
combination in the regeneration of intrabony defects. A total of 39 sites with
intrabony defects were randomly assigned into three groups: (Group I ‐ Open flap
debridement, Group II ‐ DFDBA alone, and Group III‐ DFDBA + PRF). Parameters
such as PPD, relative attachment level (RAL), and radiographic bone fill were
measured at baseline, 3 months, and 6 months. Reduction in the PPD and greater
difference in RAL was observed over the study period in all the three groups with
greater reduction in DFDBA + PRF group. Reduction in the radiographic defect
depths was observed over the study period in all the three groups with the greatest
reduction in the DFDBA + PRF group. However, no statistically significant difference
was reported by DFDBA versus DFDBA + PRF group. Combination of DFDBA and
PRF improved the clinical and radiographic parameters compared to PRF and
DFDBA alone. PRF was combined with DFDBA to produce a synergistic effect for
treating intrabony defects in chronic periodontitis patients.
52
MATERIALS &
METHOD
Materials and method
MATERIALS AND METHOD
Systemically healthy patients with moderate to severe chronic periodontitis
were selected for this study from the Outpatient Department of Periodontology,
Rungta College of Dental Sciences and Research, Bhilai, Chhattisgarh.
Study Design:
The study was double blinded, randomized controlled clinical trial. Patients diagnosed
with chronic periodontitis based on clinical and radiographical criteria proposed by
the 1999 International World Workshop were selected for the study. 111 A total of 40
sites were selected for this clinical study, and Phase I therapy was performed in all the
patients. Four weeks following Phase I therapy a periodontal re-evaluation was
performed to confirm the suitability of the sites for the study. The selected sites were
randomly divided into Group I and Group II according to the type of treatment
rendered to them.
Group I – 20 surgical sites treated with Demineralized Freeze-Dried Bone Allograft
(DFDBA).
Group II – 20 surgical sites treated with Demineralized Freeze-Dried Bone
Allograft (DFDBA) combined with Amnion membrane (AM).
Duration of study:
Study was carried out for 6 months with clinical evaluation at baseline, 90th day and
180th day and radiographical evaluation at baseline and 180th day.
53
Sample Size:
40 sites (20 sites in each group) with intrabony defects were included in the study.
Sample size determination:
Sample size was estimated using SPSS software.
The minimum sample required was calculated taking into consideration the following:
N = minimum required sample size in each of the group
D = difference in mean = 0.86
SD2 = Squared pooled deviation = 0.346
1.96 = conventional multiplier for alpha 0.05
1.26 = conventional multiplier for power 90%
2(1.96+1.26)2 𝑆𝐷2
Minimum sample size is N = =2(10.36) (0.346)/ (0.86)2
𝐷2
= 10 minimum sites
Appling Boneferroni correction for missing data management, a total of 40 sites (20
sites in each group) with intrabony defects was selected for this study.
Sampling Technique-
The sampling technique to be employed was purposive sampling technique.
INCLUSION CRITRIA
1. Age group of 25-55 years.
2. Presence of at least one radiographical detectable interproximal intraosseous
defect with a probing depth ≥ 6 mm following initial therapy.
54
3. Depth of the intraosseous vertical component of the defect ≥ 4 mm as
measured by radiographic means.
4. Presence of at least 2 mm of keratinized tissue around the teeth associated with
the defect.
5. Systemically healthy patients.
EXCLUSION CRITERIA
1. Subjects taking medications, such as corticosteroids, calcium channel blockers
or immunosuppressive drugs, which are known to interfere with periodontal
wound healing.
2. Subjects allergic to medications.
3. Subjects who are pregnant or lactating.
4. Smokers /Tobacco users.
5. Subjects showing poor oral hygiene maintenance after initial phase I therapy.
Method of Collection of Data:
The proposed study was carried out on patients with chronic periodontal disease as
assessed by clinical and radiographical findings. Patient’s verbal and written informed
consent was obtained from all the patients before the commencement of the study. A
brief history of each patient was recorded on a case history proforma. Four weeks
following phase I therapy a periodontal reevaluation was performed to confirm the
suitability of the sites for the study. (Annexures)
55
CLINICAL PARAMETERS
The clinical parameters used for assessment before and after surgical procedures
were:
1. Plaque Index (PI) (Silness and Loe, 1964)112, 113
2. Gingival Index (GI) (Loe and Silness, 1963) 112, 113
3. Probing pocket depth (PPD)114
4. Relative attachment level (RAL)115
5. Gingival recession (GR)116
Radiographic evaluation:
1. Orthopantomogram (OPG)
2. Digital Radiovisiography (RVG) with millimeter grid mount.
OPG, RVG and routine hematological investigations were done preoperatively
at baseline. At 90th day follow-up all the clinical parameters were evaluated. At 180th
day follow-up the clinical parameters as well as the radiographical parameters were
evaluated.
On the day of surgical procedure, before administering local anaesthesia,
Plaque Index (Silness and Loe, 1964) and Gingival Index (Loe and Silness, 1963) of
the tooth of interest were recorded as follows:
56
Plaque Index (Silness and Loe, 1964) 112, 113
Plaque Index was evaluated on the cervical third of the tooth with no attention
to the plaque that extended to the middle or incisal thirds. The explorer was passed
across the tooth surface in the cervical third near the entrance of the sulcus.
Table 2- Scoring criteria for Plaque Index
Score Criteria
0 No plaque in the gingival area.
1 A film of plaque adhering to the free gingival margin and adjacent area of
the tooth. The plaque may only be recognized by running a probe across
the tooth surface.
2 Moderate accumulation of soft deposits within the gingival pocket, on the

gingival margin and/or adjacent tooth surface, which can be seen by the
naked eye.
3 Abundance of soft matter within the gingival pocket and/or on the

gingival margin and adjacent tooth surface.
The evaluation of scored was done at four areas i.e. the distal-facial, facial,
mesial-facial and lingual surface in the cervical third of the tooth. The scored around
57
each tooth was added and divided by four, the plaque index score for the tooth was
obtained.
Table 3- Suggested Nominal Scale for Patient Evaluation for Plaque Index
Rating Scores
Excellent 0
Good 0.1-0.9
Fair 1.0-1.9
Poor 2.0.3.0
Gingival Index (Loe and Silness, 1963) 112, 113
The severity of gingivitis is scored on all surfaces of teeth as done for bleeding
on probing. A blunt instrument, such as a periodontal probe was used to assess the
bleeding potential of the gingiva.
Table 4- Scoring criteria for Gingival Index
Score Criteria
0 Absence of inflammation/Normal gingival
58
1 Mild inflammation; slight change in color, the gingival margin is slightly
more reddish or bluish-red than normal, slight edema, a colorless gingival
exudate may be observed, no bleeding on probing.
2 Moderate inflammation; gingival is red or bluish-red, there is moderate
glazing, there is enlargement of the margin dur to edema, bleeding is
provoked on probing.
3 Severe inflammation; marked redness and hypertrophy ulceration,
tendency to bleed spontaneously
For scoring purpose, the tissues surrounding each tooth were divided into four
gingival scoring units: distal-facial papilla, facial margin, mesial-facial papilla and the
entire lingual gingival margin. The scores around each tooth was added and divided
by 4; the Gingival Index score for the tooth was obtained.
Table 5- Suggested Nominal Scale for Patient Evaluation for Gingival Index
Gingival Index Scores Condition
0.1-1 Mild Gingivitis
1.1-2.0 Moderate Gingivitis
2.1-3.0 Severe Gingivitis
59
Probing Pocket Depth (PPD), Relative Attachment Level (RAL) &
Gingival Recession (GR):114, 115, 116
An occlusal stent was fabricated with self-cure acrylic resin on a dental stone cast
obtained from an alginate impression for positioning of the periodontal probe
(University of North Carolina No. 15 Probe, Hu-Friedy, USA). The occlusal stent was
made to cover the occlusal surfaces of the teeth being treated and were extended on
the buccal and lingual surfaces to cover the coronal third of the teeth involved. A
groove was made in the inter-dental regions, such that the position and angulation of
the probe remained the same for all pre- and post-operative measurements. An
imaginary line was drawn on the bucco-occlusal line angle of the teeth using a black
permanent marker. Using the groove as a guide, the periodontal probe was inserted
into the periodontal pocket until resistance was encountered and PPD (using the
gingival margin as reference), and RAL (using the marking in the stent as reference)
were recorded.
GR was measured from fixed reference point (marking in the stent) to the
gingival margin by UNC – 15 Probe.
PPD, RAL & GR were recorded at baseline i.e. after the completion of phase I
therapy and post-operatively at 90th day and 180th day.
Radiographic Measurements:8
Digital intraoral periapical radiographs were taken for all the sites at baseline and
180th day. The radiographs were standardized by using long cone paralleling
technique with the digital radiovisuography (RVG) with 70 KVp, 10 mA and
60
exposure time of 0.8 seconds. The radiographic linear bone growth/defect fill were
measured using millimeter grid mount.
Radiographical vertical osseous defect depth was calculated as the linear
distance (in mm) measured with the following formula at baseline and at 180th day
follow-up.
At baseline: -
vertical osseous defect depth = (FRP to BOD) – (FRP to AC)
AC = Alveolar crest
BOD = Base of the bone defect
FRP = Fixed reference point
At 180th day: -
Changes in alveolar crest level = (FRP to AC at baseline) – (FRP to AC at 180th day)
Amount of defect fill (ADF) = Initial defect depth – defect depth at 180th day
𝐀𝐦𝐨𝐮𝐧𝐭 𝐨𝐟 𝐝𝐞𝐟𝐞𝐜𝐭 𝐟𝐢𝐥𝐥 𝐚𝐭 𝟏𝟖𝟎𝐭𝐡 𝐝𝐚𝐲

Percentage of defect fill (%) = × 100
𝑩𝒂𝒔𝒆𝒍𝒊𝒏𝒆 𝒅𝒆𝒇𝒆𝒄𝒕 𝒅𝒆𝒑𝒕𝒉
61
Figure 10- Defect Landmarks
CEJ
AC
BD
Figure 11- Defect landmarks in the radiograph
62
ARMAMENTARIUM
1. Mouth mirror
2. Straight probe
3. Explorer
4. Tweezer
5. University of North Carolina (UNC-15) Periodontal probe (Hu-Friedy,
USA)
6. Disposable gloves
7. Disposable mouth mask
8. Disposable syringes
9. Local anesthetic solution (2% lignocaine HCL with 1:80,000 adrenaline)
10. Bard-Parker handles no. 3 with no. 11, 12 and 15 blades
11. Periosteal elevator
12. Hand scalers
13. Gracey Curettes (no.1-14) (Hu-Friedy, USA)
14. Universal Curettes (Columbia 4R,4L) (Hu-Friedy, USA)
15. Graft carrier
16. Membrane placement instrument
17. Castroveijo scissors
18. Goldman-Fox scissors
19. Tissue forceps
20. Needle holder
21. Suture cutting scissors
22. Curved scissors
63
23. Sterile gauze
24. Kidney tray
25. 5% Povidone iodine
26. 0.9% Saline
27. 0.2% Chlorhexidine digluconate
28. Amnion membrane (Tissue bank, TATA Memorial Hospital & Research
Center, Mumbai)
29. DFDBA bone granules (Tissue bank, TATA Memorial Hospital & Research
Center, Mumbai)
30. Dappen dish
31. Suture material (Mersilk, No. 3-0, Ethicon, Johnson & Johnson, India)
32. Periodontal dressing (Coe-pakTM, GC America Inc. Alsip, II, USA)
TREATMENT PROTOCOL
1. Presurgical Procedure:
Initial therapy consisting of full mouth supragingival and subgingival scaling and
root planning was carried out. Detailed instruction regarding self-performed plaque
control measures were given to the patients.
After four weeks, study subjects were recalled and only those maintaining
optimum oral hygiene were further subjected to the surgical procedure. The PPD,
RAL and GR were recorded for each selected site in all patients.
64
2. Surgical Procedure:
Prior to the surgical procedure, the patients were instructed to rinse with 0.2%
chlorhexidine gluconate for 1 minute. The surgical protocol emphasized complete
asepsis and infection control.
Incision and Flap Reflection: -
The operative site was anaesthetized with 2% lignocaine HCL with adrenaline
(1:80,000). After achieving adequate anesthesia, crevicular incisions were given
(extending one tooth on either side of the tooth of interest) on the facial and lingual
sides reaching the tip of the interdental papilla using B.P knife with blade no. 11 and
an interdental incision with blade no. 12. A full thickness mucoperiosteal flap was
reflected using the periosteal elevator, taking care that the interdental papillary tissue
was preserved or retained as much as possible. Meticulous defect debridement and
root planing was performed using hand instruments and area-specific curettes. No
osseous recontouring was performed. Suture was passed through the elevated flaps as
per the relevant technique prior to performing the regenerative procedure. In Group I,
the intrabony defects was treated with DFDBA allograft and material was filled up to
the level of alveolar crest. In Group II, the intrabony defects was treated with
DFDBA allograft and covered with dry AM. Prior to the regenerative material
placement an aluminum foil template was cut in to the desired amount according to
the osseous defect. Then the AM was trimmed with the help of curved scissors as the
template. After coming in contact with blood dry AM quickly became supple by
hydration and was adapted with hand instruments to cover DFDBA allograft. The AM
was extended ≥ to 3mm beyond defect border and was allowed to touch adjacent root
surfaces. If the AM barrier folded on itself, no attempt was made to unfurl the barrier.
65
After placement of AM, the mucoperiosteal flaps was repositioned and secured in
place using 3-0 non-absorbable black braided silk surgical suture. Direct loop sutures
were placed at the defect site and figure of eight in the adjacent sites. The surgical
area was protected and covered with non-eugenol periodontal dressing. Pressure was
applied to the surgical sites for 1minute with the moistened gauze.
Post-operative care:
After the surgical procedure, patients in both groups were prescribed
medications. Analgesics and anti-inflammatory drugs (Paracetamol 500mg,
Aceclofenac 100 mg, Serratiopeptidase 15mg twice a day) and antibiotics
(Amoxicillin 500mg thrice a day) for 5 days post-operatively. Then postoperative
instructions were given to the patients.
Post-operative recall visits:
Patients were recalled after one-week post operatively for removal of
periodontal dressing and the sutures. Symptoms regarding discomfort, pain and
sensitivity were recorded. Any sign of swelling, inflammation, infection, flap
displacement, hematoma and necrosis were noted and dressing was replaced for
another one week, if necessary. Recall appointments were given at 90th day and 180th
day post-surgery and the relevant clinical and radiographic parameters were recorded
during these visits. At each visit, oral hygiene instructions were reinforced.
66
STATISTICAL ANALYSIS
Statistical software: The Statistical software namely SPSS v.16.0, was used for the
analysis of the data and Microsoft office and Excel programs used to generate graphs,
tables etc.
Statistical Methods: Descriptive statistical analysis was carried out in the present
study. Results on continuous measurements were presented on Mean  SD (Min-Max)
and results on categorical measurements were presented in Number (%). Significance
is assessed at 5% level of significance. The following assumptions on data were made,
Assumptions:
1. Dependent variables should be normally distributed
2. Samples drawn from the population should be random, Cases of the samples should
be independent.
Independent t-test:
Independent student t test was used for the comparison of mean Plaque index, Gingival
index, PPD, RAL, Radiographic Defect Depth and Amount of Defect Fill.
It is applied to a data of observation for same variables between two samples. It can
also be applied to paired data of observations from one sample only when each
individual gives pair of observations.
67
For testing the significance of difference:
i. Find the difference in each set of observation for the same variables or paired
observations before and after (X1-X2=d) or (X2-X1=d)
ii. Calculate the mean difference (d)
iii. Calculate the standard deviation (SD) of difference (SD of d’s) and standard
error (SE) of difference (SD/n)
iv. Calculate the ‘t’ value by substituting the above values in the formula:
Analysis of variance (ANOVA)
Analysis of variance (ANOVA) is a collection of statistical models, and their
associated procedures, in which the observed variance in a particular variable is
partitioned into components attributable to different sources of variation.
In its simplest form ANOVA provides a statistical test of whether or not the
means of several groups are all equal, and therefore generalizes t-test to more than two
groups. Doing multiple two-sample t-tests would result in an increased chance of
committing a type I error. For this reason, ANOVAs are useful in comparing two, three
or more means.
The basic principle of ANOVA is to test the difference among the mean of
populations examining the amount of variation within each if these samples, relative to
the amount of variation between the samples. In terms of variation between the given
population, it is assumed that the observation differ from the mean of this population
only because of random effective i.e. there are influence which are unexplainable,
68
whereas in examining the difference in between the population we assume that the
difference between the mean of the jth population and grand mean is attributable to what
is called a specific factor or what is technically described as treatment effect. Thus while
using ANOVA, we assumed that each of the sample was drawn from a normal
population and that each of these population had the same variance, we also assumed
that all factors other than one being tested are effectively controlled, this in other words
means that we assumed the absence of many factors that might affect the conclusion.
ANOVA was used to determine the mean difference between the different time
intervals. (baseline, 90th day and 180th day)
FORMULA:
Fc = between samples variance
Within sample variance
Significant figures
+ Suggestive significance (p value: 0.05<p<0.01)
* Moderately significant (p value: 0.01<p  0.05)
** Strongly significant (p value: p0.01)
KRUSKAL-WALLIS H TEST:
The Kruskal–Wallis test by ranks, Kruskal–Wallis H test (named after
William Kruskal and W. Allen Wallis), or one-way ANOVA on ranks is a non-
parametric method for testing whether samples originate from the same distribution. It
is used for comparing two or more independent samples of equal or different sample
sizes. It extends the Mann–Whitney U test when there are only two groups. The
69
parametric equivalent of the Kruskal–Wallis test is the one-way analysis of variance
(ANOVA).
A significant Kruskal–Wallis test indicates that at least one sample
stochastically dominates one other sample. The test does not identify where this
stochastic dominance occurs or for how many pairs of groups stochastic dominance
obtains. For analyzing the specific sample pairs for stochastic dominance in post hoc
testing, Dunn's test, pairwise Mann-Whitney tests without Bonferroni correction, or the
more powerful but less well-known Conover–Iman test are appropriate.
Since it is a non-parametric method, the Kruskal–Wallis test does not assume a
normal distribution of the residuals, unlike the analogous one-way analysis of variance.
If the researcher can make the less stringent assumptions of an identically shaped and
scaled distribution for all groups, except for any difference in medians, then the null
hypothesis is that the medians of all groups are equal, and the alternative hypothesis is
that at least one population median of one group is different from the population median
of at least one other group.
Test statistics is given by:
 ni = is the number of observations in group i
 rij= rank of observation j from i group
 N is the total number of observations
70
MANN-WHITNEY U TEST:
A popular nonparametric test to compare outcomes between two
independent groups is the Mann Whitney U test. The Mann Whitney U test,
sometimes called the Mann Whitney Wilcoxon Test or the Wilcoxon Rank Sum
Test, is used to test whether two samples are likely to derive from the same
population (i.e., that the two populations have the same shape). Some
investigators interpret this test as comparing the medians between the two
populations. Recall that the parametric test compares the means (H0: μ1=μ2)
between independent groups.
In contrast, the null and two-sided research hypotheses for the
nonparametric test are stated as follows:
H0: The two populations are equal versus
H1: The two populations are not equal.
This test is often performed as a two-sided test and, thus, the research
hypothesis indicates that the populations are not equal as opposed to specifying
directionality. A one-sided research hypothesis is used if interest lies in
detecting a positive or negative shift in one population as compared to the other.
The procedure for the test involves pooling the observations from the two
samples into one combined sample, keeping track of which sample each
71
observation comes from, and then ranking lowest to highest from 1 to n1+n2,
respectively.
Test Statistic for the Mann Whitney U Test
The test statistic for the Mann Whitney U Test is denoted U and is
the smaller of U1 and U2, defined below.
where R1 = sum of the ranks for group 1 and R2 = sum of the ranks for group 2.
CHI-SQUARE TEST
The Chi-Square statistic is most commonly used to evaluate Tests of
Independence when using a crosstabulation (also known as a bivariate
table). Crosstabulation presents the distributions of two categorical variables
simultaneously, with the intersections of the categories of the variables appearing in the
cells of the table.
The Test of Independence assesses whether an association exists between the
two variables by comparing the observed pattern of responses in the cells to the pattern
that would be expected if the variables were truly independent of each other.
72
Calculating the Chi-Square statistic and comparing it against a critical value
from the Chi-Square distribution allows the researcher to assess whether the observed
cell counts are significantly different from the expected cell counts.
The calculation of the Chi-Square statistic is quite straight-forward and
intuitive:
2 (𝑓0 − 𝑓𝑒 )2
𝜒 =∑
𝑓𝑒
where fo = the observed frequency (the observed counts in the cells)
and fe = the expected frequency if NO relationship existed between the variables
As depicted in the formula, the Chi-Square statistic was based on the difference
between what was actually observed in the data and what would be expected if there
was truly no relationship between the variables.
73
Materials & method
Figure 12- Diagnostic cast with acrylic stent
Figure 13- Millimeter grid mount
74
Materials & method
Figure 14- Radiograph with millimeter grid mount
Figure 15- Surgical armamentarium
75
Materials & method
Figure 16- Demineralized freeze-dried bone allograft
Figure 17- Amnion membrane (AM) allograft
76
Materials & method
CLINICAL PHOTOGRAPHS SHOWING PROCEDURES IN GROUP I

(DFDBA)
Figure 18- RAL measured using UNC -15 probe preoperatively
Figure 19- PPD measured using UNC -15 probe preoperatively
77
Materials & method
Figure 20- Incision placement
Figure 21- Osseous defect between right mandibular first molar and second molar
after debridement
78
Materials & method
Figure 22- DFDBA being carried to the defect
Figure 23- Defect grafted with DFDBA
79
Materials & method
Figure 24- Flap closure with simple interrupted sutures
Figure 25- Periodontal dressing given
80
Materials & method
Figure 26- RAL measured using UNC -15 probe post-operatively
81
Materials & method
Figure 28- RVG at baseline
Figure 29- RVG at 180th day
82
Materials & method
CLINICAL PHOTOGRAPHS SHOWING PROCEDURES IN GROUP II (AM)
Figure 30- RAL measured using UNC -15 probe preoperatively
83
Materials & method
Figure 32- Incision placement
Figure 33- After debridement
84
Materials & method
Figure 34- Osseous defect between right mandibular second premolar and first
molar after debridement
Figure 35- DFDBA and Trimmed AM with template
85
Materials & method
Figure 36- Defect grafted with DFDBA
Figure 37- AM placed over the grafted defect
86
Materials & method
Figure 38- Flap closure with simple interrupted sutures
Figure 39- Periodontal dressing given
87
Materials & method
Figure 40- RAL measured using UNC -15 probe post-operatively
88
Materials & method
Figure 42- RVG at baseline
Figure 43- RVG at 6 months
89
OBSERVATION
& RESULTS
Obsevation & Results
OBSERVATION AND RESULTS
40 intrabony defects of systemically healthy patients within age range of 25-55
years, with moderate to severe chronic periodontitis were included in the study.
 Following parameters were recorded at baseline, 90th day and 180th day post
surgically for both group I (DFDBA) and group II (DFDBA and AM):
 Plaque Index (PI),
 Gingival Index (GI),
 Probing pocket depth (PPD) in mm,
 Relative attachment level (RAL) in mm, and
 Gingival recession (GR)
 Digital intraoral periapical radiographs were evaluated at baseline and 180th
day:
 Vertical osseous defect depth
 Amount of defect fill
 Percentage of defect fill
During the six months period, the wound healing was uneventful, no bone graft
and membrane was exposed, nor were any of the sites eliminated from the study. None
of the selected patients dropped out before the termination of the study.
90
PLAQUE INDEX (PI)
Table 6 - Intragroup comparison of mean Plaque Index at Baseline, 90th day and
180th day in group I:
Time N Mean SD F value p-Value
interval
Baseline 20 2.07 0.18
90th day 1.02 0.30 253.361 0.001 (H.S)

20
180th day 20 0.57 0.10
Statistical test: ANOVA; (p<0.05- significant, CI=95%), H.S - Highly Significant
Table 7 – Intragroup comparison of mean Plaque Index in group I at different
time interval:
S No. Intragroup comparison Mean difference p-value
1. Baseline – 90th day 1.045 0.001 (H.S)
3. 90th day - 180th day 0.455 0.001 (H.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), H.S- Highly
Significant
91
Table 8 - Intragroup comparison of mean Plaque Index at Baseline, 90th day and
180th day in group II:
interval
Baseline 20 2.05 0.25
257.456 0.001 (H.S)

90th day 20 0.96 0.22
180th day 20 0.61 0.13
Table 9 - Intragroup comparison of mean Plaque Index in group II at different
time interval:
3. 90th day - 180th day 0.345 0.001 (H.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), H.S - Highly
Significant
92
Table 10: Intergroup comparison of mean Plaque Index between various time
intervals:
Time Group N Mean SD Mean t- p-value
interval difference value
DFDBA 20 2.07 0.18

Baseline 0.01500 0.213 0.833
DFDBA (N.S)
20 2.05 0.25
+AM
DFDBA 20 1.02 0.30

90th day 0.06500 0.768 0.448
DFDBA (N.S)
20 0.96 0.22
+AM
DFDBA 20 0.57 0.10

180th day 0.04500 0.465 0.243
DFDBA (N.S)
20 0.61 0.13
+AM
Statistical test: Independent Sample t-test; (p<0.05- significant, CI=95%), N.S –
Non-Significant
93
PLAQUE INDEX (PI) (Table- 5, 6, 7, 8, 9 & Graph-1)
The mean PI scores at baseline, 90th day and 180th day for group I (DFDBA)
was 2.07±0.18, 1.02±0.30 and 0.57±0.10 respectively. When the mean PI score was
compared using ANOVA, the difference was found to be statistically highly significant
(P-0.001). (Table – 5) Intragroup comparison using Tukey’s post-hoc test showed a
mean difference of 1.045 and 1.500 from baseline to 90th day and baseline to 180th day
respectively, which was found to be statistically highly significant (P-0.001). The
difference between 90th day and 180th day was found to be 0.455 which was also found
to be statistically highly significant (P-0.001). (Table – 6)
The mean PI scores at baseline, 90th day and 180th day for group II (DFDBA +
AM) was 2.05±0.25, 0.96±0.22 and 0.61±0.13 respectively. When the mean PI score
was compared using ANOVA, the difference was found to be statistically highly
significant (P=0.001). (Table – 7) Intragroup comparison using Tukey’s post-hoc test
showed a mean difference of 1.095 and 1.440 from baseline to 90th day and baseline to
180th day respectively, which was found to be statistically highly significant (P-0.001).
The difference between 90th day and 180th day was 0.345 which was also found to be
statistically highly significant (P-0.001). (Table – 8)
Independent sample t test showed a non-significant result of PI scores between the
groups from baseline to 180th day (Table – 9, Graph – 1)
94
GINGIVAL INDEX (GI)
Table 11 - Intragroup comparison of mean Gingival Index at Baseline, 90th day
and 180th day in group I:
interval
Baseline 20 2.14 0.24
267.13 0.001 (H.S)

90th day 20 1.39 0.20
180th day 20 0.69 0.12
Statistical test: ANOVA; (p<0.05- significant, CI=95%), H.S- Highly Significant
Table 12 – Intragroup comparison of mean Gingival Index in group I at different
time interval:
3. 90th day - 180th day 0.700 0.001 (H.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), H.S. - Highly
Significant
95
Table 13 - Intragroup comparison of mean Gingival Index at Baseline, 90th day
and 180th day in group II:
interval
Baseline 20 2.28 0.21
424.542 0.001 (H.S)

90th day 20 1.45 0.19
180th day 20 0.66 0.15
Table 14 – Intragroup comparison of mean Gingival Index in group II at different
time interval:
3. 90th day - 180th day 0.795 0.001 (H.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), H.S- Highly
Significant
96
Table 15: Intergroup comparison of mean Gingival Index between various time
intervals:
Time Group N Mean SD Mean t- value p-value
interval difference
DFDBA 20 2.14 0.24

Baseline
-0.140 1.963 0.057 (N.S)
DFDBA
20 2.28 0.21
+AM
DFDBA 20 1.39 0.20

th
90 day
-0.060 0.972 0.336 (N.S)
DFDBA
20 1.45 0.19
+AM
DFDBA 20 0.69 0.12

th
180 day
0.030 0.698 0.489 (N.S)
DFDBA
20 0.66 0.15
+AM

Non-Significant
97
GINGIVAL INDEX (GI) (Table- 10, 11, 12, 13, 14 & Graph-2)
The mean GI scores at baseline, 90th day and 180th day for group I (DFDBA) was
2.14±0.24, 1.39±0.20 and 0.69±0.12 respectively. When the mean GI score was
The mean GI scores at baseline, 90th day and 180th day for group II (DFDBA +
AM) was 2.28±0.21, 1.45±0.19 and 0.66±0.15 respectively. When the mean GI score
was compared using ANOVA, the difference was found to be statistically highly
significant (P=0.001). (Table – 12) Intragroup comparison using Tukey’s post-hoc test
showed a mean difference of 0.945 and 1.740 from baseline to 90th day and baseline to
180th day respectively, which was found to be statistically highly significant (P-0.001).
The difference between 90th day and 180th day was 0.795 which was also found to be
Independent sample t test showed a non-significant result of GI scores between the
groups from baseline to 180th day (Table – 14, Graph – 2)
98
PROBING POCKET DEPTH (PPD)
Table 16 - Intragroup comparison of mean Probing Pocket Depth at Baseline, 90th
day and 180th day in group I:
interval
Baseline 20 6.95 0.94
90th day 20 5.15 0.74 60.263 0.001 (H.S)
180th day 20 4.10 0.78
Table 17 - Intragroup comparison of mean Probing Pocket Depth in group I at
different time interval:
3. 90th day - 180th day 1.050 0.001 (H.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), H.S -

Highly Significant
99
Table 18 - Intragroup comparison of mean Probing Pocket Depth at Baseline, 90th
day and 180th day in group II:
interval
Baseline 20 7.30 0.92
90th day 20 4.60 0.85 115.106 0.001 (H.S)
180th day 20 3.45 0.60
Table 19- Intragroup comparison of mean Probing Pocket Depth in group II at
different time interval:
3. 90th day - 180th day 1.65 0.001 (H.S)

Highly Significant
100
Table 20: Intergroup comparison of mean Probing Pocket Depth between
various time intervals:
interval difference
DFDBA 20 6.95 0.94

Baseline
-0.35000 1.190 0.241
DFDBA 20 7.30 0.92 (N.S)
+AM
DFDBA 20 5.15 0.74

90th day
0.5500 2.182 0.035 (S)
DFDBA 20 4.60 0.85
+AM
DFDBA 20 4.10 0.78

180th day
0.6500 2.953 0.005 (S)
DFDBA 20 3.45 0.60
+AM
Statistical test: Independent Sample t-test; (p<0.05- significant, CI=95%), S-

Significant, N.S –Non-Significant
101
PROBING POCKET DEPTH (PPD) (Table- 15, 16, 17, 18, 19 & Graph- 3)
The mean PPD at baseline, 90th day and 180th day for group I (DFDBA) was
6.95±0.94, 5.15±0.744 and 4.10±0.78, respectively. When the mean PPD was
The mean PPD at baseline, 90th day and 180th day for group II (DFDBA + AM)
was 7.30±0.92, 4.60±0.85 and 3.45±0.60 respectively. When the mean PPD was
(P=0.001). (Table – 17) Intragroup comparison using Tukey’s post-hoc test showed a
difference between 90th day and 180th day was 1.65 which was also found to be
Independent sample t test showed a significant reduction in PPD between the
groups at 90th day and 180th day (Table – 19, Graph – 3), Group II (DFDBA+AM)
showing a greater reduction in PPD.
102
RELATIVE ATTACHMENT LEVEL (RAL)
Table 21 - Intragroup comparison of mean Relative Attachment Level at Baseline,
90th day and 180th day in group I:
interval
Baseline 20 12.05 1.39
90th day 20 10.35 1.78 13.411 0.001 (H.S)
180th day 20 9.30 1.86
Table 22- Intragroup comparison of mean Relative Attachment Level in group I
at different time interval:
1. Baseline – 90th day 1.700 0.007 (S)
3. 90th day - 180th day 1.050 0.132 (N.S)
Statistical test: Tukey’s post-hoc test; (p<0.05- significant, CI=95%), S-

Significant, H.S - Highly Significant, N.S- Non-Significant
103
Table 23 - Intragroup comparison of mean Relative Attachment Level at Baseline,
90th day and 180th day in group II:
interval
Baseline 20 12.95 1.93
18.559 0.001 (H.S)

90th day 20 9.070 2.17
180th day 20 8.043 2.05
Table 24- Intragroup comparison of mean Relative Attachment Level in group II
at different time intervals:
3. 90th day - 180th day 1.700 0.030 (S)

Highly Significant, S- Significant
104
Table 25: Intergroup comparison of mean Relative Attachment Level between
various time intervals:
Time Group N Mean SD Mean t- p-value
interval difference value
DFDBA 20 12.050 1.39454

Baseline
-0.90000 1.689 0.099 (N.S)
DFDBA 20 12.950 1.93241
+AM
DFDBA 20 10.350 1.78517

90th day
1.2800 2.032 0.049 (S)
DFDBA 20 9.070 2.17885
+AM
DFDBA 20 9.300 1.86660

180th day
1.257 2.027 0.048 (S)
DFDBA 20 8.043 2.05196
+AM
Statistical test: Independent Sample t-test; (p<0.05- significant, CI=95%), S-

105
RELATIVE ATTACHMENT LEVEL (Table- 20, 21, 22, 23, 24 & Graph- 4)
In group I (DFDBA), mean RAL values changed from 12.050±1.39454 at
baseline to 10.350±1.78517 at 90th day and 9.300±1.86660 at 180th day. When mean
RAL was compared using ANOVA, the difference was found to be statistically highly
significant (P-0.001) (Table – 20). Intragroup comparison using Tukey’s post-hoc test
showed a mean difference of 1.700 from baseline to 90th day which was found to be
statistically significant (P-0.007) and a mean difference of 2.750 from baseline to 180th
day which was found to be statistically highly significant (P-0.001). However, mean
difference in RAL from 90th day to 180th day was 1.050 which was found to be
statistically non-significant (P-0.132) (Table – 21).
In group II (DFDBA + AM), mean RAL values changed from 12.950±1.93241
at baseline to 9.070±2.1788 at 90th day and 8.043±2.0519 at 180th day. When mean
RAL was compared using ANOVA, the difference was found to be statistically highly
significant (P-0.001) (Table – 22). Intragroup comparison using Tukey’s post-hoc test
showed a change of 2.250 and 3.950 from baseline to 90th day and 180th day
respectively which was found to be statistically highly significant (P=0.001). However,
mean difference in RAL from 90th day to 180th day was 1.700 which was found to be
statistically significant (P-0.030). (Table – 23)
Intergroup comparison of RAL at baseline was found to be statistically not
significant showing a P value of 0.099 and comparison of RAL p value was 0.49 and
0.048 at 90th day and 180th day respectively which was found to statistically significant.
(Table – 24 and Graph – 4)
106
GINGIVAL RECESSION (GR)
Table 26 - Intragroup comparison of mean Gingival Recession at Baseline, 90th
day and 180th day in group I:
Time N Mean SD Chi-value p-Value
interval
Baseline 20 0.65 0.745
4.391 0.111 (N.S)

90th day 20 1.00 0.561
180th day 20 1.05 0.686
Statistical test: Kruskal-Wallis H test; (p<0.05- significant, CI=95%), N.S- Non-

Significant
Table 27 - Intragroup comparison of mean Gingival Recession at Baseline, 90th
day and 180th day in group II:
Time N Mean SD Chi-value p-Value
interval
Baseline 20 0.70 0.732
1.582 0.45 (N.S)

90th day 20 0.80 0.615
180th day 20 0.55 0.604
Statistical test: Kruskal-Wallis H test; (p<0.05- significant, CI=95%), N.S- Non-

Significant
107
Table 28: Intergroup comparison of mean Gingival Recession between various
time intervals:
Time Group N Mean SD Mean U- value p-value
interval difference
DFDBA 20 0.65 0.74

Baseline
-0.05000 191.500 0.832 (N.S)
DFDBA 20 0.70 0.73
+AM
DFDBA 20 1.00 0.56

th
90 day
0.20000 166.000 0.369 (N.S)
DFDBA 20 0.80 0.61
+AM
DFDBA 20 1.05 0.68

th
180 day
20 0.50000 123.000 0.038 (S)

DFDBA 0.55 0.60
+AM
Statistical test: Mann-Whitney U test; (p<0.05- significant, CI=95%), S-

108
GINGIVAL RECESSION (GR) (Table- 25, 26, 27 & Graph- 5)
In group I (DFDBA), mean GR values changed from 0.65±0.74 at baseline to
1.00±0.56 at 90th day and 1.05±0.68 at 180th day. When mean RAL was compared using
Kruskal-Wallis H test, the difference was found to be statistically non-significant (P-
0.111) (Table – 25).
In group II (DFDBA + AM), mean GR values changed from 0.70±0.73 at
baseline to 0.80±0.61 at 90th day and 0.55±0.60 at 180th day. When mean GR was
compared using Kruskal-Wallis H test the difference was found to be statistically non-
significant (P-0.45) (Table – 26).
Intergroup comparison using Mann-Whitney U test of GR at baseline and 90th
day was found to be statistically non-significant showing a P value of 0.832 at baseline
and 0.369 at 90th day. However, GR 180th day was found to be statistically significant
showing a P value of 0.038. (Table – 27, Graph – 5)
109
RADIOGRAPHIC DEFECT DEPTH
Table 29: Intragroup comparison of mean Radiographic Defect Depth at Baseline
and 180th day in group I:
Time N Mean SD Mean t-value p-value

interval diff
Baseline 20 4.95 1.01 2.84000 15.858 0.001

(H.S)
180th day 20 2.11 0.57
Statistical test: paired t test; (p<0.05- significant, CI=95%), H.S- Highly

Significant
Table 30: Intragroup comparison of mean Radiographic Defect Depth at
Baseline and 180th day in group II:
Time N Mean SD Mean t-value p-value

interval diff
Baseline 20 4.77 0.496 2.92000 20.964 0.001

(H.S)
180th day 20 1.85 0.552
Statistical test: paired t test; (p<0.05- significant, CI=95%), H.S- Highly

Significant
110
Table 31: Intergroup comparison of mean Radiographic Defect Depth between
various time interval:
interval difference
DFDBA 20 4.9550 1.01643

Baseline 0.18000 0.712 0.481
DFDBA 20 4.7750 0.49617 (N.S)
+AM
DFDBA 20 2.1150 0.57425
0.26000 1.460 0.153

180th day (N.S)
DFDBA 20 1.8550 0.55201
+AM

Non-Significant
111
RADIOGRAPHIC DEFECT DEPTH (Table- 28, 29, 30 & Graph- 6)
In group I (DFDBA), the mean radiographic defect depth was 4.9550±1.0164
at baseline. At 180th day, the mean radiographic defect depth was 2.1150±0.5742. The
difference in depth was 2.8400 which was statistically highly significant (P-0.001).
(Table – 28)
In group II (DFDBA + AM), the mean radiographic defect depth was
4.7750±0.4961 at baseline. At 180th day, the mean radiographic defect depth was
1.8550±0.5520. The difference in depth was 2.9200 which was statistically highly
significant (P-0.001). (Table – 29)
Intergroup comparison of the defect depths showed no significant difference at
baseline. Comparison of the defect depth using independent sample t test was also
found to be non-significant when evaluated at 180th day postoperatively. (P-0.153).
(Table – 30, Graph- 6)
112
AMOUNT OF DEFECT FILL
Table 32: Intergroup comparison of mean Amount of Defect Fill at 180th day:
interval difference
DFDBA 20 2.8400 0.80092

180th day
-0.08000 -0.353 0.726
DFDBA 20 2.9200 0.62290 (N.S)
+AM

Non-Significant
AMOUNT OF DEFECT FILL (Table- 31 & Graph- 7)
Comparison of the amount of defect fill using independent sample t test was
found to be non-significant when evaluated at 180th day postoperatively. (P-0.726).
(Table – 31, Graph- 7)
113
PERCENTAGE OF DEFECT FILL
Table 33: Intergroup comparison of Percentage of Defect Fill at 180th day:
Time interval Group N Chi-value p-value
DFDBA 20
180 days 23.70 0.04 (S)
DFDBA +AM 20
Statistical test: Chi-square test; (p<0.05- significant, CI=95%), S- Significant
PERCENTAGE OF DEFECT FILL (Table- 32 & Graph- 8)
Intergroup comparison of the percentage of defect fill using Chi-square test
showed statistically significant difference at 180th day (P-0.04). (Table –32, Graph- 8)
114
DFDBA
2.5
DFDBA+AM
2.07 2.05
2
1.5
MEAN
1.02 0.96
1
0.57 0.61
0.5
0
BASELINE 90TH DAY 180TH DAY
TIME INTERVAL
Graph- 1. Comparison of mean Plaque Score Index between group I and group II
at baseline, 90th day and 180th day
DFDBA
2.5
2.28 DFDBA+AM
2.14
2
1.5 1.39 1.45

MEAN
1
0.69 0.66
0.5
0
TIME INTERVAL
Graph- 2. Comparison of mean Gingival Score Index between group I and group II
at baseline, 90th day and 180th day
115
DFDBA
8 DFDBA+AM
7.3
7 6.95
6
5.15
5 4.6
4.1
MEAN
4 3.45
0
TIME INTERVAL
Graph- 3. Comparison of mean Probing Pocket Depth between group I and group
II at baseline, 90th day and 180th day
DFDBA
14
12.9 DFDBA+AM
12 12
10.3
10 9.07 9.3
8.04
8
MEAN
0
TIME INTERVAL
Graph- 4. Comparison of mean Relative Attachment Level between group I and

group II at baseline, 90th day and 180th day
116
DFDBA
1.2 DFDBA+AM
1.05
1
1
0.8
0.8 0.7
0.65
MEAN
0.6 0.55
0.4
0.2
0
TIME INTERVAL
Graph- 5. Comparison of mean Gingival Recession between group I and group II at

baseline, 90th day and 180th day
BASELINE
5 4.955 180TH DAY
4.775
3
MEAN
2.115
1.855
2
0
DFDBA DFDBA+AM
GROUP
Graph- 6. Comparison of mean Radiographic Defect Depth between group I and

group II at baseline and 180th day
117
180TH DAY
2.92 2.92
2.9
2.88
MEAN
2.86
2.84
2.84
2.82
2.8
DFDBA DFDBA+AM
GROUP
Graph- 7. Comparison of mean Amount of Defect Fill between group I and group
II at 180th day
118
DISCUSSION
Discussion
DISCUSSION
The ideal goal of periodontal therapy is the restoration of the periodontium by
predictable regeneration of bone and connective tissue attachment, which has been
destroyed by periodontal disease. Conventional periodontal therapy has limited scope
and results are not predictable. Healing following conventional therapy is most likely
to occur mainly through the formation of a long junctional epithelium. 117
The aim of the regenerative procedure is to displace the epithelial attachment at
a more coronal position than before treatment, allowing cells from periodontal ligament
and bone to repopulate the root surface and to form a new periodontal attachment. 118
Alveolar bone and cementum have good potential of regeneration provided that
the necessary cell types and cell signals are present. The same is true for periodontal
ligament, but for this to form a functional attachment, the collagen fibers must become
enclosed by newly formed bone on one surface and cementum on the other. This
requires the regeneration of the three tissues to be finally integrated. Also, for any new
attachment to form, junctional epithelium which proliferates over exposed connective
tissue, must be excluded from the wound.119
A successful outcome of periodontal tissue engineering requires appropriate
cells, signals, scaffolds, blood supply, mechanical loading and pathogen control. Cells
provide the machinery for new tissue growth and differentiation, whereas growth
factors and other molecules modulate the cellular activity and provide stimuli for cells
to differentiate and support tissue neogenesis. A three-dimensional template structure
is provided by scaffolds to support and facilitate these processes that are critical for
tissue regeneration. New vascular networks promoted by angiogenic signals provide
the nutritional base for tissue growth and homeostasis, whereas appropriate mechanical
119
Discussion
loading is essential for the development of highly organized, functional PDL fibers.
Finally, because of the microbial load at the periodontal lesion, strategies to control
infection and host response are required to optimize periodontal regeneration. 120
Recent advances in molecular and cellular biology have increased our
understanding of tissue regeneration and of interactions between extracellular matrix,
growth factors and cells during wound healing. In bone regeneration, osteoblasts
proliferate and produce ECM components and local growth factors which play key roles
in regulating cellular activities.121
An increased knowledge of cellular response and function within the
periodontium has led to the development of numerous treatment modalities exhibiting
different degree of success. Treatments including modification of root surface, grafting
with bone or bone substitute, stimulation of cells with growth factors, hormones or
extracellular matrix proteins and cell occlusive barrier membrane have all been
explored for their ability to predictably regenerate the periodontium. 122
Treatment of periodontal intrabony defects via isolation for selective cell
repopulation has been an accepted treatment modality for nearly 30 years. In 1976,
Melcher123 discussed the compartmentalization of tissues to achieve regeneration, and
the clinical application of this concept was first demonstrated by Nyman et al.124 then
in 1986, Gottlow et al. coined the term guided tissue regeneration (GTR) to describe
this treatment modality, which allows for the formation of bone, cementum, and
periodontal ligament in degranulated periodontal defects.125 Since its introduction,
GTR therapy has evolved and changed. Initially, non-resorbable occlusive barriers were
used to simply isolate degranulated periodontal intrabony defects. Over the years, bone
or bone substitutes were used to fill these defects, and when covered by a barrier, the
120
Discussion
term combination GTR was used. Non-resorbable barriers were eventually replaced
with bioabsorbable barriers and, more recently, biologic growth factors have been used
to enhance healing. The evolution of regenerative therapy through evaluation of
placental-based amnion allograft membranes (AM), which have inherent biologic
properties that actively promote wound healing in lieu of simply providing an occlusive
barrier for selective cell repopulation.21
Various regenerative materials have provided clinicians with a reliable method
of achieving defect fill and defect resolution. The autogenous bone graft is considered
as ‘gold standard’ but limited amount of intraoral donor bone, need for second surgical
site for graft procurement and potential risk for root ankylosis and resorption with iliac
crest graft was its drawback. Alloplasts as graft materials have an unlimited supply and
provide greater defect fill and less crestal resorption than debridement alone.
Histologically, the alloplasts becomes encapsulated by connective tissue and a long
junctional epithelium is present along the tooth surface with no evidence of
cementogenesis or new periodontal ligament formation. Allografts can be obtained
from a tissue bank in the form of FDBA or DFDBA. The use of allografts in the
treatment of periodontal defects has become popular since studies have reported defect
fill of greater than 50% in the majority of treated sites. Histologically, when placed in
intrabony defects and compared to nongrafted defects, DFDBA demonstrate
significantly more new cementum, new connective tissue, and bone formation in
intrabony defects grafted with DFDBA than in nongrafted sites. 81
Furthermore, bone grafts materials that are needed in periodontics should be
osteoinductive, have good handling characteristics, and have physical properties
providing appropriate stiffness for the treatment site. Bone graft material commonly
used for these procedures are DFDBA and FDBA. The osteoinductive properties of
121
Discussion
DFDBA have made it the grafting material of choice as compared to FDBA, xenografts
and alloplasts. The use of DFDBA has seen successfully proven in a histologic study
wherein 80% of test sites showed complete regeneration. The demineralization process
of DFDBA exposes its BMP’s that makes it osteoinductive in nature. 83,84
Periodontal osseous grafting techniques are directed towards the restoration of
the lost periodontium and specifically the regeneration of alveolar bone. Bone healing
in grafted sites has been characterized as occurring by several mechanisms. Of these,
the term “osteoinduction” implies bone formation through the active recruitment of less
differentiated host cells by bioactive diffusible mediators and their eventual expression
as bone tissue. The presence of an inductive signal in demineralized cortical bone
eliciting osteogenesis from precursor cell populations has been termed “Bone
Morphogenetic Protein” (BMP). In addition to the inducer substances, an organic
matrix is required. Both these elements are made available through the demineralization
of bone tissue. DFDBA is a material with related osteoinductive potential as realized in
various in vitro and in vivo model systems. 88
In present study, chronic periodontitis patients with a total of 40 intrabony
defects were selected. Patients with good systemic health and no contraindication to
periodontal surgery were selected, since patients suffering from systemic diseases like
uncontrolled diabetes mellitus or those patients on immunosuppressive therapy, almost
always show a compromised wound healing response. 126
Variation in the assessment of pocket depths and attachment levels may occur
if the probing site and the direction of the probe insertion differ from one measurement
to another.127 Reliability of these measurements depends on the stability of the structure
used as a fixed reference point. CEJ is frequently used as a fixed reference point is often
difficult to identify. To eliminate these problems acrylic occlusal stent with groove and
122
Discussion
reference marking was used to provide a landmark for accurate and reproducible
measurements and guide for the probing position.128
Clinical parameters including plaque index and gingival index were recorded
and probing pocket depth, relative attachment level and gingival recession were
measured from the selected patients at baseline, 90th day and 180th day post-operatively.
All those patients maintaining efficient oral hygiene in recall appointments during
phase I therapy, were considered for the study because of local and behavioral factors
influence the regenerative outcome.
Radiographic evaluation was done at baseline prior to surgery and at 180th day
postoperatively by digital intraoral periapical radiographs with millimeter grid mount
standardized with long cone paralleling technique with 70 KVp, 10 mA and exposure
time of 0.8 second.
After completion of phase I therapy and the attainment of surgical
manageability of tissues, the selected sites were randomly treated with DFDBA (Group
I) and DFDBA with AM (Group II). All patients in the study showed good compliance
and the healing period was uneventful for both test and control group.
The study did not have non-graft site as control group because numerous studies
have been reported statistically significant results in favor of bone replacement grafts
as compared to non-grafted sites.129-131
To the best of our knowledge there are no clinical studies reported using
DFDBA with AM in the treatment of intrabony defects, the present study was done to
evaluate and compare the clinical efficacy of Amnion Membrane with DFDBA bone
granules in the treatment of intrabony defects in terms of clinical and radiographic
parameters.
123
Discussion
All patients participating in the study demonstrated good oral hygiene level and
a healthy gingival condition throughout the study period as indicated by plaque index
(PI) and gingival index (GI) scores. The reduction in PI & GI score was statistically
highly significant in both the groups. This improvement in PI and GI was attributed to
the maintenance of optimum oral hygiene by the patient and regular supportive
periodontal care program. The results obtained in this study were similar with previous
study by Katuri et al. who reported a significant improvement in GI as compared to
baseline while treating Infrabony defects.132
A statistically significant improvement in gingival index may be attributed to
the resolution of inflammation and return of the gingival tissues from a diseased to
health state. Numerous reports based on short term and long-term data indicate that
good oral hygiene reflected by low plaque scores, was associated with enhanced
periodontal regeneration and stability.133-135
On comparison between the two groups at 90th day and 180th day post-surgery,
mean plaque index and gingival index was statistically non-significant. Similar findings
were observed in previous studies.136-138
Mean change in gingival margin position was not statistically significant for the
Group I (DFDBA) from baseline to 180th day post-surgery. Mean change in gingival
margin position was not statistically significant for the Group II (DFDBA+ AM) from
baseline to 180th day post-surgery.
On comparison between two groups at 90th day post-surgery mean change in
gingival margin position was statistically not significant and at 180 th day post-surgery
mean change in gingival margin position was statistically significant. Results of this
study are similar to findings of Kiany et al.139
124
Discussion
Reduction in probing pocket depth is considered to be the most important
outcome of periodontal procedure, including periodontal regeneration, because it
directly affects the ability of a clinician to maintain a treated site. In this study, mean
reduction in probing pocket depth was statistically highly significant for the Group I
(DFDBA) from baseline to 180th day. The results of this study are similar to the findings
of Iorio-Siciliano et al.140 and Camargo et al.141 who demonstrated notable
improvement in the reduction of pocket depth. In Group II (DFDBA+ AM) mean
reduction in pocket depth was also statistically highly significant 180 th day post-
surgery, again comparable to the findings of Chen et al.141,142
The primary reason for reduction in probing pocket depth after treatment can be
attributed to the reduction in inflammation, shrinkage of the pocket wall and gain in
attachment level. As well as reattachment of collagen fibers and bone fill also impede
penetration of probe.
On comparison between the two groups at 90th day and 180th day post-surgery,
mean reduction in pocket depth was statistically significant in Group II (DFDBA+AM)
as compared to Group I (DFDBA). Results of this study are similar to findings of
previously mentioned studies.136,137
The most practical clinical parameter for assessing the results of
regenerative periodontal treatment methods is RAL. The assessment of attachment
levels due to its fixed reference point (FRP) provides better information relating to gain
or loss of attachment to the root surface and in assessing the disease progression as
compared to pocket depth measurements. Though attachment level measurements too
have certain inherent shortcomings like its high level of tactile sensitivity and time
consumption, it still represents “Gold Standard” by which clinicians record disease
status.143
125
Discussion
In this study, mean relative attachment level was statistically highly significant
for the Group I (DFDBA) from baseline to 180th day. The results of this study are
similar to the findings of Iorio-Siciliano et al.140 and Kukreja et al.137 who
demonstrated gain in clinical attachment level. In Group II (DFDBA+ AM) mean
relative attachment level was also statistically highly significant at 180th day post-
surgery. The results of this study are similar to the findings of Camargo et al.141 and
Kher et al.144 who demonstrated notable improvement in the gain in attachment level.
On comparison between the two groups at 90th day and 180th day post-surgery, mean
relative attachment level was statistically significant. Results of this study are similar
to findings of previous studies.136,145,146
The results of the study with respect to mean defect fill for Group I and Group
II were quite similar. Though the value was higher in Group II than in Group I
mathematically, there was no statistically significant difference found between the two
groups. In accordance the present study, some other similar type of clinical trials also
shows no statistically significant difference between combination and bone graft alone
type of therapy.147-149 These studies were in agreement with our study which records no
additional benefit of GTR membrane together with bone graft alone. However, there
are certain studies which favor the combination therapy with respect to bone
fill.145,150,151
The uneventful healing response of the periodontal tissue proved the
biocompatibility of AM. Despite the lack of histologic clues and with respect to
clinical healing, it can be claimed that, cellular adhesion to the membrane surface,
blood clot stabilization, and integration of the membrane with the proliferating
connective tissue of the gingiva occurred. To date, there are no published data on the
use of AM in conjunction with DFDBA for the treatment of intrabony defects. Also,
126
Discussion
there are several variations that make it difficult to compare the results of this clinical
study with those of other studies that examined other biodegradable membranes.
During the immediate postoperative period, no membrane exposures were
observed. In both groups, uneventful healing and subsequent integrity of soft tissue
were noted.
Since its introduction there have been immense alterations in the processing
of AM, which have resulted in improved clinical outcomes. The lyophilized (freeze-
dried) method of preparation of AM induces minimal changes in its biologic
properties. This method results in loss of epithelial cells and minimizes
immunogenicity, as it does not require refrigeration, the product can be stored at
room temperature and has an extended shelf life of 3 years. 152
AM appear to be suitable for GTR, as they are chemotactic for periodontal
ligament fibroblasts and can serve as fibrillar scaffolds for early vascular ingrowth. AM
has unique characteristics, including bacteriostatic effects, antiadhesive properties,
wound protection, and epithelialization effects, that make it different from routinely
used barrier membranes.153
It has been shown that AM, through adhesion to the wound surface, can act as
an antibacterial barrier and reduce bacterial infiltration. 154 Thus, it has been speculated
that AM, because of its antibacterial properties, could decrease the risk of infection and,
based on the presence of growth factors, promote healing. AM contains growth factors
that hasten formation of granulation tissue by stimulating the growth of fibroblasts. 155
In the meantime, AM vascularizes healthy granulation tissue and stimulates
neovascularization in the neighboring tissues. 156 Also, AM provides a protein-enriched
bioactive matrix that facilitates cell migration. 157 Hence, it can be speculated that the
use of AM as a membrane for GTR could stimulate vascularization of the granulation
127
Discussion
tissue in the defects and promote cell migration and wound healing.
The presence of laminin-5 in high concentrations throughout AM, with its high
affinity for gingival epithelial cells, could accelerate healing and integration of the
membrane with gingival tissue.156,158 In other words, it has been claimed that AM has
the ability to form an early physiologic “seal” with the host tissue. This precludes
bacterial contamination.159 This quality of good integration of AM with the overlying
gingiva may account for the smaller amount of REC observed in the Group II
(DFDBA+ AM). For confirmation of this hypothesis, further studies with histologic
examination at different points during the healing period are necessary.
It can also be claimed that the presence of various growth factors in AM, such as
platelet-derived growth factors alpha and beta and transforming growth factor beta, is
likely to induce faster sealing of the defects and limited loss of the grafting material in
the Group II (DFDBA+ AM).160
Thickness of normal AM is 0.02 to 0.5 mm, which is equivalent to six to eight
layers of cells.161 One of the major advantages of AM, in comparison to other bio-
degradable membranes, is its thinness (320 μm) and good adaptability. The significant
lack of GR observed in the Group II (DFDBA+ AM) might be a result of the thinness
of the AM, which resulted in better adaptation of the membrane over the bony defect
and consequently better coverage of gingiva over the membrane. AM is suggested for
use in areas with limited thickness and height of gingiva, as full coverage of membrane
is more easily accomplished.
Considering the advantages of AM, it can be introduced as an inexpensive and
readily available membrane. Its unique physical nature permits the clinician to ignore
many of the recognized guiding principles for the application of customary barrier
membranes. From a clinical and practical viewpoint, the ease of handling, trimming,
128
Discussion
and adapting of this form of AM renders it an easy-to-use and operator-friendly
membrane. The AM used in this study, after being hydrated, needs less defined
trimming and adapts tightly to the underlying grafting material, bony margin, and tooth
surface. It is naturally self-adhesive to the underlying surfaces. However, it must be
noted that this physical property of AM does not afford any space maintenance
capabilities, because of a lack of stiffness. Thus, after becoming dampened by the tissue
fluid, it adapts to the surgical field. Therefore, some kind of space-saving grafting
material was needed underneath membrane.
In an attempt to further improve the clinical outcomes of GTR, this study was
designed to employ a combined periodontal regenerative technique. 162 It means that the
study did not include a nongrafted group. There was no defect selection according to
the number of walls or configuration of the defect in the protocol of the study, so it was
expected that unfavorable and large defects might be encountered during surgery.
According to the rationale for combined periodontal regenerative therapy, the bone
material that is used in conjunction with membranes enhances the stability of the
coagulum, sustains the membranes in the presence of non-contained defects, and
prevents collapse of the membranes onto the root surface or into the defects during
wound healing, thereby preserving the space necessary for regeneration. Reduced or
limited space might result in compromised healing as a consequence of inadequate
space for tissue ingrowth.163 In this study, it was assumed that DFDBA would
enhance and facilitate the proliferation of osteogenic cells through its osteoinductive
quality.
The provision of physical support by the graft material under such
membranes allows the flaps to be sutured over the defects without exerting pressure
on the membranes or displacing them. Owing to the pliability and fragility of AM,
129
Discussion
the presence of DFDBA as a graft material in the defect provided good support for
AM and prevented its collapse into the defect.
One of the important factors in the outcome of GTR is the speed at which the
biodegradable membranes are absorbed. Researchers disagree on the precise
bioabsorption time of barrier membranes. There is not enough evidence to determine
the time to degradation of AM when it is used as graft, wound dressing, or membrane.
In fact, it is difficult to determine how long the barrier effect of the AM lasts. It has
been claimed that the protective function of AM, acting as a skeletal substructure,
diminishes by the 14th to 21st days as a result of mucoid degeneration.164,165 It can be
hypothesized that placement of AM as a membrane under a periodontal flap and
prevention of exposure to the oral cavity might lead to longer degradation time. The
gains in RAL and reductions in PPD in this study make it safe to speculate that the
absorption of AM was slow enough to produce the desired effects.
Despite the promising findings of this study, it has a few limitations to be
considered. Although acrylic resin guides were used to confirm the reproducibility of
the measurements, the application of pressure-sensitive probes might have resulted in
a smaller margin of error. Reentry was not considered in this study, so the changes
in crestal bone levels and bony defect depths could not be measured directly.
Certainly, the nature of the attachment between the newly regenerated tissue and the
root surface cannot be determined without biopsy of the treated teeth. Histologic
studies are needed to claim true periodontal regeneration. Because none of the teeth
included in this study were candidates for extraction, a histologic study was not
performed. Further studies with a larger sample size over a longer postoperative
follow-up period are needed to conclusively proclaim the efficacy of this
membrane.
130
Discussion
The results of this investigation indicated that AM as a novel barrier membrane,
in conjunction with DFDBA, is quite predictable and comparable to DFDBA alone
when used in intrabony defects in bringing improvements in clinical parameters. In
addition to its various biologic properties, AM is easy to use as it could be folded and
compressed into narrow spaces between the roots. AM, with its diverse characteristics,
can influence the clinician’s decision.
131
SUMMARY &
CONCLUSION
Summary & conclusion
SUMMARY AND CONCLUSION
The present study was undertaken with the aim of this study is to evaluate
clinically and radiographically the efficacy of Amnion Membrane (AM) allograft
barrier with DFDBA and DFDBA alone in treatment of periodontal intrabony defect in
chronic periodontitis patients and observing the findings at baseline, 90th day and 180th
day postoperatively.
The following results were observed:
Clinical parameters:
 There was reduction in the mean plaque and gingival indices scores from
baseline to 180th day postoperatively, which was statistically significant. It
suggests improvement in patient’s oral hygiene throughout the study period.
 In both the groups there was a reduction in mean PPD and gain in RAL which
was statistically significant. The mean GR score was statistically significant in
Group II.
 When reduction in PPD and gain in RAL was compared between both the
groups, the Group II showed a statistically significant improvement over Group
I.
Radiographic Evaluation:
 In both the groups there was decrease in mean defect depth and increase in
percentage of defect fill compared to baseline which were statistically
significant.
 However, no significant difference was observed in intergroup comparison.
132
Summary & conclusion
From the analysis of the clinical and radiographic results the following conclusions
were drawn:
I. Individually both groups showed promising results in treatment of periodontal
intrabony defects.
II. However, better results were observed with Group II (DFDBA + AM) in clinical
parameters (PPD, RAL, and GR) as compared to Group I (DFDBA).
Use of AM as a GTR membrane seems to be promising for the treatment of
infrabony defects. Future studies with more critically designed protocols, larger sample
size and inclusion of histological examination as a criterion for periodontal
regeneration, are warranted to further explore this potential of the AM.
133
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153
ANNEXURES
Annexures
154
Annexures
155
Annexures
RUNGTA COLLEGE OF DENTAL SCIENCES & RESEARCH,

BHILAI
PATIENT’S CONSENT FORM
Title of the study “A COMPARATIVE EVALUATION OF THE EFFICACY OF

AMNION MEMBRANE (AM) ALLOGRAFT BARRIER WITH
DEMINERALISED FREEZE-DRIED BONE ALLOGRAFT (DFDBA) AND
DFDBA ALONE IN TREATMENT OF PERIODONTAL INTRABONY DEFECT
IN CHRONIC PERIODONTITIS PATIENTS: A CLINICO-RADIOGRAPHICAL
STUDY.”
Principal investigator- Dr. Nida Sultana
OPD No.:
I the undersigned Mr./Ms./Mrs.____________________________________have

been informed about the study and its related procedure that will be carried out in the
Department of Periodontology at “Rungta College of Dental Sciences and Research,
Bhilai” in the language which I understand.
I have understood the procedure completely and I willingly give my consent to

Dr. Nida Sultana to carry out the procedure for her dissertation. I have also been
informed that the confidentiality of the names will be maintained and the recorded
data and photographs will be used for the record keeping, dissertation, scientific
discussion, presentations, publications, etc. I do not have any objection for all the
above.
Signature of Patient Signature of

parents/Guradians
Signature of Investigator Signature of Staff
156
Annexures
RUNGTA COLLEGE OF DENTAL SCIENCES AND RESEARCH,

BHILAI
CASE HISTORY PROFORMA
“A COMPARATIVE EVALUATION OF THE EFFICACY OF AMNION
MEMBRANE (AM) ALLOGRAFT BARRIER WITH DEMINERALISED FREEZE-
DRIED BONE ALLOGRAFT (DFDBA) AND DFDBA ALONE IN TREATMENT OF
THE PERIODONTAL INTRABONY DEFECTS IN CHRONIC PERIODONTITIS
PATIENTS: A CLINICO-RADIOGRAPHICAL STUDY.”
Name: Date:
Age: OPD No:
Sex: Case No:
Occupation: Telephone No:
Address:
Chief Complaint:
History of Present Illness:
Past Dental History:
Personal History:
157
Annexures
1) PARAMETERS CONSIDERED FOR EVALUATION:
AT BASELINE:
DATE:
Plaque Index (PI) Silness and Loe (1964)
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
PI SCORE:
Gingival Index (GI) Loe and Silness (1963)
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
GI SCORE:
AFTER 90th DAY:

DATE:
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
158
Annexures
PI SCORE:
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
GI SCORE:
AFTER 180th DAY:

DATE:
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
PI SCORE:
8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8
GI SCORE:
2) DATA RECORDED FROM SURGICAL SITE:
 Location of infrabony defect:

 Group I:
159
Annexures
 Group II:
Clinical measurements:
Clinical BASELINE 90th DAY 180th DAY

parameters
Site Group I Group II Group I Group II Group I Group II
Probing
Pocket
Depth
(PPD)
Relative
Attachment
Level
(RAL)
Gingival
recession
(GR)
Radiographic measurements:
Parameters BASELINE 180th DAY

Site Group I Group II Group I Group II
Bone Loss
(from CEJ –
base of the
defect)
Defect Depth
(from crest of
bone – base of
the defect)
Crestal Height
(from CEJ –
crest of bone)
160
Annexures
Percentage of
Defect Fill
(area of defect)
3) PROVISIONAL DIAGNOSIS:
4) INVESTIGATIONS:
 Complete blood count:
 Blood sugar:
 Orthopantomograph (OPG):
 Radiovisiography (RVG):
 Study models:
5) FINAL DIAGNOSIS:
STAFF SIGNATURE
161
Annexures
MASTER TABLE 1
Group I (DFDBA) Group II (DFDBA WITH AM)
Name Age/ Plaque Index Gingival Index Name Age/ Plaque Index Gingival Index
Sex Baseline th
90 day th
180 day Baseline th
90 day th
180 day Sex Baseline th
90 day th
180 day Baseline 90th day 180th day
Pankaj Patil 31/M 2.1 1.4 0.7 1.8 0.2 0.5 Pankaj Patil 31/M 2.2 1.2 0.6 2.3 1.3 0.8
Bharti Sahu 36/F 2.2 1.5 0.9 2.3 1.1 0.7 I Sunil 26/M 2.4 1.5 0.7 1.9 1.1 0.7
Shailendri Dhahariya 38/F 2.5 1.8 0.8 1.9 0.8 0.5 I Sunil 26/M 2.1 1.2 0.5 2.2 1.2 0.7
Pankaj Patil 31/M 2.2 1.6 0.7 2.1 1.2 0.6 Bharti Sahu 36/F 2.2 1.6 0.8 2.1 1.3 0.6
Rituraj Goswami 40/M 2.4 1.4 0.5 2.3 1.1 0.6 Shailendri Dhahariya 38/F 2.6 1.8 0.8 2.3 1.1 0.5
D Vamsi 25/M 2.3 1.1 0.6 1.9 0.9 0.5 Shailendri Dhahariya 38/F 2.5 1.5 0.6 1.3 0.7 0.4
Rituraj Goswami 40/M 2.4 1.7 0.8 2.1 1.2 0.7 T Anita 31/F 2.8 1.7 0.7 2.3 1.1 0.6
D Vamsi 25/M 2.2 1.2 0.6 1.9 0.8 0.5 Pramila Banjare 35/F 2.5 1.4 0.6 2.1 0.9 0.6
Mohini Sahu 48/F 2.4 1.3 0.7 1.9 0.7 0.5 Bharti Chandrakar 26/F 2.2 1.6 0.6 2.1 1.1 0.7
Shiv Kumari 29/F 1.9 1.2 0.5 1.8 0.8 0.4 Mohini Sahu 48/F 2.3 1.2 0.6 1.9 0.7 0.5
Shiv Kumari 29/F 2.3 1.6 0.9 2.2 1.1 0.8 Shiv Kumari 29/F 2.5 1.5 0.8 1.8 0.8 0.5
Chandana Rao 40/F 1.8 1.1 0.6 1.9 0.9 0.6 Bharti Chandrakar 26/F 2.3 1.2 0.7 1.9 0.7 0.4
Usha Chaubey 42/F 1.8 1.1 0.6 1.8 0.7 0.5 Chandana Rao 40/F 2.5 1.6 0.8 2.1 0.7 0.6
Ganga Das 32/M 2.1 1.3 0.8 2.2 1.2 0.5 Ganga Das 32/M 2.8 1.4 0.9 2.2 0.8 0.5
V Uma 34/F 2.2 1.6 0.9 2.3 1.2 0.7 Dhanesh Ku. Sahu 27/M 2.2 1.3 0.6 1.6 0.7 0.6
Usha Chaubey 42/F 2.5 1.5 0.7 2.2 1.1 0.6 Beena Yadav 31/F 2.6 1.5 0.8 2.2 1.1 0.8
Nandlal Kalet 46/M 1.9 1.4 0.7 2.2 1.2 0.4 Dhanesh Ku. Sahu 27/M 2.4 1.4 0.6 2.2 0.9 0.6
Usha Chaubey 42/F 1.8 1.2 0.5 2.2 1.6 0.7 K Saibabu 54/M 2.6 1.7 0.8 2.2 0.8 0.7
Dhanesh Ku. Sahu 27/M 1.9 1.5 0.6 2.2 1.4 0.6 Vijay Badge 51/M 2.1 1.2 0.5 2.2 0.9 0.6
Bhagraji Yadav 55/F 1.9 1.3 0.7 2.2 1.3 0.5 Vijay Badge 51/M 2.2 1.6 0.2 2.2 1.3 0.9
162
Annexures
MASTER TABLE 2

Site
PPD RAL GR PPD RAL GR
no.
th th th th th th th th th th
B 90 d 180 d B 90 d 180 d B 90 d 180 d B 90 d 180 d B 90 d 180 d B 90th d 180th d
1 7 4 3 11 8 7 0 1 1 7 5 3 12 10 8 0 1 0
2 7 4 4 13 10 10 1 1 0 8 6 4 15 13 11 0 0 0
3 8 5 5 13 10 10 2 2 1 7 5 3 15 13 11 0 0 1
4 6 4 3 13 11 10 0 1 1 7 4 3 15 12 11 1 1 1
5 6 5 4 13 12 11 1 1 2 8 5 3 14 11 9 1 0 0
6 6 5 4 15 14 13 0 1 1 9 7 4 16 14 11 1 0 0
7 7 6 5 11 10 9 1 2 2 8 5 4 10 7 6 2 1 1
8 6 4 3 10 8 7 1 1 1 7 4 3 10 7 6 2 1 1
9 7 5 4 10 8 7 2 1 1 6 5 4 13 12 11 0 1 1
10 8 6 4 12 10 8 0 2 1 7 5 3 11 9 7 0 1 0
11 7 5 5 11 9 9 0 1 1 6 4 3 12 10 9 1 1 1
12 6 6 4 11 11 9 1 1 2 6 5 4 11 9 7 1 1 0
13 8 6 5 12 10 9 2 1 1 7 7 5 12 12 10 0 0 0
14 7 5 3 12 10 8 0 1 2 8 5 3 12 9 7 1 2 2
15 6 5 4 11 10 9 0 0 0 7 5 4 13 11 10 0 1 0
16 8 6 5 13 11 10 1 1 0 6 5 3 15 14 12 0 1 1
17 6 6 5 15 15 14 1 1 2 7 4 3 14 11 10 0 0 0
18 8 6 5 12 10 9 0 0 1 8 5 3 16 13 11 1 1 0
19 7 5 3 11 9 7 0 1 0 9 5 3 12 8 6 1 1 1
20 6 5 4 12 11 10 0 0 1 8 6 4 11 9 7 2 2 1
163
Annexures
MASTER TABLE 3

Site
Defect depth Amount of defect fill Percentage of defect fill Defect depth Amount of defect fill Percentage of defect fill
no.
th th th th th
Baseline 180 day 180 day 180 day Baseline 180 day 180 day 180th day
1 4 2 2 50 4.5 1.8 2.7 60
2 4.2 2.5 1.7 40.4 5 1.2 3.8 76

3 5 1.2 3.8 76 4.8 2 2.8 58.3
4 4 1.2 2.8 70 5.2 2.8 2.4 46.1
5 8 3 5 62.5 5 2 3 60
6 5 1.5 3.5 70 4 1.2 2.8 70
7 6 3 3 50 4.2 2.5 1.7 40.4
8 4.5 2.2 2.3 51.1 5 1.5 3.5 70
9 4.1 1.5 2.6 63.4 5 2 3 60
10 4 2 2 50 4.8 2.5 2.3 47.9

11 6 2.8 3.2 53.3 4.2 2.2 2 47.6
12 4.5 2 2.5 55.5 5 1.8 3.2 64

13 6 2.8 3.2 53.3 5 1.2 3.8 76
14 4.8 2 2.8 58.3 5 1.2 3.8 76
15 4.8 2 2.8 58.3 4 1.2 2.8 70

16 4 2 2 50 4.8 2 2.8 58.3
17 4.2 2.5 1.7 40.4 5 1.5 3.5 70
18 5 1.5 3.5 70 6 3 3 50
19 6 2.8 3.2 53.3 4 2 2 50

20 5 1.8 3.2 64 5 1.5 3.5 70
164

Dr. Nida Sultana Thesis

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Dr. Nida Sultana Thesis

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PANDIT DEENDAYAL UPADHYAY MEMORIAL HEALTH

SCIENCES & AYUSH UNIVERSITY OF CHHATTISGARH

“A COMPARATIVE EVALUATION OF THE EFFICACY OF

Under the guidance of

Writing this thesis has been fascinating and extremely rewarding. As a

or indirectly in shaping up my academic career. It was hardly possible for me to

thrive in my thesis work without precious support of these personalities. Here is

the small tribute to all those people.

To commence with, I bow my head in deep reverence to the ALMIGHTY

At this moment of accomplishment, I am greatly indebted to my guide

Dr. KARTHIK KRISHNA M Professor and Head of Department of Periodontology at

pathway full of hurdles. Whenever I digressed, his amazing gift of explaining

complicated things in a simplified manner helped me in regaining my focus. His

passion, unflinching courage and conviction has always inspired me to do more. He

is one teacher who truly made a difference in my life. No matter what I do to

owe him, my sincere thanks.

I humbly acknowledge Mr. SANJAY RUNGTA, Chairman, Rungta College of

highly advanced research institute and allowed me to do this research.

My earnest thanks to Dr. V GOPINATH and Dr. PADMA R Professor &

I would like to express my heartfelt gratitude and deep appreciation to

Dr. AENA JAIN PUNDIR Professor in the Department of Periodontology at Rungta

to work which made me realize the worth of discovering my own capabilities.

I am grateful to my prized staff Dr. SONIKA BODHI and Dr. SHRUTI

BHATNAGAR Senior Lecturers in the Department of Periodontology at Rungta

also for their constant guidance.

I also express my deep thankfulness to Dr. BALASUBRAMANYAM VASANT

have been meaningless and incomplete.

I am extremely thankful to Tissue bank of TATA Memorial Centre, Advanced

me the Amniotic Membrane and Bone Graft used in the study.

encouragement in shaping this thesis.

I owe thanks to a very special person, my husband Dr. SHAHZAD AHMAD

at times I thought that it is impossible to continue, he helped me to keep things

love and unconditional support.

I am bound to express my thanks to my colleagues Dr. SAMARPITA NANDA

and Dr. SMRITI GUPTA for constant encouragement, motivation, unconditional

support and positive appreciation throughout my post-graduation period. Even in

wholeheartedly, I couldn’t appreciate it enough.

It’s my fortune to gratefully acknowledge the support of my juniors

Dr. SABREENA WILLIAMSON, Dr. KANCHAN AGRAWAL and Dr. LAWANYA

CHANDRAKAR for helping and enabling me comprehend and overcome the

hardships and hurdles throughout the course of my post-graduation.

I am also thankful to my sub juniors Dr. ABHISHEK HIRWANI, Dr. SYEDA

I wish to acknowledge the help and cooperation from our non-teaching

staff Mrs. JAYA DAS and Mrs. KAMLAWATI DEVI.

values and principles in my life.

proud and I owe everything to them.

I extend my sincere thanks to my brother NAHEED KHAN, my sister

Narrow border of language could never express my respect and gratitude to

success of this dissertation.

unknowingly helped me in the successful completion of my dissertation.

Dr. NIDA SULTANA

ACRONYMS FULL DESCRIPTION

ABM Allogenic Bone Matrix

ACM Amnion Chorion Membrane

AIAM Antibiotic Impregnated Amniotic Membrane

ANOVA Analysis of Variance

BMP Bone Morphogenic Protein

BOP Bleeding on Probing

CAL Clinical Attachment Level

CBCT Cone Beam Computed Tomography

CPS Calcium Phosphosilicate

DBM Demineralized Bone Matrix

DFDBA Demineralized Freeze-Dried Bone Allograft

DMSD Digital Media Sustainable Development