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SoDM Masters Theses
School of Dental Medicine
June 1999
TNF- a Levels in the Gingival Crevicular Fluid of
Teeth with External Apical Root Resorption
Robert Babak Marzban
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Recommended Citation
Marzban, Robert Babak, "TNF- a Levels in the Gingival Crevicular Fluid of Teeth with External Apical Root Resorption" (1999).
SoDM Masters Theses. 86.
http://digitalcommons.uconn.edu/sodm_masters/86
TNF- c LEVELS IN THE GINGIVAL CREVICULAR FLUID.
OF TEETH WITH EXTERNAL APICAL ROOT RESORPTION
Robert Babak Marzban
B.S. George Mason University
D.D.S. Medical College of Virginia
A Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Masters of Dental Science
at the
University of Connecticut
1999
APPROVAL PAGE
Masters of Dental Science
TNF-ot LEVELS IN THE GINGIVAL CREVICULAR FLUID OF TEETH
WITH EXTERNAL APICAL ROOT RESORPTION
Presented by
Robert Babak Marzban, D.D.S.
Major Advisor
Associate Advisor
Edward F. Rossomando
Associate Advisor
Kamran E.
Associate Advisor
Ravindra Nanda
University of Connecticut
1999
ii
ACKNOWLEDGMENTS
My ultimate goal to become a practicing member of the health profession will
finally come to fruition. It took only 24 short years of education. Despite previous
degrees and graduations, the completion of this Master’s Thesis is the true mark for the
end of my formal education. The long list of people deserving thanks for aiding me
throughout this process should begin with my family.
No words could describe the appreciation that my family deserves. My wife
Tourang, who remained by my side for the last eight years, despite turbulent and trying
times. You provided me with insight, support, and love in those time when it was needed
most. My father, who instilled the desire and the ethics to work, learn, and succeed by
example. My mother taught me to have passion for life and family. And my sister who
taught me how to be kind and have compassion (I still need a great deal of work in this
area).
I would like to thank Dr. Norton, my major advisor, for providing me with
direction throughout this project. Without you, Dr. Rossomando, and Dr. Safavi, I would
still be working on my lit review. For giving me the opportunity to obtain the best post-
graduate education possible and for getting my name in Medline, I would like to thank
Dr. Nanda. Not only did you accept me into your program, but also into your family and
circle of friends. Fighting through the long line of past and future residents I would like
to extend my sincere gratitude to Dr. Andrew Kuhlberg who is the true resident’s
advocate, who taught me how to stay on the straight and narrow, who helped keep my
iii
sanity, and who shared his office with me despite my poor choice in music. Thanks also
to Drs. Pae and Burleson who, along with Dr. Kuhlberg, kept statistical anarchy at a
minimum.
Second to my great looks, my most cherished attribute remains to be my group of
Mends. Aside for being the sounding board for my complaints and brain storms, my coresidents who have grown to be excellent friends have encouraged me to work hard while
playing hard. A special thanks goes to Dr. O’Hea, without whom my sample size could
be enumerated on a single hand. I would also like to thank Dr. Wadhwa for providing me
with access to his computer and for making sure that my nutritional requirements are not
neglected. Finally, thanks to Drs. McKenna, Priebe, and Nadeau for their help in
improving our sampling technique.
iv
TABLE OF CONTENTS
ABSTRACT
1
INTRODUCTION
2
REVIEW OF LITERATURE
TERMINOLOGY
INCIDENCE
INDIVIDUAL SUSCEPTIBILITY
BIOLOGY OF ROOT RESORPTION IN RESPONSE TO TOOTH MOVEMENT
4
Hyalinization
Odontoclasts
Initiation ofRoot Resorption
Restoration of resorbed root structure
Resistance to resorption
CLINICAL CORRELATIONS
Force magnitude
Treatment duration
Age
Systemic factors and diet
Alveolar bone density
Root canal treatment, trauma, and pernicious habits
Vulnerability ofspecific teeth
Mechanics
Retention and post-retention
DENTAL HEALTH AFTER ROOT RESORPTION
CYTOKINES AND HARD-TISSUE METABOLISM
lnterleuln-1
Tumor Necrosis Factor
Involvement in dental pathology
TOOTH ERUPTION MODEL
CYTOKINE INVOLVEMENT IN ORTHODONTICS
GINGIVAL CREVICULAR FLUID
RATIONALE
A NON-INVASIVE TECHNIQUE TO SAMPLE THE PDL IN HUMANS
PREVIOUS DATA
SPECIFIC AIMS
RESEARCH METHODS
STUDY POPULATION
DATA COLLECTION
4
5
6
8
8
9
10
12
13
15
15
17
18
19
20
20
21
22
23
24
25
26
28
29
31
33
36
]9
39
40
41
43
43
43
CLINICAL DETERMINATION AND SCORING
SAMPLING FROM THE GINGIVAL SULCUS
QUANTIFICATION OF BEAD-BOIYND ANTIBODIES
DATA ANALYSIS
DIFFICULTIES
45
47
48
49
SIGNIFICANCE
4
RESULTS
SAMPLE SELECTION
DESCRIPTION OF THE SAMPLE
DETERMINATION OF ROOT LOSS
TUMOR NECROSIS FACTOR-o: MEASUREMENTS
ZERO TNF- MEASUREMENTS
CORRELATION TO TNF-c
PATTERNS SPECIFIC TO PATIENTS
STEPWISE MULTIVARIABLE REGRESSION MODEL
55
55
56
58
59
60
61
61
62
DISCUSSION
DESCRIPTION OF THE SAMPLE
DETERMINATION OF ROOT LOSS
TNF-c LEVELS IN THE GINGIVAL SULCUS
THE ASSOCIATION BETWEEN TNF-tz AND CLINICAL VARIABLES
The association between root loss and TNF-z values
Gingival conditions and TNF-cr levels
TNF-z in the gingival crevicular fluid
GENERAL DISCUSSION
FUTURE STUDIES
65
66
67
69
70
71
72
74
76
CONCLUSION
78
APPENDIX I: FIGURES
APPENDIX II: TABLES
................................................................................................98
APPENDIX III. CONSENT FORMS
INFORMED CONSENT FOR ADULTS
INFORMED CONSENT FOR DEPENDENTS
BIBLIOGRAPHY
vi
161
161
164
167
LIST OF FIGURES
FIGURE 1. BONE METABOLISM. CONTRIBUTION OF CYTOKINES, HORMONES, AND
VARIOUS GROWTH FACTORS IN REGULATION AND STIMULATION OF BONE
METABOLISM
79
FIGURE 2. METHODS #1 AND #2 FOR DETERMINING ROOT LOSS
80
FIGURE 3. IMMUNOMAGNETIC BEADS, 5tM IN DIAMETER, ARE COATED WITH
ANTI-TNFct ANTIBODIES AND USED TO CAPTURE TNF-c MOLECULES FROM
THE GINGIVAL SULCUS
81
FIGURE 4. ELISA IS USED TO QUANTIFY THE AMOUNT OF TNFo HARVESTED
FROM THE GINGIVAL SULCUS
82
FIGURE 5. DISTRIBUTION OF TEETH IN OUR SAMPLE
83
FIGURE 6. DISTRIBUTION OF NORMAL AND DEVIATING ROOT SHAPES
84
FIGURE 7. DISTRIBUTION OF THE PLAQUE INDEX
84
FIGURE 8. DISTRIBUTION OF THE GINGIVAL INDEX
85
FIGURE 9. DISTRIBUTION OF THE PERIODONTAL INDEX
85
FIGURE 10. TIME SINCE SUBJECTS LAST BRUSHED THEIR TEETH UNTIL THE TIME
OF SAMPLING (IN HOURS)
86
FIGURE 11. TYPES OF TOOTH MOVEMENT ATTEMPTED DURING THE PERIOD
PRIOR TO SAMPLING
87
FIGURE 12. FORCE APPLICATION DETERMINED AT THE VISIT PRIOR TO SAMPLING
(APPLIED) AND AT THE TIME OF SAMPLING (REMAINING)
FIGURE 13. TIME IN TREATMENT FROM INITIAL BANDING VISIT (MONTHS)
88
89
FIGURE 14. AMOUNTS OF ROOT LOSS IN OUR SAMPLE AS DETERMINED FROM
METHOD #1 (RL)
90
vii
FIGURE 15. AMOUNTS OF ROOT LOSS AS DETERMINED FROM METHOD #3 (RRI)..91
FIGURE 16. TNF-c DISTRIBUTION IN OUR SAMPLE (PICOGRAMS)
92
FIGURE 17. TNF-et VALUES TABULATED AS AN INDEX
93
FIGURE 18. TNF-a VALUES TRANSFORMED USING THE NATURAL LOGARITHM
94
FIGURE 19. DISTRIBUTION: SAMPLES WITH ZERO TNF-a VALUES ARE OMITTED..95
FIGURE 20. DISTRIBUTION OF TABULATED DATA USING THE TNF INDEX AND
OMITTING SAMPLES WITH ZERO VALUES
96
FIGURE 21. DISTRIBUTION OF TRANSFORMED DATA USING NATURAL
LOGARITHM AND OMITTING SAMPLES WITH VALUES OF ZERO
viii
97
LIST OF TABLES
TABLE 1. INCIDENCE OF ROOT RESORPTION IN NON-ORTHODONTICALLY
TREATED INDIVIDUALS
98
TABLE 2. INCIDENCE OF ROOT RESORPTION SUBSEQUENT TO ORTHODONTIC
TREATMENT
99
TABLE 3. REPORTED AMOUNTS OF ROOT RESORPTION SUBSEQUENT TO
ORTHODONTIC TREATMENT
100
TABLE 4. CYTOK1NES WITH EFFECTS ON BONE METABOLISM
101
TABLE 5. ACTIONS COMMON TO BOTH INTERLEUKIN -1 AND TUMOR NECROSIS
FACTOR
102
TABLE 6. MOLECULES FOUND IN GINGIVAL CREVICULAR FLUID
103
TABLE 7. COMPOSITION OF THE GINGIVAL SULCUS. 236
104
TABLE 8. CRITERIA FOR PATIENT SELECTION AND DISQUALIFICATION
105
TABLE 9. SAMPLE SUMMARY. THE ABOVE FORM WILL BE USED TO COLLECT
DATA AT EACH VISIT
106
TABLE 10. ROOT RESORPTION INDEX
107
TABLE 11. THE PERIODONTAL INDEX (PI). 237
107
TABLE 12. GINGIVAL INDEX (GI). 224
108
TABLE 13. PLAQUE INDEX. 225
108
TABLE 14. DISTRIBUTION OF TEETH INCLUDED IN OUR SAMPLE. ALSO REFER TO
FIGURE 5
109
TABLE 15. DISTRIBUTION OF NORMAL AND DEVIATING ROOT SHAPES. ALSO
REFER TO FIGURE 6
110
ix
TABLE 16. DISTRIBUTION OF THE PLAQUE INDEX. ALSO REFER TO FIGURE 7
110
TABLE 17. DISTRIBUTION OF THE GINGIVAL INDEX. ALSO SEE FIGURE 8
111
TABLE 18. DISTRIBUTION OF THE PERIODONTAL INDEX. ALSO SEE FIGURE 9
111
TABLE 19. TIME SINCE SUBJECTS LAST BRUSHED THEIR TEETH UNTIL THE TIME
OF SAMPLING (IN HOURS). ALSO REFER TO FIGURE 10
112
TABLE 20. TYPES OF TOOTH MOVEMENT ATTEMPTED DUR1NG THE PERIOD PRIOR
TO SAMPLING
112
TABLE 21. FORCE APPLICATION DETERMINED AT THE VISIT PRIOR TO SAMPLING
(APPLIED) AND AT THE TIME OF SAMPLING (REMAINING). ALSO REFER TO
FIGURE 11
TABLE 22. TIME IN TREATMENT FROM INITIAL BANDING VISIT (MONTHS)
113
113
TABLE 23. DESCRIPTIVE STATISTICS OF THE THREE METHODS USED TO ASSESS
ROOT RESORPTION
114
TABLE 24. DESCRIPTIVE STATISTICS OF THE DIFFERENCE BETWEEN REPEATED
MEASURES
115
TABLE 25. REPEATABILITY (ERROR) OF THE THREE METHODS FOR
DETERMINATION OF ROOT LOSS
116
TABLE 26. CORRELATION BETWEEN THE THREE METHODS USED TO ASSESS ROOT
LOSS
116
TABLE 27. DISTRIBUTION OF TUMOR NECROSIS FACTOR ALPHA (TNF-a) VALUES. 117
TABLE 28. DISTRIBUTION OF RAW TNF-c VALUES IN OUR SAMPLE (PICOGRAMS). 118
TABLE 29. TNF-ct VALUES TABULATED USING AN ORDINAL INDEX 0-5
119
TABLE 30. TNF-c VALUES TRANSFORMED USING THE NATURAL LOGARITHM 119
TABLE 31. T-TEST FOR TOOTH SELECTION
120
TABLE 32. T-TEST FOR ROOT SHAPE
121
TABLE 33. T-TEST FOR TIME SINCE BRUSHING
122
TABLE 34. T-TEST FOR THE TYPE OF TOOTH MOVEMENT
123
TABLE 35. T-TEST FOR THE TIME IN TREATMENT
124
TABLE 36. T-TEST FOR THE PLAQUE INDEX
125
TABLE 37. T-TEST FOR THE GINGIVAL INDEX
126
TABLE 38. T-TEST FOR THE PERIODONTAL INDEX
127
TABLE 39. T-TEST FOR ROOT LOSS (RL)
128
TABLE 40. T-TEST FOR CORRECTED ROOT LOSS (RL*)
129
TABLE 41. T-TEST FOR THE ROOT RESORPTION INDEX
130
TABLE 42. DISTRIBUTION WITH SAMPLES CONTAINING ZERO TNF VALUES
OMITTED
131
TABLE 43. DISTRIBUTION OF TNF VALUES WITH SAMPLES OF ZERO OMITTED... 131
TABLE 44. DISTRIBUTION OF TNF VALUES SORTED ACCORDING TO THE TNF
INDEX WITH ZERO VALUES OMITTED. FOR COMPARISON, REFER TO TABLE
29
132
TABLE 45. DISTRIBUTION OF TNF DATA WITH NO SAMPLES OF ZERO WHEN
TRANSFORMED USING NATURAL LOGARITHM. FOR COMPARISON, REFER TO
TABLE 30
132
TABLE 46. CORRELATION COEFFICIENTS (TABLES 46-48)
133
TABLE 47. CORRELATION COEFFICIENTS (TABLES 46-48)
134
TABLE 48. CORRELATION COEFFICIENTS (TABLES 46-48)
135
TABLE 49. CORRELATION COEFFICIENTS, OMITTING SAMPLES WITH TNF VALUES
OF ZERO (TABLES 49-51). FOR LEGEND SEE TABLE 51
xi
136
TABLE 50. CORRELATION COEFFICIENTS, OMITTING SAMPLES WITH TNF VALUES
137
OF ZERO (TABLES 49-51)
TABLE 51. CORRELATION COEFFICIENTS, OMITTING SAMPLES WITH TNF VALUES
138
OF ZERO (TABLES 49-51)
TABLE 52. CORRELATION COEFFICIENTS BETWEEN ROOT LOSS AND RAW TNF
SCORES WEP CALCULATED FOR EACH PATIENT (N=19)
139
TABLE 53 DISTRIBUTION OF CLINICAL VARIABLES (TABLES 53-55)
140
TABLE 54. DISTRIBUTION OF CLINICAL VARIABLES (TABLES 53-55)
140
TABLE 55. DISTRIBUTION OF ROOT LOSS MEASURES AND TNF VALUES (TABLES
141
53-55)
TABLE 56. REGRESSION MODEL: Y= TNF, X= GI + MVMT + RRI
142
TABLE 57. REGRESSION MODEL: Y= TNF, X= GI + RRI
143
TABLE 58. REGRESSION MODEL" Y= TNF, X= GI
144
TABLE 59. REGRESSION MODEL" Y= TNF, X= RRI
1.45
TABLE 60. REGRESSION MODEL: Y= RANKED TNF, X= PLI + GI + RRI
146
TABLE 61. REGRESSION MODEL: Y= RANKED TNF, X= GI + RRI
147
TABLE 62. REGRESSION MODEL: Y= RANKED TNF, X GI
148
TABLE 63. REGRESSION MODEL: Y= RANKED TNF, X= RRI
149
TABLE 64. REGRESSION MODEL: Y= LN (TNF+ 1), X PLI + GI + RRI
150
TABLE 65. REGRESSION MODEL: Y= LN (TNF+ 1), X GI + RRI
151
TABLE 66. REGRESSION MODEL: Y= LN (TNF+ 1), X GI
152
TABLE 67. REGRESSION MODEL: Y= LN (TNF+ 1), X RRI
153
TABLE 68. REGRESSION MODEL: Y= TNF INDEX, X PLI + GI + RRI
154
TABLE 69. REGRESSION MODEL: Y=TNF-0’S, X=GI
155
xii
TABLE 70. REGRESSION MODEL: Y=TNF-0’S, X=RRI
156
TABLE 71. REGRESSION MODEL: Y= RANKED TNF-0’S, X=GI + BRS + RRI
157
TABLE 72. REGRESSION MODEL: Y= RANKED TNF-0’S, X=GI
158
TABLE 73. REGRESSION MODEL: Y= RANKED TNF-0’S, X=BRS
159
TABLE 74. REGRESSION MODEL: Y= RANKED TNF-0’S, X=RRI
160
xiii
ABSTRACT
Despite exhaustive research, the etiology and mechanism for the progression of
extemal apical root resorption (EARR) subsequent to orthodontic treatment still remains
evasive. The literature is replete with statistical correlates of clinical variables and root
resorption while there is little understanding of the cell biology of the underlying
mechanism, particularly in the human. This study evaluated the relationship between a
cytokine, which is known to be a significant participant in hard tissue metabolism, and
the incidence of root resorption in a population of patients who received comprehensive
orthodontic treatment. The levels of Tumor Necrosis Factor- Alpha (TNF-a) in the
gingival crevicular fluid of 85 teeth were measured and evaluated against the amount of
EARR and an array of clinical variables. The rationale for the use of an indirect
technique in monitoring root resorption is discussed. With respect to gingival
inflammation, the TNF-o data from this study reflected patterns seen in previous research
involving other cytokines. Also noted was the possible contributory role of gingival
inflammation as a confounding variable when evaluating the levels of cytokines which
are also inflammatory mediators. Regression analyses did not show a consistent model
where EARR and TNF-ot levels display a linear relationship. However, differences with
regard to the severity of root resorption were found between those samples which had a
measurable level of TNF-c and those where TNF-ct was absent (p<0.001).
INTRODUCTION
Root resorption is a common sequela of orthodontic treatment. Although a small
degree of resorption is almost universally seen, the longevity and the function of the
affected teeth remains uncompromised. The benefits provided through orthodontic
therapy typically outweigh the risk of slight root modification. Infrequently, the degree
of root resorption exceeds what is expected from conventional orthodontic therapy and
modification of treatment goals becomes a necessity. This progressive resorptive pattern
is rare, and although there are some statistical correlations, it is largely considered
unpredictable.
Over the last eighty years, a great deal of effort has been exerted in search of the
etiology and predictability of root resorption through clinical variables. A review of
literature uncovers a jumble of, sometimes conflicting, clinical information aimed at
finding the one clinical procedure or combination of perameters responsible for eliciting
the process. However, a reliable cause and effect relationship has not been found. Pure
clinical studies have provided information regarding directions in which to further
investigate.
Histologic studies are now becoming more commonplace in the root resorption
literature indicating a more finite approach. With the advent of new technology in
molecular biology and the existing database, it is possible to examine root resorption as it
occurs and to describe the precipitative events. The biology behind bone metabolism,
orthodontic tooth movement, and tooth eruption is better understood and may serve as a
model from which to study root resorption. These processes require the interplay of
various, but common, cells and their products. Admittedly, the list of biologic variables
is even more vast than the clinical variables which may be examined in orthodontic root
resorption. The purpose of this study is to examine if levels of a mediator which is
important in all the above mentioned processes is predictive for the incidence and
severity of root resorption.
REVIEW OF LITERATURE
TERMINOLOGY
The investigation of the process of root resorption began with primary teeth. This
research was extended to permanent teeth in the mid 1800’s. The relationship between
orthodontic treatment and root resorption of permanent teeth was first documented by
Ottolengui in 1914.1 Other studies soon followed in hopes of uncovering a specific
etiology. 2-7 An enumeration of the processes which cause root resorption includes:
normal physiologic tooth movement (drift), pressure fi’om an adjacent impacted tooth,
periapical or periodontal inflammation, tooth implantation or replantation, continuous
occlusal trauma, tumors or cysts, metabolic or systemic disturbances, local functional or
behavioral problems, untoward forces secondary to orthodontic treatment, and
idiopathic. 8-15
It would seem that any force, whether natural, pathologic, or iatrogenic, produces
activation of osteoclastic activity. Andreasen defines three types of external root
resorption. Surface resorption is a self limiting process that occurs on small areas of the
root surface. This is the resorption commonly seen after orthodontic treatment. It may be
undetectable by x-ray and transient, when the damage is minimal. Surface resorption is
followed with immediate repair by adjacent parts of the PDL where vital. Repair occurs
by way of restoration of the root surface with a cementum like tissue. Inflammatory
resorption indicates the progression of the initial resorption into dentinal tubules which
lead to an infected necrotic area, either the pulp or a leukocyte zone. Replacement of
resorbed tooth substance by bone is termed replacement resorption and results in sites of
ankylosis. 16, 17 Tronstad further defines inflammatory resorption as transient or
progresSive depending on the degree of the stimulus. Transient inflammatory resorption
would be the response to minimal stimulation resulting in a situation similar to
Andreasen’s surface resorption. Long-term stimulation from increased pressure,
infection, sharp edges, or a disease state is described as progressive inflammatory
resorption. 18
Surface or transient inflammatory resorption occurs with orthodontic tooth
movement. However, both of these events are by definition transient, mostly
undetectable by demal x-rays, and soon repaired. This cannot fully describe the majority
of external root resorption which occurs with orthodontic tooth movement. Tronstad
provides a more practical approach to describing root resorption in three categories.
Progressive inflammatory resorption is tissue-pressure related. Upon removal of the
stimulus, the process is halted. This process may results in apical root shortening and
may be severe in some cases. Cervical resorption occurs commonly, but is infrequent
subsequent to orthodomic therapy. The process is thought to be a result of injury to the
cervical attachment apparatus. Replacement resorption is described as stated above by
Andreasen. 16, 17
INCIDENCE
Approximately 90% of non-orthodontically treated permanent teeth present with
small areas of external root resorption. 19 Harris and Robinson found that 7-10 percent of
their non-treated sample exhibited obvious signs of apical root resorption, with severe
occurrences in 1-2 percent. 20 The incidence of root resorption, as reported in the
literature, is highly variable for non-treated and orthodontic patients (Tables 1-3). This is
partly due to the variation in the criteria for identifying root resorption between
investigators. For example, histologic resorption can easily be found even in the smallest
amount, in comparison to detection by dental x-rays. When detected, investigators differ
in their criteria for severity of resorption to qualify for incidence. In addition, there
differences in the site of examination (apical, cervical, etc.).
Root resorption incurred from orthodontic tooth movement occurs on all surfaces
of the root. However, it is most frequently noticed at the apex due to detection by way of
dental x-rays, hence the term extemal apical root resorption (EARR). Also, most
resorption sites are subsequently restored, except when occurring at the apex of the root.
Mild generalized resorption throughout the dentition is seen in almost every
orthodontically treated case ranging 0-3 mm. 19, 21-38 Most resorption sites are
relatively small (1 mm wide and 0.1 mm deep) and are soon repaired. Resorption beyond
3mm occurs in approximately 5-10% of orthodontically treated patients.
INDIVIDUAL SUSCEPTIBILITY
Individual susceptibility continues to be the most commonly cited reason for the
variability seen in degrees of root resorption after orthodontic treatment. There are
differences in susceptibility in relation to macroscopic tooth shape and size, microscopic
root shape, and differences in tissue response and activity. Even the process of root
resorption seems to vary in the same individual from site to site and from time to time.
Some researchers attribute the variability in the extent of EARR to genetic
predisposition. A heritable component for crestal bone loss has been found in twin
studies. 39 A few studies have described genetic inheritance of the predisposition for root
resorption, however a specific mode of inheritance was not found. 13, 31 A recent study
found evidence for a "substantive genetic factor in susceptibility to EARR." Although,
the authors admit that there is an obvious genetic relationship of malocclusions between
the siblings studied, and therefore, a common regimen of treatment. The similarity in
treatment between the siblings may also be a contributing factor to EARR in addition to
an "innate genetic predisposition". The authors discussed two independent pathways
effecting EARR. They speculated that the most important determinant comes from innate
genetic susceptibility, possible involving genotypically determined biochemical events.
The type of malocclusion requiting a specific treatment regime is a lesser, but also
important determinant. 40
Conflicting conclusions have been drawn regarding gender and its correlation to
root resorption. Many studies found a greater severity of resorption in females. 8, 13, 31,
41 Other studies cite an even ratio of incidence and severity between the two sexes and
attribute the increased severity found in females to their earlier maturation. 8, 9, 30, 42
BIOLOGY OF ROOT RESORPTION IN RESPONSE TO TOOTH MOVEMENT
Hyalinization
Tooth movement requires bone remodeling. In orthodontic treatment we rely on
appliances which deliver forces and create mechanical stresses which are transferred from
the tooth to the PDL and in turn, to alveolar bone. This stress induces biochemical
responses which evoke structural changes allowing for tooth movement by way of bone
remodeling.
As a tooth is displaced in the socket, early movement occurs by PDL compression
and tension. This elicits an acute inflammatory response characterized by periodontal
vasodilatation and migration of leukocytes out to the periodontal ligament capillaries. 43-
45 Cellular and vascular changes are followed by fibrillar alterations. Due to intolerable
pressure, cessation of circulation occurs and degenerative changes of the periodontium
leads to hyalinization.
With continued application of pressure, cells soon surpass the point of irreversible
degeneration. 46-48 The adjacent undamaged PDL in the border areas undergoes active
hyperemia characterized by high oxygen pressure with rapid blood flow, minimal
lymphatic flow, and minimal loss of oxygen, vitamins, and proteins. These conditions
are well suited for hard tissue resorbing cells, in contrast to passive edematous hyperemia
which occurs with high protein content in the tissue fluid inducing osteoblastic
activity.49, 50
The primary hyalinized zones in humans seldom last longer than three weeks.
The continuity between fibrils and cementum is not disrupted with normal orthodontic
treatment unless there is a localized focus of heavy pressure where hyalinization is
prolonged. 50 Pulpal blood flow disturbances and, very rarely, pulp necrosis may occur.
However, neither have been found to potentiate or elicit resorption of the root. 29, 51
Odontoclasts
Odontoclasts are the cells responsible for the resorption of roots. They are similar
to the osteoclast, large, pleomorphic (50-100 um), multinucleated cells, and most
probably ofhematopoietic origin. 58, 59 The cytoplasm is strongly basophilic,
granulated, and contains two to ten centrally located nuclei of various shapes and sizes.
Their presence in bone is infrequent, and when found, they are almost always near
mineralized matrix. Extensive golgi complexes reside near each nucleus with
mitochondria, lysosomes and free ribosomes abundant in the cytoplasm. These motile
cells also feature a ruffled border, similar to the osteoclast, constituting a complex folding
of the membrane, which is separated by a narrow space from mineralized matrix the
sealing zone. They are attracted to mineralized tissues like denuded root areas where
vital cementoblasts have been lost. Osteoclast have the capability of demineralizing
calcified tissue and degrading organic matrix. 18, 60 Odontoclasts, however, are unable
to resorb unmineralized matrix. 29, 51
The cytodifferentiation of the odontoclast has been studied by Sahara et al. They
found that tartarate resistant acid phosphatase (TRAP) activity correlates with
cytodifferentiation and function of the odontoclast. When following this process, it has
been noted that TRAP positive mononuclear cells make contact with predentin surfaces
10
on primary teeth. These cells spread out, fuse with each other, and develop into the
familiar multinucleated cells with specialized cellular components such as raffled borders
and clear zones. Upon termination of resorption, the ruffled border is lost and the cells
lose their attachment to the dentin surface. The authors concluded that odontoclasts
differentiate from TRAP positive progenitor cells. After attachment to the resorption
surface, these cells differentiate into mature odontoclasts which are able to resorb
predentin as well as detain.
Initiation
ofRoot Resorption
Orthodontic root resorption occurs in areas which are more sensitive to local
changes and where physiologic bone resorption originates. Specifically, root resorption
begins around hyalinized tissue, and is mediated by cells from the adjacent healthy
periodontal ligament. 52-55 The population of cell (first three days) involved in this
response are different than those involved in the later phase. Around the periphery of the
hyalinized zone, fibroblast-like cells from the adjacent vital parts of the PDL begin
removal of necrotic tissue. Multinucleated giant cells infiltrate and remove the main part
of the necrotic tissue, including the outer necrotic layer of the root surface. 56, 57
During the earlier phase of resorption, thinning of the cementum occurs more
rapidly directly beneath the hyalinized zone than further out to the periphery. Resorption
and repair of the PDL and root surface in the periphery is presumably mediated by cells
originating from the vital PDL. These cells have lower resorbing potential than those.
originating from alveolar bone and marrow. Initiation of a resorptive attack on the root
11
surface experimentally by creating mechanical injury, has been shown to occur more
frequently and aggressively if cells repopulating the area come from alveolar bone and
marrow. Removal of alveolar bone in this area apparently necessitates repopulation by
cells from the PDL and causes less resorption of the roots. 61-64
After removal of the alveolar bone wall and superficial cemental resorption in the
peripheral areas, root resorption stops and repair begins even in the presence of continued
orthodontic forces. By contrast, necrosis of the whole width of the PDL in the central
parts of the hyalinized zone and bone resorption necessary for tooth movement requires
presence of clast cells from the marrow. This theory of competitive healing and
observation of different cell populations at various times and location indicates that the
factor that promotes extensive clastic activity on the root surface may be the source of the
cells that repopulate the damaged area. 57
Direct root resorption begins with a hyalinized zone of the periodontium around
which areas of resorption soon appear. Indirect resorption begins from Howship lacunae,
undermining its way to the surface of the root. 24-26, 46-48, 65-67 Elimination of the
hyalinized PDL tissue takes 20-25 days, but if still stimulated, the resorption of root
structure may continue after this time. 50 At one week, the zone of hyalinization is at its
maximum after a single force application. The extent of the of the zone corresponds to
the size of resorbed root surface by three weeks. 57 It takes 10 to 35 days for resorption
lacunae to appear on the roots, however, this process is not yet visible in radiographs. 23,
28, 29, 50, 51
12
Restoration
of resorbed root structure
Upon removal of the applied forces, the resorptive process ceases. The repair
process which ensues provides a cementum fill for the lacunae. 25, 50 This repair process
delays further resorption. Complete healing may take up to 6 weeks in absence of
orthodontic forces. 26 It has been found that by allowing a tooth to relapse, the resorption
process can be halted. 50 By contrast, continued resorption occurs two to three weeks
after application of active force when the tooth is retained in the new position. This may
be due to passive stress in the PDL, which in the presence of remaining necrotic tissue,
elicits continued resorption. The clinical implication is that reactivation of an appliance
in the presence of necrotic tissue would likely cause continued resorption of the roots. 57
When the damage to root structure is small, cementoblasts from the vital portions
of the periodontal ligament provide repairs. In response to excessive forces, the large
amount of damage overwhelms the reparative ability of the cementum and PDL, forcing
bone marrow cells and alveolar bone to provide healing. The healing process, when
hosted by cell populations from marrow and alveolar bone leads to osteoclastic
activation.41, 42
Progressive insults to the root will develop areas of thinner cementum and in
instances where root resorption has already begun, the root surface may be devoid of
cementum altogether. 68 Until the repair process replenishes the cementum coveting for
the root surface, the process which has overwhelmed the repair mechanism will continue
to do irreversible damage. 24-26 This is exhibited in teeth with previous resorption.
13
Harris observed an increase of 15 % more root loss in teeth with previous signs of
resorption prior to beginning orthodontic treatment. 69
In examination of the reparative potential of resorbed roots, 28% of the teeth
studied showed signs or repair by the first week. By 8 weeks, 75% had begun repair.
The cementum was almost exclusively of the cellular type. During the first 4 weeks of
retention, repair with the resorption cavity walls only partially covered with cementum
was the most frequently seen. Between 4 to 8 weeks, functional repair with coverage of
the total surface of the resorption cavity walls dominated. Individual variation in healing
potential was great. 70
Deep lacunae were covered by a layer of cementum which was not thick enough
to restore the root to its original anatomy. This results in development of an irregular root
shape. When the apical portion of the root was resorbed, the repair cementum did not
restore the original length of the tooth. Instead, rough edges are remodeled and a layer of
cementum was laid over the apex. The PDL space undergoes bone remodeling in the
alveolus following the new root contour. 25, 26, 49, 50, 52-57, 65, 71
Resistance to resorption
The process of resorption proceeds more readily through dentin and bone than
cementum. The cementum lining the roots of teeth is somewhat resistant to remodeling
and resorption. Matrix tissues such as precementum, predentin, and osteoid have been
thought to provide resistance to resorption, however, recent data show that continuous
pressure can cause resorption of these areas also. 10, 25, 26, 65, 72 The effectiveness of
14
Sharpey’s fibers as a prevemative structure is debatable but corroborated by some. 60, 73
Root surfaces are covered with mounds which are formed by .continued mineralization of
attached periodontal fibers after the production of cementoid has stopped. Acellular
cementum has 100 percent Sharpey fiber content versus actively forming cellular
cementum with 50 percent.
Most bisphosphonates inhibit bone resorption. Hydroxyethylidene-1, 1bisphosphonate (HEBP) inhibits both bone formation and resorption. A single injection
in HEBP into rats can inhibit formation of acellular cementum. In its stead, an atypical
hyperplastic cementum is formed. In comparison to roots with acellular cementum, these
roots resorbed readily in response to orthodontic forces with no differences in the
numbers of multinucleated cells present. 74, 75
Rygh provides plausible explanations for the resistance of cementum to
resorption. First, there is less vascularity in the cementum when compared to bone.
Bone is constantly remodeled and thus the cells responsible for resorption are always
present in the PDL. On the cementum side of the PDL this is not true, as cementum is
constantly deposited throughout life. Also, cemental tissue is more mature than bone
tissue and thus less susceptible to chemical changes. Furthermore, unmineralized
cementum or cementoid appears to provide resistance to resorption by odontoclasts. 50
During the removal of the hyalinized zone, the cementum needs modification to
re-establish connection with the PDL. This requires removal of cementoid and the more
mature collagen, allowing a small breakthrough in the compromised area. The degree of
breakdown products from the hyalinized tissue which may have importance as a signal
15
for the development of resorbing cells is unknown. If rapid progression of root resorption
is noted, a rest period with removal of orthodontic forces is recommended. 26, 50, 76, 77
Studies by Andreasen show evidence that the cells in the innermost layer of the
PDL provide a protective mechanism against root resorption. The PDL consists of
cementoblasts, fibroblasts, osteoblasts, endothelial, and perivascular cells that supply the
various protective mechanisms and the potential for repair of the tissues adjacent to it.
The vitality of the PDL seems to provide protection for cementum. 16, 17 This
protection is also extended to the epithelial rests of Mallassez which are also present in
the PDL in close proximity to the cementum and may be responsible for substances
which prevent fusion of cementum to bone. 16, 17, 78 The mechanism becomes apparent
when examining autotransplantation studies. The greatest likelihood of success in an
autotransplantion is obtained when the PDL remains undisturbed. Those teeth are least
likely to undergo resorption if the PDL is left unharmed. 79
CLINICAL CORRELATIONS
Force magnitude
In theory, concentration of stress at the apex of the tooth would be closely related
to resorption of roots. This would explain why intrusive mechanics are commonly
blamed for severe incisor resorption. 34, 66, 80 However, other movements have also
been shown to cause resorption. 3, 10, 26, 35-38, 81, 82 Generation of high forces has
been noted to closely correlate with the appearance of resorption lacunae. 24-26, 28, 41,
42, 51, 65, 83, 84 This is part of the reasoning for the use of optimal forces in tooth
16
movement. Many early studies investigating the relationship of force to tooth movement
advocated the use of light and continuous force systems. Their findings established that
heavier forces did not provide any advantage in the rate of tooth movement and would
instead lead to a larger amount ofhyalinization, undermining resorption, and possibly a
greater degree of root resorption. 22, 28, 29, 51, 85-87
In review of a an excellent series of studies, force was found to increase the rate of
tooth movement but not the degree of resorption. 88 In comparison to a continuous 50
gram force, a 100 gram force applied by use of the same mechanism was not found to
have a significant difference in regards to tooth movement or EARR. 89 At 200 grams, a
significantly increased amount of tooth movement was seen. However, the degree of
EARR still was not significantly different than what was seen with the 50 gm force. 90 A
comparison of continuous and interrupted continuous forces showed that continuous
forces were more effective in producing tooth movement but again, there was no
difference in the amount of root resorption. 91 The experimental period in these studies
was limited to 7 weeks. Perhaps a longer examination time or a larger differential in the
amount of applied forces may have provided more insight into the effects of force
application with respect to root resorption.
When examining the experimental teeth at various time points (1 to 7 weeks),
increasing amounts of resorption was noted as time increased. Early signs were visible
by one week and increased to 20 times more by seven weeks. Resorption half way to the
pulp was found by the third week. The authors noted a great amount of individual
17
variation in EARR irrespective of the amount of tooth movement that occurred with the
continuous 50 grn force. 92
In studying palatal expansion where high force systems are generated Vardimon
et al found that compression of the PDL is excessive, thereby inhibiting "physioimmune
system" and potentiating EARR. They concluded that EARR has three determinants:
impulse, critical barrier, and environmental density. Impulse was found to be the
predominant factor with two components, force and time. During short time periods,
force magnitude is largely responsible for EARR severity in a logarithmic function. With
longer treatment, the time during which force is applied becomes the deciding factor. 83
Treatment duration
There seems to be some relationship between clinical treatment length and root
resorption.4, 5, 11, 12, 25, 29, 33, 35, 41, 42, 51, 76, 80 As early as two weeks after
force application, resorption lacunae develop in almost half of orthodontically treated
teeth.28, 29, 51 Levander and Malmgren found that if radiographic resorption is not
detected after 9 months of treatment, the amount resorbed by the end of treatment will
most likely not by excessive. 76, 77, 93 Because orthodontic appliances may be present
for long periods without applying pressure to specific teeth, many researchers are hesitant
to claim a cause and effect relationship when considering overall treatment time. 10, 34,
94
18
Age
The progression of age causes many changes in periodontal tissues. Alveolar
bone increases in density, the PDL space narrows, and both undergo a decrease of
vascularity and aplasia. Bone remodeling and osteoblastic activity are decreased in adults
and thus the initiation of tooth movement is delayed in comparison to the adolescent
patient. This is attributable to the increased time to reach the mobilization phase in
response to hyalinization during tooth movement. 25, 26, 65
In a study of alveolar bone tumover in rats, older rats were found to have higher
numbers of osteoclasts (5 per mm) in comparison to younger rats (2.5 per mm). The rate
of skeletal tumover, gauged by levels of alkaline phosphatase and acid phosphatase, was
higher in the younger rats. The authors concluded that in an older system there is a
higher recruitment of bone cells to compensate for a slower skeletal turnover. 95
The layer of non-calcified cementiod coveting the root surfaces of younger teeth
was found to be thicker on less mature roots and thus providing a protective barrier
against resorption. 22, 24, 50 These factors, in part, explain the findings of studies which
show correlation between early treatment and smaller degrees and decreased prevalence
of root resorption. 4, 5, 11, 21-26, 35-38, 60, 65, 80, 96
Orthodontic movement of teeth with incompletely formed apices has been
associated with decreased root length upon completion of tooth formation and
dilaceration.4, 5, 27, 29, 51, 96 Other researchers dispute the resistance of less mature
roots to resorption. 9, 80 Harris and Baker found that prior to commencing orthodontic
therapy, adults had shorter roots than adolescents. This corroborated the findings of
19
earlier research which noted root loss progression with age under normal conditions. 31 In
application of identical treatment techniques to samples of adolescents and adults, no
significant difference was found in the amount of further root resorption. The association
between age and root loss from tooth movement was superficial and could be attributed to
other dental factors (loss of dentition or periodontal problems). 97 In another study,
Harris stated that "loss of stability from adjacent teeth, use of fewer remaining teeth, and
loss of anchorage in bone are significant predictors of external apical root resorption". 20
Systemic factors and diet
Immunologic factors, systemic disease, and diet have been related to the
resorption of roots. The role of the immune system in root resorption has been
investigated, however, conclusive evidence has not yet been found. 98 Early
investigators believed systemic disease such as endocrine imbalances and metabolic
diseases of bone to be the major etiologic factor associated with root resorption. 6, 99
More recent data indicates an association of abnormal hormone levels from endocrine
diseases of the pituitary, thyroid, and parathyroid with root resorption but only as a
secondary causative factor. 5, 25, 100
Even a secondary relationship between hormone
level and root resorption is disqualified by some research. 99, 101 Deficiency of dietary
calcium and vitamin D were originally thought to cause the resorption of roots. 102
Recently it has been concluded that nutrient imbalances may also be only a secondary
influencing factor. 35, 36 In a controlled study, lactating rats with a calcium deficiency
were noted to have an expected increase in parathyroid hormone secretion and thus a
20
decrease in bone density. Tooth movement in these rats was greatly facilitated and low
levels of root resorption was noted. 103
Alveolar bone density
Movement through denser bone was found to correlate with higher amounts of
root resorption. Lamellar bone or cortical bone is difficult to resorb, in addition increased
amounts of marrow space in less dense bone allows easier recruitment of resorptive cells
for bony remodeling. 26, 33, 103-106 Kaley and Phillips found that the chance for root
resorption is 20 times greater when the maxillary incisors are in close proximity to the
palatal cortex. 107 Other researchers have found no relation between root resorption and
proximity to the palatal cortex. 37, 38, 108 In a study of the effects of rapid palatal
expansion on tooth roots, Barber and Sims noted that although severe resorption occurred
over the entire buccal root surface, over 35% of the root surface, even heavier resorption
occurred in the cervical third. The authors speculated that the close apposition of this part
of the root against the buccal cortical plate could be to blame. 109
Root canal treatment, trauma, and pernicious habits
Although a few early studies indicate otherwise, 110 current belief indicates that
teeth treated endodontically undergo decreased amounts of resorption, presumably due to
the increased density of dentin. 37, 38, 111 A common reaction to traumatic injury is
external root resorption and recommended treatment of these teeth includes calcium
hydroxide treatment followed by instrumentation and obturation at a later time. Internal
21
resorption is treated the same way, in hopes to increase dentin hardness and thus increase
resistance to resorption.
Roots of teeth which experience resorption in response to trauma are at increased
risk for further resorption when moved orthodontically. 31, 32, 106 However, no
increased resorption is seen in orthodontically teeth which remain unaffected by trauma.
A waiting period of 4-5 months is recommendable for those teeth which have
experienced trauma, during which no signs of inflammatory resorption become present.
112 Occlusal trauma from restorations, prosthetics, and even on natural teeth with
interferences and prematurities produce jiggling forces which will elicit thinning of the
cemental layer over roots with possible progression to root resorption. 113 Nail-biting,
mouth breathing, tongue thrust, clenching have all been positively associated with
increased root resorption. 33, 35, 69, 113, 114
Vulnerability ofspecific teeth
Although all teeth have been shown to display some degree of root resorption,
some teeth experience greater amounts of shortening than others. Many studies relate this
variability to difference in sensitivity. 2, 3, 9, 10, 31, 80, 81, 115 The amount of root
movement is thought to be a great contributing factor. 7, 10, 34, 82, 94, 116 In order of
decreasing severity, the most frequently affected teeth are maxillary incisors, mandibular
incisors, distal root of the mandibular first molars, and mandibular and maxillary second
premolars.10, 12, 26, 30, 94, 116 Also, root shape has been determined to be an
important factor in susceptibility. Roots which are dilacerated, blunt, or pipette-shaped,
22
undergo significantly more resorption than their normally shaped counterparts. 13, 34-38,
76, 80, 93 Teeth with naturally short roots those which are not resorbed were
previously thought to be more apt to experience resorption than longer teeth. In an
evaluation of force systems required for tooth movement, it becomes clear that longer
teeth need greater force values to be moved and the actual displacement of the root apex
is larger during tipping or torquing movemems. Newer studies corroborate this logic. 37,
38, 42, 76, 93
Mechanics
A great deal of controversy exists regarding the method and mechanics of tooth
movement in regards to root resorption. Studies involving correlations between root
resorption to orthodontic mechanics are phenomenological in nature. They are statistical
and do not describe the cause. The only consistent finding from this vast literature is that
applications of high moments delivered over long periods of time seem to increase the
chance of clinical root resorption. As stated before, maxillary incisors are the most
commonly affected tooth with regard to root resorption. This may be due to an inherent
sensitivity of these teeth to resorption as a.result of more extensive treatment, i.e.
intrusion, extent of movement, and movements which concentrate forces at the apex. 2124 But, even the correlation between the extent of root movement and intrusion to root
resorption is debated by some. 10, 34 Gottlieb did not observe root shortening or apical
blunting during incisor intrusion up to 4 mm (mean of 2.36 mm) using 15 to 20 grams of
force per incisor. 117 Using 25 grams of force per incisor, Dermaut and DeMunck
23
obtained 3.6 mm of intrusion. They observed 2.5 mm of root resorption but found no
correlation between the amount of intrusion or duration of applied imrusive force with
increased amounts of root resorption. 34 In addition, jiggling forces created from repeated
trauma by functioning on inclined planes or by using intermaxillary elastics and
removable appliances, have been shown to contribute to root resorption. 5, 7, 11, 35, 36,
41 Early research indicated that jiggling forces caused by removable appliances may be
harmful to the roots. 7 However, by use of fixed appliances, roots cannot function
individually which is also hypothesized to be harmful. 2, 35, 36
Comparisons of Begg versus edgewise techniques have been conflicting as to
which is less likely to illicit root resorption since both techniques have been reported to
be culpable. 41, 104-106, 112 Although more tipping movements are expected by use of
Begg mechanics, an accurate comparison between these two techniques is impossible
since throughout treatment so many different variables become apparent no matter which
technique is employed. Orthodontic treatment with or without extractions has not been
found to differ with regard to the potential for root resorption. 80, 82
Retention and post-retention
If severe root resorption is seen during treatment, either modification or
termination of treatment should be considered. Upon termination of active treatment,
cessation of the resorption process should occur. 10, 18, 19, 24, 82, 105, 118-120 At this
point repair begins, which in itself may cause a minor reduction in root length as the
rough edges at the root apex are remodeled. 26, 111, 121 This process may take
24
approximately 6 weeks after active forces are discontinued 26, however, instances where
active resorption continues for a prolonged time have been reported. 122 Traumatic
occlusion would be highly suspect. 11 Retainers with active forces should also be
examined when continued resorption is seen. Movement as a result of orthodontic
relapse has been de-valued as a contributor to resorption26, however, with no other
contributing factors, even light muscle forces should draw some attention. 105
As mentioned above, if radiographic resorption is not detected by 9 months of
treatment, the amount or EARR at the end of treatment will most likely not be
excessive. 76 However, if rapid progression to EARR is noted in mid-treatment, a pause
with removal or orthodontic forces is recommended. 26, 76, 93, 111 To demonstrate
this, a group of investigators compared incisors where EARR was noted after 6 months of
treatment. The control group continued the original treatment plan whereas in an other
group treatment was paused for 2-3 months. The experimental group had significantly
less resorption at the end of treatment than those in which the original treatment plan was
continued without a pause. 77
DENTAL HEALTH AFTER ROOT RESORPTION
It is well accepted that some root resorption occurs in every orthodontically
treated tooth. Most resorption sites are repaired to restore the original anatomy of the
root. Although defects are filled in by cementum, the initial reattachment of Sharpey’s
fibers is not as extensive as original which may indicate a less effective attachment
apparatus. 109 In the cases with apical root shortening, root loss is rarely greater than 2
25
mm. Loss of 2 mm of apical root length would cause loss of total attachment area of the
root by 5-10 percent. 10 Zachrisson concluded that 2 mm of root shortening is not
detrimental to the longevity and of the tooth. 123 More severe root shortening, 4 to 6 mm,
is equivalent to 20 to 35% decrease of attachment. 10 Marginal attachment loss,
specifically, alveolar bone height is considered more significant to support and function
that loss of root length. 10, 29, 32, 94, 115, 116, 123-125
CYTOKINES AND HARD-TISSUE METABOLISM
The term cytokine can include interleukins, tumor necrosis factors, interferons,
colony stimulating factors, growth factors, neuropeptides, monokines, and lymphokines.
Cytokines are a group of generally glycosylated peptide regulatory factors, produced
transiently by most nucleated cells. Even at low molecular weights (<80kDa), they are
very potent, acting in the 10-13 to 10-10 M range. They interact in a complex cell-tissue
communication network modulating growth and differentiation. 126 They have
pleiotropic overlapping biological activities, which are mainly autocrine or paracrine. 127
High affinity receptors, constitutively or inducible, are expressed on target cell surfaces
usually in low numbers and may be cytokine-specific or shared by distinct cytokines.
The chemical affinity of receptors for a wide range of cytokines has been measured to be
generally around 10-50 pM. 128 This corroborates studies that measure cytokine
concentrations required for 50% of maximal growth rate for cell cultures which are also
in the 10-50 pM range. 129, 130
26
The interaction between cytokines and cells is dependent on a number of factors.
This mode of communication is governed by cell secretion rates, transport processes, and
ultimately by genetic and biochemical processes. Secretion rates may be also be
modulated by many different means. The maximum secretion rate is an important factor
and is estimated by evaluation of molecules with specific half-lives and functions to
perform. In an analysis of mRNA and RNA polymerase, the maximum secretion rate of a
cell was estimated to be 2300-8000 molecules per second. Based on the above variables,
the effective intercellular communication distance is mathematically estimated to be 250
um or approximately 50 cell radii. This extrapolates to a 10-30 minute time constant for
intercellular communication by cytokines. 131
Originally a single molecule was thought to mediate bone resorption. Osteoclast
activating factor (OAF) was named as the factor released by cultured peripheral blood
mononuclear cells which had been activated with dental plaque antigens and stimulated
bone resorption in organ cultures. 132 Later it was found that this effect was mediated by
a number of molecules from various cell types. More recently, the number of cytokines
which are involved in bone metabolism has grown extensively. Table 4 and figure 1
illustrate some of the factors which play significant roles in bone metabolism.
lnterleukin-1
Interleukin -1 (IL-1) was the first immune cytokine positively identified as having
an action in the metabolism of bone. The name interleukin was conferred due to
27
regulatory functions on the immune system. Since then, many other functions have been
attributed to this family of proteins.
Two related proteins have been identified, IL-1 ot and IL-1
,
with almost identical
actions. The two proteins come from distinct genes on chromosome 2. Both ha,e 3 l kDa
precursors and share 26% amino acid homology. The mature IL-1 ot molecule contains
159 amino acids and weighs 17.5kDa with pI 5. IL-1 weighs in at 17.3 kDa, with 153
amino acids and pI 7. In most cell types IL-11 is synthesized and secreted in a greater
amount over IL-lc. 133
IL-I is more active in bone metabolism.
It may stimulate bone
resorption while inhibiting bone formation. 134 Although the amino acid homology
between the two forms is limited, both interact with the same plasma membrane
receptors. The affinity of the receptor for IL-1 does not correlate with the magnitude of
the biological response and the post-receptor events are unclear. Cyclic AMP (cAMP)
accumulation, protein phosphorylation, and other events have been measured following
stimulation of cells. 135 A third member of the IL-1 family has been discovered, and
although containing 26% homology with the other two members, it performs antagonistic
functions, thus named IL-1 receptor antagonist protein (IRAP or IL-lra). 136
As noted earlier, IL-1 has pronounced effects on the immune system. Its effects
on T and B cells mainly include induction of growth and differentiation other factors such
as IL-2, IL-4, IL-6 and interferon gamma. Interleukins may activate natural killer (NK)
cells when working in concert with other factors. Chemotactic effects have also been
found in these family of proteins. Apart from immune functions, IL-I has been found to
have major roles in the synthesis of acute phase proteins in the liver, in the coagulation
28
process in the endothelium, induction of fever at the level of the hypothalamus by
prostaglandin E-2 (PGE2) production, involvement in hemopoiesis, and various
metabolic effects. Powerful metabolic effects on cartilage, bone, and fibrous connective
tissue are well documented. 137, 138
IL-1 induced stimulation of bone resorption is seen as an increase of
multinucleated cells in marrow spaces. This resorption can be inhibited by calcitonin, but
is unaffected by indomethacin, suggesting that the resorption is osteoclast mediated but
that prostaglandin production is not required. 139 Other studies have found various
results regarding indomethacin inhibition in bone resorption. The role of prostaglandins
in bone resorption is well accepted, however, a prostaglandin-independent mechanism of
IL-1 induced resorption is also possible. In fact IL-1 is the most potent resorbing agent of
bone, active at concentrations of 3 X 10. 139
Tumor Necrosis Factor
Tumor necrosis factor (TNF) also exists in an o and [ forms. Both proteins are
coded for on chromosome 6 and share a 30% amino acid homology. TNFo is cleaved to
form a 17 kDa mature protein which, in the active form, exists as a trimer. 140, 141 TNF
has been found to interact with two receptors. Again, the post-receptor activities are
unclear.142 A major source of TNF is believed to be the monocyte and macrophage,
although it may be produced by a variety of cells. TNF production may be stimulated by
lipopolysaccharides, interferons, IL-1, and many other cytokines.
29
The pleiotropic actions of TNF are similar and sometimes overlapping with IL-1
(table 5). Both cytokines are produced by the same cell types and have synergistic
effects. TNF is involved in most of the pathways listed above for IL-1. In addition,
much attention has been attributed to TNF because of its cytotoxic effects on certain
tumor lines. 139-141, 143
TNF stimulates bone resorption in a way similar to IL’ 1. At very low
concentrations IL-113 and TNFc are implicated in bone remodeling through specific
receptors on bone cell populations. 45, 138, 144 In vitro studies show increased numbers
of osteoclasts in marrow cultures in response to TNF. 144 Both IL-1 and TNF have been
shown to induce osteoclastic activity in vivo. 145 In addition to osteoclast recruitment,
IL-1 and TNF have been found to inhibit osteoblast maturation, collagen synthesis and
osteocalcin production.
Involvement in dental pathology
Both IL-1 and TNF are implicated in the destruction of supporting structures in
periodontal disease. 146 This is in part due to the ability of these molecules to directly
induce secretion of collagenase by fibroblasts, and their facilitation of bone and cartilage
resorption. 144 Increased levels of both molecules are present in the periodontal tissues
and/or gingival crevicular fluid of sites exhibiting periodontal disease. 147-152
Following treatment of diseased sites, levels of these, and other cytokines levels
decrease. 153
30
Infection of the dental pulp may result in the formation of a periapical lesion and
bone resorption. The impetus for this process is thought to be the presence of immune
complexes, lipopolysaccharides, or other bacterial components any of which may trigger
the production of inflammatory.cytokines. 154, 155 Macrophages, fibroblasts,
neutrophils, and osteoclasts in the area of the infected pulp are stimulated to synthesize
and secrete cytokines, specifically, IL-1 a and TNF tx. 156
Generally, TNF is also a potent inducer of bone resorption, with the ct form being
more potent than the
13 form. Although, neither are as potent as IL-1. An increase in IL-1
may be a later and more significant potentiator of bone resorption. However, an increase
in TNF may be incipient and portend an IL-1 increase. Furthermore, IL-1 and TNF
stimulate endogenous PG production (and a slew of other cytokines) which would
abrogate the metabolic process. 157
IL-1 and TNF are synergistic, both stimulating PGE-2 production and bone
resorption (table 5). Many important synergistic effects are shared by these two
molecules. This includes regulation of phospholipid metabolism, induction of acute
phase proteins, prostacyclin synthesis, promotion of growth in fibroblasts, expression of
human major histocompatibility complex antigens, induction of fever, and synthesis of
hematopoietic growth factors, activation of inflammatory leukocytes, modification of
vascular permeability, and induction of bone resorption. 143, 158-160
Previous studies using in vitro models illustrate the interactive effects of cytokine
combinations on PGE production by fibroblasts. Using murine calvarial organ cultures, a
ten-fold increase in stimulation of PGE-2 production is seen when stimulated by IL-1 and
31
TNF in concert. This is in comparison to stimulation by 1 uL ofIL-1 or 50 ng/mL of
TNF which produced 5-7 mg/mL of PGE-2 when used individually. 161 Unfortunately,
quantitative evaluation of PGE production in response to cytokines in the in vivo model
remains a difficult task, but these data would help describe feedback mechanisms in other
cell types. 162
In summation, IL-1 and TNF are one of the earliest cytokines to be associated
with bone metabolism. The vast amount of research indicates that these molecules induce
bone resorption by affecting proliferation and migration of osteoclasts, by inducing the
release of other factors to aid in the process, and by influencing the differentiation of
osteoblasts. 138 The previously mentioned effects in addition to the synergistic effects
that these molecules have with other proteins/cytokines (PG’s) which also may induce
bone resorption make them invaluable markers for bone metabolism.
TOOTH ERUPTION MODEL
In an examination of the cell biology of root resorption, the resorption of
deciduous roots during tooth eruption may serve as a model. Marks and Cahill noted that
the demal follicle is necessary for the eruption process. 163 The follicle itself, consists of
a connective tissue sac which envelops the tooth before its eruption. If the follicle is
removed, eruption does not occur. Conversely, if the contents of the follicle are
substituted with inert material, leaving the follicle itself, eruption still occurs. 164 During
the process of eruption, monocytes and macrophages influx into the follicle and their
numbers generally peak at the time of active eruption. Osteoclast numbers also increase,
32
collecting around the coronal area of eruption crypt. As the monocyte population
decreases so does the osteoclast numbers. 165 Therefore, to allow tooth eruption through
bone, monocyte and macrophage influx is necessary for osteoclast formation. 166
Cells participating in tooth eruption and root resorption need to have a method of
communicating with each other. They do this by the creation of biochemical molecules,
chemical signals that target specific cell surface receptors. In their investigation of the
molecular signaling pathway for tooth eruption, Wise and Lin found four possible
candidates.166
The first and most likely trigger for tooth eruption is colony stimulating
factor 1 (CSF-1), also called macrophage colony stimulating factor. Kodama found that
CSF-1 played an essential role in osteoclast differentiation. 167, 168 In a study using
osteopetrotic rats, where a reduction of the number of osteoclasts prevented tooth
eruption, injection of CSF-1 triggered an osteoclast increase resulting in tooth
eruption.167, 169
Normal rats injected with CSF-1 experienced accelerated tooth
eruption and increase of osteoclast and monocyte numbers. 170 The CSF-1 gene is
expressed in the rat dental follicle and once translated, has an autocrine effect on the
CSF- 1 gene. 171
The presence of interleukin-1 alpha (IL-1 ct) has been noted in the stellate
reticulum. Because of its ability to enhance CSF-1 gene transcription, it is hypothesized
that a diffusion ofIL-1 ct into the dental follicle may be the trigger for CSF-1
transcription. 166 Epidermal growth factor (EGF) is suspected to be the regulator of IL-1 ct
expression. Following EGF injections in rats, levels oflL-let in the stellate reticulum
increase. 172 Furthermore, these injections also enhance the expression of IL-la
33
receptors in the dental follicle. Although EGF does not stimulate CSF-1 synthesis
directly, its effect on IL-1 a transcription and IL-1 ct receptors provides an indirect
mechanism for tooth eruption. Transforming growth factor beta-1 (TGF-1) is also
considered as an initiator of tooth eruption. This is due to its ability to enhance IL-1 c
translation and its role in chemoattraction of monocytes. It has also been
immunolocalized in the stellate reticulum. 173 Its transcription has found to be enhanced
incubation with EGF. 174 The signal for tooth eruption could begin with TNFc, EGF, or
TGF-131, any or all of which could enhance IL-1 c synthesis. It should be noted that
TNFet is a proximal molecule in the cytokine cascade leading the expression of the
interleukins. TNFct is a product of macrophages which, as was mentioned, are an
essential part of the normal dental follicular eruption and root resorption of deciduous
teeth. Therefore, osteoblastic activation could occur from the effects of CSF-1, IL-1
,
or
both. 166, 172
CYTOKINE INVOLVEMENT IN ORTHODONTICS
The importance of Prostaglandin E (PGE) in bone and cementum remodeling has
been described by Raisz and others. 175, 176 Prostaglandins, in general, are potent
modulators of physiological and pathological reactions. In animal studies, local
administration of PGE increases the rate of tooth movement. 177, 178 Conversely,
administration of prostaglandin inhibitors may slow tooth movement presumably by
inhibiting cyclooxygenase activity and thus PG synthesis. 179, 180 In a study of rats
injected with PGE2, tooth movement was significantly enhanced, however, findings
34
regarding its role in root resorption were inconclusive. 181 A variety of cells have been
observed to increase synthesis and secretion of PGE in response to mechanical or
chemical stimuli. 134, 182-184 Under these conditions gingival fibroblasts have been
observed to synthesize PGE and cAMP. Increased PGE production is also seen when IL1 [ is administered to stressed PDL fibroblasts. 134 The link between cytokines mediating
bone remodeling during tooth movement has been frequently demonstrated.
Orthodontic appliances apply forces to teeth and in turn, to the periodontal
ligament. These mechanical events illicit a biochemical process which allows bone
remodeling for tooth movement. This process may be regulated by acid phosphatase,
hormones
185, neurotransmitters 44, and cytokines (IL-1 c and [, IL-2, tumor necrosis
factor c, and interferon ,). 43-45, 134, 162, 183-191 In examination of human gingival
crevicular fluid, increased levels ofIL-l, IL-6, TNF-ct, and EGF were seen 24 hours
after force application. These cytokine levels plateaued suggesting down regulation after
initial stress. 191 Grieve also found an increase in cytokine levels in the gingival
crevicular fluid of orthodontically moved teeth. At one and twenty four hours after initial
activation, IL-1 levels rose to 8.9 pg and 19.2 pg respectively. The IL-1 levels at the
control teeth measured 2.0 and 2.9 pg. PGE rose to 108.9 and 97.9 pg at 24 and 48
hours compared to control levels of 61.8 and 70.8 pg.192 The lagging peak of PGE
compared to IL-1 indicates a stimulatory effect by IL-1 which has been also noted in
other studies. 162, 183, 184, 186, 187 The drop in cytokine levels with time can be
attributed to lack continuous force systems used in these studies.
35
It is speculated that these cytokines originated from cells in the PDL in their
efforts as mediators of early tooth movement. In order to trace the origins of cytokines
involved in tooth movement, distalized molars from cats were immunohistochemically
examined at different time intervals. Levels of calcitonin gene-related peptide, IL-1 et,
IL-113, and TNF-x increased significantly in the alveolar bone marrow cavity cells near
the sites of orthodontically stressed PDL, particularly where compressed. After one
week, the channels leading from the PDL into neighboring marrow cavities were
populated by cells with high levels of these cytokines. After four weeks, cessation of
bone remodeling was seen, and the marrow cells resumed baseline levels of cytokine
formation. This would indicate that marrow cells respond to the orthodontic forces
expressed onto the PDL by way of cytokine production which enables participation in
bone remodeling and thus tooth movement. 45 In summation, early sources for these
cytokines may be fibroblasts, macrophages, osteoclasts, osteoblasts, cementoblasts, and
cementoclasts. All of these cells stain for cytokines at 12 to 24 hours, however
osteoclasts and adjacent mononucleated cells are the only active cells 14 days later in the
process. 45 Again, the lack of continuous forces may explain the drop in the number of
mediators.
In evaluation of stimulated blood monocytes taken from individuals with severe
EARR and control subjects, no difference was found in the levels ofIL-1 [ and TNFc. A
wide range of stimulated and unstimulated IL-113 and TNFct production was seen. It was
concluded that no intrinsic difference exists in peripheral monocyte production of these
molecules between patients whom exhibit severe EARR and those who do not. 193
36
In another study, root resorption was inhibited in rats by topical application of a
bisphosphonate. 194, 195 The use ofbisphosphonate will suppress fibroblastic activity
and cause a decrease in periodontal ligament width. 196 Finally, a third study established
a relationship between adhesion molecules and aggressive osteoclastic activity on root
surfaces in a feline model. 197
The cytokine levels may be the same for bone resorption or root resorption in the
PDL and ultimately in the crevicular fluid. There appears to be no specific marker for
resorbing cementum as opposed to bone. It is likely that the array of markers would
change or present a different profile for normal tooth movement as compared with
concomitant extensive root resorption. This would be seen in the PDL and could be seen
in a crevicular fluid assay.
GINGIVAL CREVICULAR FLUID
A thin sulcular epithelium allows passage of fluid from gingival connective tissue
into the gingival crevice. This fluid aids in cleansing material from the sulcus, contains
plasma proteins which promote epithelial attachment to the tooth, and contains antibodies
for antimicrobial protection. Gingival crevicular fluid contains various elements, mostly
derived from surrounding periodontal tissues, which may not always correlate to levels
found in blood serum (table 7).
In 1958, Brill and Krasse demonstrated that chemicals injected into the
bloodstream can be collected from the gingival sulcus in three minutes. 201 They
concluded that there is a passage of material from the blood vessels into the connective
37
tissue and through the epithelium into the sulcus. They noted that in health, gingival
crevicular fluid is scarce in volume and presents as a transudate thereby being difficult to
collect. 202 In the Brill study fluorescein was used, however, a number of molecules have
been shown to be able penetrate the sulcus. Those substances include large or charged
molecules such as albumin203 endotoxin204 and histamine205
Passage of these
substances indicates permeability to substances of molecular weight up to one
million.206
Later researchers reclassified gingival crevicular fluid (GCF) as an inflammatory
exudate. They claimed that increased amounts of GCF from the Brill studies 203,207
could have been elicited by an increase in capillary permeability due to damage from
intrasulcular insertion of paper strips for the collection of GCF. 208, 209 Different
methods have been explored in quantifying the amount of GCF and its coments at various
normal to pathological states. Paper strip assays of slightly inflamed gingiva measured
0.1 mg of GCF in 3 minutes. 210 Isotope dilution method used in the interproximal
molar areas with a mean gingival index less than 1 measured 0.43 to 1.56 ml. 211 As
stated before, excess amounts of GCF is present as an exudate. Presence in a sulcus
which appears normal could be explained by the fact that even a clinically healthy sulcus
will exhibit inflammation when examined microscopically. GCF measurements in
individuals with periodontitis measured at 0.52 ml at diseased sites and 0.28 ml at nondiseased sites in the same individual. These measurements were taken with the paper
strip method over 30 seconds. 153 Using the same method over 20 seconds, GCF
measurements were made on healthy, gingivitis, and periodontitis patients. The volumes
38
were 0.255, 0.540, and 0.370 ml respectively. 212 Increased fluid production could also
be due to mastication of coarse foods, toothbrushing, gingival massage21 O, ovulation213
contraceptives 214, and smoking215.
RATIONALE
A NON-INVASIVE TECHNIQUE TO SAMPLE THE PDL IN HUMANS
The orthodontic literature is replete with clinical studies dealing with statistical
correlates of root resorption and a host of independent variables. There is little
understanding of the cell biology of root resorption, particularly in the human. Without
extraction and histologic evaluation, the only method of diagnosis for root resorption is
the radiograph.
Unfortunately, significant modification of root shape is required to be detectable
by x-ray. 198, 199 Early detection would require periodic radiographic screening with
periapical radiographs of the incisors taken at least every year. 12, 26, 29, 35, 94, 119 A
full mouth periapical series is required to ascertain the pattern and severity of resorption.
Resorption of the facial or lingual surfaces may still escape radiographic detection.
Therefore, development of a non-invasive method for evaluation should be very useful.
This would provide a more detailed accounting of the balance between bone and root
resorption, and because of its benign nature, would allow for early detection of more
pernicious resorption. In addition, it would allow for more frequent monitoring without
untoward side effects.
Because the site of the root resorption is at the apex or along the surface of the
dental roots, monitoring for changes in biochemicals would require either tissue samples
or development of an indirect technique. The co-investigators have developed an indirect
39
40
technique. The details of our previous findings with this technique have been
described. 200 The site for obtaining the samples is the gingival sulcus.
As the sulcus is relatively remote from sites where resorption is active, the
question becomes how to assay cytokines and growth factors formed at these sites.
Absorption assays may be performed on filter paper strips
190-192, or by the more
efficient immunomagnetic technique. 200 This project will allow us to directly study cell
biological changes in an average human population undergoing orthodontic treatment.
We have selected a molecular signal that is known to play a significant role in bone
metabolism (table 6).
PREVIOUS DATA
Assuming that the clast cells are involved in resorption are in sufficient number to
initiate the process, production of TNF, other cytokines and phosphatase should be an
early event in this resorptive process. 216-220 Therefore, for example, one should
observe an increase in TNF during active root resorption beyond that seen in a control
Previous studies using IL-1 as a representative marker molecule have shown IL-1
is present in the normal healthy sulcus. Upon application of an orthodontic force, the
level of IL- 1 and other molecules increased significantly. 183, 184, 186, 192 TNF is
present in the normal healthy sulcus in a range of 17.1-482.8 pg with a mean of value of
159.4 SD+/- 25.4. 216, 217 Orthodontic tooth movement has been shown to increase
these levels up to two fold. 200
41
Another study employed the immunobead technique to follow TNF levels in
deciduous teeth on a group of patients in a private dental practice. Seventeen deciduous
tooth sites were sampled, thirteen with normal exfoliation (root resorption) and four
believed to be ankylosed by clinical criteria. The TNF values found in the patients with
normal exfoliation ranged from 29-149pg/site with a mean of 66pg/site. The positive
values in the teeth considered ankylosed ranged from 12-82pg/site with a mean
concentration of 38pg.
A larger study is now being conducted using the same criteria. After comparison
of the data collected so far, a difference is already obvious. Statistical analysis has not
yet been completed, but the difference between experimental and control samples show
an encouraging trend that differences can be determined using this immunobead
technique.
SPECIFIC AIMS
Present models suggest that the root resorption process should be preceded or
accompanied by an increase in the level of biochemical modulators. Based on these
models, we hypothesize that if the resorptive process is increased, there should be a
measurablle change in the level of these biochemical modulators. The following are our
specific research aims for this project:
1) To measure TNFc levels present in gingival crevicular fluid using the immunobead
technique.
42
2) To validate the sampling technique using clinical situations where a difference in TNF
levels are known to exist.
3) To compare crevicular fluid levels of TNFa in various clinical settings among
orthodontic patients.
4) To compare differences in TNFc levels in different stages of orthodontic treatment.
5) To determine if the TNFa levels of in the crevicular fluid correlate with the degree of
root resorption.
6) To determine if this process can be useful in diagnosis of root resorption.
RESEARCH METHODS
STUDY POPULATION
Subjects will be asked to participate from the University of Connecticut School of
Dental Medicine. Race, ethnicity, or gender are not exclusionary criteria. We will
attempt to include a minimum of 20 human subjects in the permanent or late mixed
dentition.
Table 8 comains the full listing for exclusion and inclusion criteria. Exclusion
criteria will preclude patients with a history of chronic inflammation, chronically taking
anti-inflammatory medication (i.e. aspirin), smokers, and patients with an elevated caries
index because subclinical periodontitis may be more likely and mask the specificity of
our results. 190, 191,200
Subjects will be identified as having undergone some degree of root resorption
during orthodontic treatment. The extent of resorption will be evaluated by use of a
periapical radiograph and scaled as described below (clinical determination of root
resorption). The involved teeth, their adjacent, and contralateral counterparts will all be
evaluated.
DATA COLLECTION
A host of variables will be examined for each patient at each visit. In addition to
nominal and continuous data, some variables will be tabulated using ordinal indices. The
43
44
criteria used for categorization is described below. Information will be collected at each
visit as shown in table 9.
Although not all collected data will be relevant to the currem investigation, some
of the variables have been found to statistically correlate with root resorption and will be
recorded for furore examination. Demographic data will include patient name and date of
collection, patient number, age 4, 5, 11, 21-26, 35, 36, 60, 65, 80, 96,
97, sex 8, 13, 31,
41, contributing medical history 5, 6, 25, 35, 36, 96, 102, 103, familial history of EARR
39, and time since last tooth brushing.
The latter will be recorded as recent tooth
brushing may alter the composition of gingival crevicular fluid.
Information about each sampled tooth will also be collected. Description of root
morphology 13, 34, 37, 38, 76, 80, level of EARR 76, history of trauma 35, 36,
previous root canal treatment 37, 38, 110,
112,
111, presence of pathology, restorations,
periodontal index221, and sulcus depth.
Information about orthodontic treatment will also be collected. This will include
the type of movement 3, 10, 26, 34-38, 66,
80-82, force applied 24-26, 28, 29, 41, 42, 65,
83, force remaining at the time of assay, time since force application 83, whether the
force was continuous or interrupted 22, 28, 29,
85-87, occlusal trauma 113, use of
intermaxillary elastics 5, 7, 11, 35, 36, 41, use of rapid maxillary expander and time since
83, presence of an openbite, digital or other pernicious habits 35, 42, 70, 84, 113, 114,
222, 223, treatment time so far 4, 5, 11, 12, 25, 29, 33, 35, 41, 42, 51, 76, 80,
116, and
45
expected treatment time remaining. Also, the measured TNF level for each tooth will be
recorded following the appropriate laboratory assays.
CLINICAL DETERMINATION AND SCORING
Radiographic assessment of root morphology will be made from periapical
radiographs. Quantification of root loss will be performed by use of three methods.
Method #1 involves the direct measurement, to the nearest tenth of a millimeter, of entire
length of the tooth as viewed from pre and post treatment periapical x-rays. The
difference in tooth length from pre to post treatment is defined as root loss (RL). In
method #2 we will attempt to correct for magnification error between x-rays by
multiplying the ratio of crown heights against the post treatment tooth length. Corrected
root loss (RL*) will be calculated as follows (figure 2):
Method #2:
RL*
T T (C/Cz)
where T is the measurement of the pre-treatment tooth length, T2 is the post treatment
tooth length, and C/Cz is a ratio of pre-treatment to post treatment crown lengths
(correction factor). A third method will also be used for the assessment of root
resorption. 35 Method #3 employs a subjective measurement, the root resorption index
(RRI) based on an ordinal scale of 0-4. 76, 107, 112, 116 A score of 0 indicates the lack
of any visible root modification, whereas scores of 1-4 indicate increasing amounts of
root resorption as determined from periapical x-rays (table 10). Although not as precise,
this scale allows for a simple classification system of severity. The subjective assessment
46
will attempt to categorize the degree of root modification for each tooth in an ordinal
scale. The first seventy measurements and all root resorption index determinations will
be repeated by the same investigator at a different time. The new values will be
compared against the original recordings to determine measurement error.
The Periodontal Index (PI) estimates the severity, pocket formation, and
masticatory function of each tooth. 221 The scale has a true biologic gradient but lacks
objective specificity about the degree of inflammation. The PI is the most common
classification system used with epidemiological studies and is currently used in the
National Health Survey. X-rays or probes are non used so the actual periodontal problem
may be underestimated (table 11).
The Gingival index was developed solely for the purpose of assessing the severity
of gingivitis and its location in four areas: the distofacial papilla, facial margin,
mesiofacial papilla, and entire lingual gingival margin (table 12). A periodontal probe is
used to assess bleeding potential. It is a subjective assessment of inflammation, and
requires manipulation to assess bleeding. 224
The Plaque Index is a subjective assessment of plaque accumulation on the
dentition. Although in its inception, it is intended for use on a per patient basis. A
specific tooth in each quadrant of the mouth is to be evaluated by running probe across
tooth surface. A total score is then assigned for the patient in order to evaluate overall
oral hygiene. 225, 226 In this study, a plaque score will be recorded for each tooth (table
13).
47
SAMPLING FROM THE GINGIVAL SULCUS
Paramagnetic beads (M-450 coated with sheep anti-mouse antibodies) will be
obtained from Dynal, Inc., Lake Success, NY (figure 3). The beads are manufactured so
as to be spherical shape and relatively uniform in size, each being 5 um in diameter.
Anti-TNFot antibodies will be attached to the beads and to be used for capture. The
procedure for attachment of antibodies to the beads has been described. 218,227
The immunomagnetic sampling procedure has been previously described. 200 The
sulcus of the tooth to be tested will be gently irrigated with water at 370 C for thirty
seconds and air dried with a dental syringe. To determine the levels of the markers, a
stock solution will be prepared containing lx106 of beads coated with antibodies for the
markers. The immunobeads are introduced into the sulcus as a slurry prepared with a
known number of beads. Approximately 2 mi of the slurry is deposited into the sulcus by
use of a pipette with a plastic tip with care so as not to damage or irritate the gingiva.
The flat tip of the pipette should be placed just at the free gingival margin and the slurry
dispensed slowly. The slurry has a brown color allowing the dispensing to be easily
monitored by the appearance of the brown color along the neck of the tooth. This
procedure will sample the entire gingival sulcus since the beads quickly distribute
themselves in all four areas of the crevice (mesial, distal, buccal and lingual).
The slurry will remain in the sulcus for 1 minute, the mean time it takes for the
gingival sulcus to fill with crevicular fluid. 200 A special instrumem has been devised for
removal of the beads from the sulcus. This harvester has a sterilizable spatula tip and a
shank which houses four disk shaped permanent magnets. The magnets generate a field
48
of 185-188 gauss at the tip. The immunobeads will be recovered with a gentle sweeping
motion of the tip at the gingival margin. 228
QUANTIFICATION OF BEAD-BOUND ANTIBODIES
After harvesting, the beads appear as a brown mass on the tip. The tip is inserted
into a 1.5 ml Eppendorf microcentrifuge tube containing PBS/BSA buffer and gently
vortexed to suspend the beads. The beads are easily separated from the solution by
placing a magnet against the side of the tube. 200, 216
The immunobeads are cleaned of detritus by repeated resuspension in fresh
solution and separation using a permanent magnet. At this point, a purified concentration
of beads is accumulated with their attached monoclonal antibodies bound to their
respective molecules.
Recovery may be reduced by loss of beads in the sulcus, during the purification
process, and in the laboratory phase (assay). More than 99% of the beads are routinely
recovered from the sulcus. 216 Remaining beads will be either dislodged and lost during
post harvesting irrigation or washed out GCF. A few remaining beads may be
phagocytosed by macrophages (Kupfer cells).
Recovered beads are counted using a haemocytometer and analyzed for antigen by
an ELISA (figure 4). The general procedure for the ELISA assay uses the reagents
provided in commercially available kits (Predicta TNF alpha kit, Genzyme Corporation,
49
Cambridge). As part of our validation process, we have compared the results obtained
with TNF in a plate ELISA to those of the bead ELISA. The results of the comparison of
the two methods show close similarity both in quantity and quality.
The amount of the antigen will be determined by calibration of the recorded
absorbance to a standard curve made for each assay. The lower limit sensitivity is
determined by_adding two standard deviations of all zero standards to the mean
absorbance of all zero standards. This value is used to separate positive measurements
from zero in each assay. If the sample absorbance is greater than this value, the
measurement is considered positive. If the measurement is less than or equal to this
value, it is considered to be zero. The level of antigen is expressed in pg antigen/sample
site.
DATA ANALYSIS
As this method captures the antigen directly from the sulcus without the collection
of sulcular fluid, it is not necessary to measure fluid volume, and the antigen values will
be expressed directly as an amount (pg) per site. Also, the total number of beads
recovered from each sulcus will be determined (see above). Thus, the amount of antigen
will be expressed as pg antigen/number of beads recovered/site. These data will be
analyzed for differences in the cytokine profile of the crevicular fluid in our sample of
patients. We will then compare levels of antigen in various clinical conditions to
determine if correlations exist.
All measured variables will be first treated with descriptive statistics. As
described above, raw root resorption amounts and the root resorption index will each be
50
measured twice at on separate occasions. The difference in measurements between the
two time intervals will describe measurement error from the periapical films. The three
methods will be compared against each other for measurement error and reproducibility
(reliability).
The various clinical (independent) variables will be similarly treated with
descriptive statistics. TNF data will also be analyzed descriptively. The data will be
evaluated to check for the possibility of pooling samples in order to differentiate between
subsets of the sample population. Differences between groups will be evaluated by
ANOVA and t-tests. Separate means will also be constructed for data collected for
contralateral and opposing teeth. We will test the equalities of the means and, if not
equal, evaluate their differences. This will be performed by a one-way analysis of
variance (ANOVA) to test the equality of this mean data across the three study groups.
Should the ANOVA reveal statistically significant results, we will use multiple
comparison procedures to isolate specific pairwise differences. Prior to implementing the
ANOVA, we will perform data transformations if the extent of variation across groups
indicate this to be appropriate. The clinical variables will be separately evaluated for
correlation to TNF values. Stepwise linear regression models will be used to evaluate the
contribution of each variable to TNF found in the sulcus.
As TNF values are expected to be highly variable, grouping of data for t-tests may
not be sufficient to analyze this data. Instead, each subject will serve as both the
experimemal and control. The differences in root resorption index and amount will be
evaluated against TNF values in each patient. The remaining clinical variables will be
51
treated the same way as necessary. All statistical analyses will be with the advice and
assistance of the University of Connecticut Health Center Biostatistical Facility.
We have investigated the power of the ANOVA model to be used in our study.
By way of example, existing normative pilot data for TNF (159 pg/106 beads +/-25)
indicate that it will be possible to detect small to moderate effects of sizes with high
power. For example, specifying an alpha level of 0.05 for the ANOVA, we should detect
a mean difference of 20% with 52 subjects per group; a mean difference of 30% with 21
subjects per group; a mean difference of 40 % with 14 subjects per group; and a mean
difference of 50% with 8 subjects per group. The percent mean difference is based on
comparing the largest and smallest of the group means with the power at least 0.70 for all
tests. These values depend upon the reliability of the pilot data.
DIFFICULTIES
The potential problems of recruiting patients for clinical research also apply to our
project. Locating patients who meet our criteria with the appropriate radiographs is
difficult. Compounding the problem of sustaining an adequate sample size is the
requirement of locating enough patients with the more severe resorption pattem. The
literature indicates that mild resorption should be common in orthodontic patients.
However, those patients with a more severe resorption pattern are rare. Their presence in
our sample will help to stratify our results and demOnstrate possible differences.
The basic science phase of this project presents with its own difficulties.
Originally, our intent was to examine a panel of cytokines from the gingival crevicular
fluid. IL-1, PGE-2, and acid phosphatase levels were to be measured along with TNF, as
they are also important mediators in bone metabolism, tooth movement, and tooth
eruption. Each mediator has a significant and, to some degree, unique role.
Incorporating the other cytokines into the existing study would provide useful
information and increase the significance of this study. However, time requirements and
funding for a separate laboratory phase each mediator prohibited the greater venture.
TNF was chosen as the study molecule for reasons previously described.
Previous research involving TNF values from gingival crevicular fluid have
shown variability which could not be explained by clinical conditions. The same
difficulty exists for incidence of root resorption. Investigators attribute this variability to
individual variation within their respective samples. By comparing differences with each
52
53
patient separately, we hope to eliminate the problem of variation between individuals.
However, intra-subject variation still exists as a problem.
The method used to capture and quantify the TNF molecules, the immunobead
technique, presents with its own difficulties. Loss of a significant portion of the beads
may result in extreme TNF values. Unless the recovered beads are counted in every
sample, extreme outliers may have to be discarded. TNF measurements of zero or those
above 1500pg should be judiciously interpreted if at all.
Finally, the presence of TNF in the sulcus as a result of an event ongoing at the
apex of the root seems unlikely. If the TNF is synthesized and secreted for a specific
purpose (root resorption, bone remodeling, etc.), then it should be used for that purpose
rather than being dumped out into the sulcus. Cells with receptors for this molecule are
far more abundant elsewhere. Also, if the presence of TNF in the sulcus is intentional, its
removal by our sampling technique should cause a deficiency somewhere.
SIGNIFICANCE
Clinically, the use of bead-bound antibodies would be an accurate test to
determine levels of any number of molecules in the gingival sulcus and an index of what
may be occurring metabolically in the periodontal membrane. Also, it could be a
sensitive and definitive method for the diagnosis of root resorption and its possible
severity. Our results will be evaluated to determine if the technique allows for the
discrimination of root resorption in various phases of orthodontic treatment. Finally, and
most important, if specific molecules can be identified that participate in or precipitate
root resorption, one can envision the use of antagonistic drugs as a pharmacologic
prevention of root resorption in the future.
Three outcomes are possible: 1) an elevation of TNF levels is observed in relation
to x-ray detectable root resorption, 2) there is no change in cytokine levels as detected by
our methods, 3) there is a spectrum of increases and decreases in relation to the time of
onset of the untoward root resorption.
54
RESULTS,
SAMPLE SELECTION
The original sample consisted of fifty nine subjects selected on the basis of the
inclusion criteria as previously described. Forty eight of these subjects were also
involved in another project to evaluate the progression of root resorption. For the
purposes of the other project, these subjects had periapical radiographs taken with a
standardized measurement jig on a three month basis. The other eleven patients were
selected on the basis of the inclusion criteria and mid-treatment periapical radiographs
indicating extemal apical root resorption on at least one tooth. All patients were involved
in comprehensive orthodontic treatment in our clinic, had appropriate periapical
radiographs at the start of treatment, and presented for treatment in the permanent or late
mixed dentition.
Of the sample of fifty nine subjects, nineteen patients were included in the final
patient sample pool. The remaining were excluded either on the basis of their refusal to
participate in our study (twenty eight), termination of orthodontic treatment (four), or lack
of evidence of radiographic root resorption on any teeth (seven). Our final sample
consisted of ten males and nine females. The mean age of the sample was 14.2 years
with a standard deviation of 4.9 years. The youngest patient was 11 years old, the eldest
was 26, and the median age was 12.
A total of 110 samples were taken from the nineteen subjects, averaging 5.8 teeth
per patient. Since only effected teeth- those with radiographic evidence of root
55
56
resorption- and comparable control teeth were sampled, the number of samples per
patient varied. At a minimum, four samples (4 teeth) were evaluated from each patient.
The maximum was 10 samples taken from the same patient. All sampled teeth were
maxillary teeth, and most (78%) were incisors. Table 14 and figure 5 display the
distribution of the teeth included in our sample.
DESCRIPTION OF THE SAMPLE
A multitude of independent variables were recorded for each tooth as described
previously in table 9. The emire sample had no familial history of root resorption and no
significant medical history. None of the patients were treated with rapid maxillary
expansion, reported any pernicious habits, or displayed an openbite malocclusion. Only
one tooth in our sample (110 teeth) had a history of previous trauma (2.5 years prior to
treatment). Three teeth had been previously restored (minimum of one year prior to
sampling) with moderate to conservative restorations. None displayed any signs of
periapical pathology or had been treated with root canal therapy.
From pre-treatment periapical x-rays, most teeth (80%) displayed an
unremarkable root anatomy whereas 20% demonstrated abnormal root morphology (table
15 and figure 6). The most frequent type of deviation in root form was blunting (15.3%).
Pipette shaped roots and dilacerations were rare in this sample at 2.4% and 1.2%
respectively.
Various indices were used to assess gingival conditions. Three teeth (two
percent) had sulcus depths beyond three millimeters. In all three instances, probing
depths measured at 4mm and were in sites of gingival inflammation (pseudopocketing).
57
No teeth in our sample showed loss of attachment or bony support. Tables 16-18 and
figures 7-9. describe the plaque index, gingival index, and periodontal index for the
sample. Only one tooth displayed severe plaque accumulation while none displayed
severe gingival or periodontal index scores. Most teeth had mild to moderate plaque
scores (86%), gingival index scores (84%), and periodontal index scores (82%).
The time between when patients reported brushing their teeth and when the tooth
was sampled is displayed in table 19 and figure 10. All patients had brushed their teeth
between two to eleven hours prior to sampling.
A subjective assessment of the type of orthodontic movement and the mechanics
involved directly prior to sampling was perfolxned to evaluate for possible correlation
with dependent variables. Table 20 and figure 11 depict that 81% of the samples were
involved in some form of orthodontic tooth movement directly prior to sampling, the
most common type being tipping (53%). Twenty six percent of the samples were
translated directly prior to being sampled while only 2% were rotated.
Force application to the sampled teeth was recorded both at the appointment prior
to sampling and also at the time of sampling, representing the force remaining from the
activated appliance (table 21 and figure 12). Nineteen percent of the sample was not
orthodontically moved and thus had no force application. Moderate forces of 100-250
grams were applied to 59% of the sample and light forces (<100grns) were used for the
remaining 22%. Heavy forces (>300grns) were not used throughout the sample. At the
time of sampling, only light forces (74%) or no force at all (26%) still remained.
The time in treatment in our patient sample ranged from 0 to 36 months (table 22,
figure 13). The majority of the sample (85%) had been in treatment for a year or less.
58
The remaining 15% of the sample were into the third year of treatment. Forty percent of
the sample was in their 12th month of treatment.
DETERMINATION OF ROOT LOSS
Three methods were employed in measuring root loss (table 23, figures 14 and
15). Method # 1 involved direct measurement of the entire tooth length from pre and post
treatment periapical x-rays. The difference in length from pre to post treatment was
defined as root loss (RL). In our sample, the mean root loss as determined from method
# 1 was 0.69 mm, with a standard deviation of 0.63 mm (standard error 0.0684), and a
range from -1.7 to 2.8 mm. In method #2, the length of the crown, which should remain
relatively constant throughout treatment, was used to correct for magnification error. The
corrected root loss (RL*) was calculated to be 0.69 +/-0.63 mm (SE 0.0687). The range
was -1.69 to 2.86 mm. The mean of the root resorption index (RR1) from method #3 was
0.86 with a standard deviation of 0.80, standard error of 0.0872, and range from 0 to 3.
Direct measurements of the crown and/or the entire tooth for the first 70 samples,
as in methods #1 and #2, were repeated at a separate interval by the same investigator to
assess the reliability (reproducibility). Subjective assessment of root loss in method #3
(root resorption index), was performed for the entire sample at two separate intervals.
Reliability between the two intervals, where the same measuremem was made,
was assessed by measuring the correlation between the two sets of measurements. Table
24 displays the descriptive statistics and correlation coefficients for the difference in
repeated measures. The correlation between all direct repeated measures was greater than
0.98. Repeated RRI measures showed a correlation of 0.89. Descriptive statistics and
59
correlation coefficients were also calculated for the difference in repeated RL and RL*
measures (table 25). RL and RL* both display a slightly higher error than RRI (0.81 and
0.82). Correlation coefficients between the three methods were calculated. The
correlation between the corrected (RL*) and the uncorrected (RL) root loss was 0.99.
The correlation coefficients between the root resorption index and method 1 and method
2 were both 0.72.
TUMOR NECROSIS FACTOR-a MEASUREMENTS
Of the 110. samples taken, only 85 .were used for data analysis regarding TNF-c
levels from the gingival crevice. Data from the other twenty five samples were discarded
because of technical problems encountered in the laboratory phase during quantification
of the captured TNF-ct. The first column of numbers in table 27 displays descriptive
statistics for TNF-ct levels. The mean for the sample of 85 was 232.9 pg +/- 422.4 (SE
45.81). Zero TNF-ct values were found for 47% of the sample. The remaining 53%
were distributed in a highly skewed manner ranging up to 2944 pg (table 28 and figure
16).
Multiple methods were employed to evaluate the TNF data. TNF values were
transformed to ordinal data using an index ranging from 0 to 5. The TNF index reduced
the skewness of the data (table29 and figure 17) having a mean of 1.27 +/- 1.57 (SE
0.1702). Transformation of TNF data, using its natural logarithm, created difficulties in
that the samples with values of zero could not be included (zero has no natural log). To
overcome this problem, all TNF values were increased by one (picogram). This allowed
60
calculation of the natural logarithm for all samples (table 30, figure 18) further reducing
the skewness.
ZERO TNF-ct MEASUREMENTS
As mentioned above, zero TNF-a values were found in 40 of the 85 samples
(47%). In order to evaluate for discriminatory variables in this subset of the samples,
student’s t-tests were carried out for a number of variables (Tables 31-41). With respect
to the Gingival Index, the Periodontal Index, and all three methods of assessing root loss,
samples with positive TNF-a levels showed a significant difference from those with none
at all (p<0.01). Due to the differences found between the subsets in our sample, some
statistics were calculated separately for the subset with positive TNF values.
In calculating the descriptive statistics displayed in table 42, the samples with
TNF values of zero were omitted. This decreased the sample size to 43, increased the
mean to 437.5 pg, with an increase in standard deviation and error to 508.5 and 77.55
respectively. The same procedure was carried out for TNF index values producing a
mean of 2.35 +/- 1.39 (SE 0.2128). With the samples containing zero TNF values
removed, the natural logarithm could be calculated directly. The frequency tables and
histograms for all three methods are illustrated as tables 43-45 and figures 19-21.
61
CORRELATION TO TNF-tx
Correlation coefficients for TNF data and clinical variables are displayed in tables
46-48. As the distribution of TNF values was not normal (Poisson distribution),
Spearman’s ranked correlation coefficients were also calculated. Correlation coefficients
for RL, RL*, and RRI to raw TNF-c levels were 0.22, 0.21, 0.37 respectively. Moderate
to mild correlations were also found for the Gingival Index (0.49), the Periodomal Index
(0.42), and the type of tooth movement (0.22). When using Spearman’s ranked
correlation, the coefficients for the GI, RL, and RL* increased to 0.67, 0.35, and 0.43.
The PI and RRI coefficients dropped to 0.42 and 0.36, whereas the type of tooth
movement showed no correlation to ranked TNF data.
Similar calculations were made, omitting the samples containing TNF values of
zero (tables 49-51). More variables displayed moderate levels of correlation with raw
and ranked TNF data. The time since the patient last brushed their teeth, force
application, the type of tooth movement, and the time in treatment had coefficients of
0.29 to 0.49). RL and RL* showed almost no correlation with TNF (raw or ranked data).
The correlation of RRI to TNF dropped to 0.32 and 0.29.
PATTERNS SPECIFIC TO PATIENTS
The variation in samples, specifically with regards to TNF values, created
difficulties in pooling data and finding baseline measurements to use for comparison. As
described in the methods section, we attempted to analyze data collected for each patient
individually. A more complete regression model could not be constructed due to the lack
62
of data points for each patient. Also, some variables were constant across samples from
the same patient (time in treatment, brushing, etc.). An analysis of the association of
TNF to root loss was performed for each patient by measuring the correlation between
root loss (RL) and raw TNF values (table 52). Of the 19 patients in our sample, 5 showed
a moderate to high correlation between TNF and RL. The remaining 14 patients had
mild, none, or a negative correlation between the variables.
STEPWISE MULTIVARIABLE REGRESSION MODEL
As it was the purpose of this project to evaluate the effect of root resorption, and
other clinical variables, on the levels of TNF-ot in the gingival sulcus, it was deemed that
a regression model would be more appropriate for data analysis.
By using a stepwise
multivariable regression model, the variation in the TNF-et data (the dependent variable)
attributable to the independent variables could be calculated. Thirteen clinical variables
and all three methods of assessing root loss were used as the independent variables (tables
53-55):
Variables were dropped in a stepwise backwards elimination manner if the p-
value of their coefficient of determination exceeded 0.05. The GI, RRI, and type of
movement remained to explain for 37% of the variation in raw TNF-c values
(table 56).
The coefficient of determination for GI and RRI alone was 31% (table 57). The Gingival
Index and the Root Resorption Index explained for 29% and 14% of the variability in
TNF levels respectively (table 58 and 59). Collinearity of the variables was examined
63
from correlations already performed (tables 46 48). No redundancy could be attributed
to the R values from the three variables (GI, RRI, and movement).
As mentioned before, TNF-a values were not normally distributed. Traditionally,
data transformation for regression of non-normal data involves using the natural
logarithm of either both variables, or only the independent variable(s). For the purposes
of our data, we required transformation of only the dependent variable. Again, several
methods were used to evaluate the legitimacy of our regression model.
The first method involved the use of ranked TNF values, followed by stepwise
regression as before. The Gingival index and the RRI still retained a significant p-value.
However, the type of tooth movement was not found to retain a significant p-value,
whereas the Plaque Index did (table 60). Combined, the three variables explained 56% of
the variation in ranked TNF-c scores. The Plaque Index was dropped, lowering the Rz
value to 0.52 (table 61). Individually, GI and RRI had Rz values of 0.31 and 0.20 (tables
62 and 63).
The second method to normalize the data involved using the natural logarithm
(In)of the TNF-ot values in the regression model. Table 64 displays that, PLI, GI, and
RRI were left with p-values less than 0.05 once again, and explained 54% of the of the
variation in lnTNF values. GI and RRI retained a Rz value of 0.51 together, and 0.42
(GI) or 0.19 (RRI) individually (tables 65-67).
The TNF index, described above, was used in our third method. Table 68 and
further analysis showed that although the R value for PLI, GI, and RRI is slightly lower
64
at 0.49, use of the TNF index showed essentially the same pattem as using lnTNF or
ranked TNF values.
When performing multivariable stepwise regression on the data without the
samples with zero TNF- values, no combination of variables could be found which
would retain a significant coefficient of determination. GI and RRI could explain for
36% and 10% of the TNF variation when examined individually (tables 69 and 70).
When the positive TNF data are ranked, GI, RRI, and the time since brushing retained
significant R2 values of 0.58 (table 71). Individually, they could explain for 43%, 8%,
and 16% of the variation in ranked TNF values (tables 72-74). Using the lnTNF and TNF
index data, a similar pattern was found where GI maintains a moderate association with
TNF and the association between RRI and TNF wavers between weak to almost none.
DISCUSSION
DESCRIPTION OF THE SAMPLE
The lack of severe apical resorption in our sample precludes conclusions
regarding the association of increased TNF-ct levels in the face of fulminating root
resorption. According to the literature, this type of resorption (beyond 3mm) may be
expected in approximately 10% of orthodontic patients. Furthermore, selection of
particular teeth, maxillary incisors and/or teeth with deviating root forms, may increase
the likelihood of locating the teeth which experience a more aggressive resorption
pattern. Our sample consisted mainly of maxillary incisors (80%) and included a fair
number of teeth with deviated root forms (20%) Aside from the selection of patients with
other possible contributory factors such as familial history of root resorption, significant
medical history, pernicious habits, and recent trauma, not a great deal more may be done
in order to produce a sample that should to include more patients with severe root
resorption. Furthermore, the possible contributory factors listed above, and those
mentioned in the introduction, do not guarantee location of those severely effected teeth.
Most patients in our sample were adolescents. Some studies indicate that younger
patients are less prone to root resorption as their roots have only recently completed
formation or are still in the process. 4, 11, 22-24, 35-38, 60, 65, 96 This explains why a
few samples showed an increase in root length throughout treatment, and possibly why
the amounts of root resorption throughout our sample were small. It must be mentioned
65
66
that there is no conclusive evidence regarding an association between age and severity of
root resorption in the literature. 97
The simplest and most direct means to increase the number of teeth with severe
resorption in our sample is to select a larger sample. In our study, the original 59 patients
selected would have yielded in excess of 250 samples (4-5 samples per patient). If the
5% incidence seen in other studies held true for ours, we would still only find 13 teeth
with severe resorption. Enforcement of the exclusion criteria, termination of treatment,
and disinterest for participation in the study by the patient (usually the parent) reduced
our patient pool to 19. Furthermore, of the 110 samples collected, 25 had to be discarded.
Some were due to the loss of too many beads (and captured TNF-ct) during the
purification or the collection phase. Others were simply discarded due to errors in
technique during the assay. Laboratory difficulties reduced our sample size to 85 teeth.
Again, applying the 5% incidence rate for severe root resorption, we would expect
approximately 4 teeth with the more severe resorption pattern. We found two teeth with
resorption near or just beyond 2.5mm.
DETERMINATION OF ROOT LOSS
Root loss was calculated by three means The first method was a direct
comparison of pre-treatment to post-treatment root length. In the second method, we
attempted to correct for magnification error by using the length of the crown (which
should be more stable than the root) to establish a ratio to aid in calculation of the relative
tooth length in either film. These two methods produced very low measurement error and
67
greater than 80% correlation between repeated measures of the first 70 samples. The
measurement error in this study resembles similar studies which also employ periapical
films. The two methods also correlate highly with each other, producing essentially the
same error and the same data. In light of this, the first method is preferred as it is quicker
and simpler. Method #3 incorporated a subjective assessment of root loss using a
calibrated ordinal index. Repeated measures of the Root Resorption Index actually
produced a higher correlation (0.89) between repeated measures than the direct root loss
methods #1 and #2. This method provided a simple but effective way to assess root
resorption which may be invaluable in epidemiological studies.
TNF-c LEVELS IN THE GINGIVAL SULCUS
The samples which were used to collect data provided a high range of TNF-c
levels with almost 50% registering at zero. For the purpose of data analysis, the highly
skewed TNF values were transformed using multiple techniques. An index was first used
to compress the range of TNF values. The natural logarithm provided a more normal
distribution across our sample. The transformations were similar, both reducing the
skewness and compressing the data by reducing the range. TNF values were also ranked,
which is the preferred method for evaluating correlations in a sample which is not
normally distributed. Samples with zero values were removed in order to view the
positive data separately and the previous data transformations were performed to repeat
similar results. Removal of the zero values normalized the data somewhat, mostly in the
case of the TNF natural logarithms.
68
The high number of samples with zero TNF values creates some concem regarding
the contribution of these samples to our data analysis. A reading of zero TNF-c in a
sample from the gingival crevicular fluid may occur by various means.
1) Sampling error and lab error accounted for the loss of twenty-five samples. It is
possible that samples which registered at zero were also affected by lab error.
Our sampling technique may have failed to register the presence of TNF-c in the
sulcus, reading as a false negative. This may reflect errors in the collection
technique from the sulcus, a loss of the bead-bound antibodies during purification
or processing, or an error during the assay.
2) There may have been no TNF-c in the sulcus at the time of sampling. This can
occur if the TNF was washed away by tooth brushing or rinsing (still a false
positive). A student’s t-test failed to find a significant difference between the zero
TNF samples and those which registered positive for TNF with respect to when
the patient last brushed their teeth (table 33).
3) Finally, we may have had readings of zero simply because there was no reason for
TNF to be present in those samples (an accurate reading). A statistical difference
(p < 0.01) was found between the zero and positive TNF samples with respect to
GI, PeI, RL, RL*, and RRI (tables 37-41). These variables, alone or in
combination, may contribute to the presence, or lack of TNF-c in the gingival
sulcus. Previous research involving TNF-ot harvested from gingival crevicular
fluid describes similar results where zero or very low levels of the marker were
found. 153, 212, 229 In light of these findings, clinical perameters such as
69
gingival inflammation and root resorption provide the most plausible explanation
for the finding of zero TNF levels. All five of these variables correlate with one
another to a mild degree requiting further analysis to pinpoint the most significant
contributor to the dichotomy in our data (tables 46-48).
THE ASSOCIATION BETWEEN TNF-c AND CLINICAL VARIABLES
Although correlation coefficients were calculated for all recorded variables, in
order to investigate the relationship between them and TNF-et (the dependent variable)
levels a regression model was deemed more appropriate. Several models were
constructed in which a backwards stepwise analysis was performed. Four methods were
used in evaluating TNF data: raw TNF-ot levels, ranked TNF values, transformed values
using natural logarithms, and the TNF Index.
In all models, the Gingival Index maintained a level of significance with a
coefficient of determination of 0.29-0.45 The Root Resorption Index contributed to 14-
20% of the variation in TNF levels when considering the entire sample. When omitting
the samples with values of zero TNF, the RRI failed to be consistent, or provided a low
coefficient of determination (R < 0.10) in the various methods of interpreting and
transforming the TNF data. A few other variables became significant in the various trials,
but failed to prove as consistent as GI and RRI.
7O
The association between root loss and TNF-t values
The direct measures of root loss, RE and RL*, failed to show a significant
coefficient of determination in relation to TNF data. The correlation between either
direct measure and the RRI was moderate to high and significant (0.72, p<0.001). All
three methods were designed to measure root loss. If they correlate with one another and
are essentially different rulers to measure the same variable, they should have the same
relationship with TNF- levels. Due to the fact that R values for RRI with respect to
TNF data were low and the correlation with other measures of root loss (which failed to
show a statistical relationship with TNF) were high, the association between RRI and
TNF values is questionable.
According to the previously mentioned t-tests (tables 39-41), all three methods of
determining root loss showed statistically significant difference when disceming between
no TNF and some TNF in the sulcus. It would be difficult to explain the significance in
all three methods and the significant R2 value for the regression model of RRI and TNF
as a result of type I errors. When considered together, the data precludes a conclusion of
a linear dependent relationship between the degree of root loss observed in this study and
the TNF-c values observed. However, the coincidence of data between zero and positive
samples in relation to root loss indicates some association.
Scientifically, the relationship between root loss and TNF- in the gingival
crevice is not fully explained in the past literature. Root resorption occurs in the
periodontal ligament, which is sealed off from the gingival crevice by junctional
epithelium. Although this attachmem is not impervious to molecules like TNF-c, the
71
most likely source for its presence in an otherwise healthy sulcus remains to be the
gingiva (and its blood supply).
Gingival conditions and TNF-tz levels
Several studies have found that molecular signals harvested from the crevicular
fluid reflect the progression and severity of periodontal conditions (table 6). The high
correlation between TNF-tx levels and the Gingival and Periodontal index in our study
validates our sampling technique. The correlation was higher for the gingival index
probably due to the greater range with which it presented throughout our sample. This
range helped to stratify the data in respect to the gingival index and in comparison to the
periodontal index (or other variables).
Regression models indicated a moderately strong dependent relationship between
TNF-ct levels in the crevicular fluid and the Gingival Index. Depending on the how the
TNF data was used, the Gingival Index alone could be used to explain 30-42% of the
variation in TNF values.
If TNF-ct levels in the gingival crevicular fluid were totally dependent on
gingival conditions only, then, most of the data points relating the two variables should
lie on the regression line. In that case, we should be able to predict gingival
inflammation, or have a quantitative measure of its severity by measuring TNF-a levels.
Perhaps, the Gingival Index is not totally accurate in describing gingival inflammation.
This may easily be true, as it is a simple index based on four categories. Because TNF-ct
may be measured on a continuous axis, as it was in this study, the linear dependent
72
relationship between the two may be "fogged" and display a lower coefficient of
determination. The only other explanation would be that other variables may contribute
to TNF-t levels in the gingival sulcus and only part (maybe most) of the variability is
due to the variation in gingival conditions.
TNF-a in the gingival crevicularfluid
Periodontal literature confirms the relationship between periodontitis and
crevicular molecular signals. 153, 230 The actions of molecules such as IL-1 and TNF are
well recognized in bone metabolism, specifically, in the destructive process involving the
breakdown of alveolar bone in periodontitis. Similar to root structure, alveolar bone is
relatively remote from the sulcus. Albeit, radiographic root resorption occurs at the apex,
and alveolar bone loss occurs at the crest of the bone, very near to the sulcus. However,
the process of transfusion from the PDL to the sulcus can and does occur in periodontitis,
and may occur (over a slightly longer distance) during root resorption. Furthermore, root
resorption occurring at the apex is probably also progressing on the axial walls of the root
at all levels, and cytokines mediating this process may be produced at these higher levels
thus closing the gap to the gingival crevice. Finally, the markers which appear in the
sulcus need not originate or even be intended for the PDL. Stimulus to increase
production of these factors is a local phenomena, however, it may reach slightly further
than intended. Nearby cells in the gingiva may be similarly affected and begin
production of mediators for metabolic functions in the periodontium related to the
73
processes occurring at the level of the bone. In other words, the presence of TNF-c in the
gingival sulcus, in relation to root resorption, is far from remote.
The more interesting question involves the rationale for the presence of TNF-o in
the gingival sulcus. When stimulated, cells synthesize and secrete molecules such as
TNF-tx for purposes of communication and metabolic function. These molecules have an
intended purpose and target. They should be taken up by target cells or broken down
when not utilized. The rationale for the presence of"excess" cytokines in the gingival
sulcus may provide answers regarding metabolic processes and pathologies in the region
in and by itself.
1) Human physiology does allow room for errors and excess. These molecules
are secreted for the purposes of metabolism and communication. A cell
population in a person who is overly susceptible (genetically) to specific
stimuli may act overzealously to minor insults. A decreased threshold to
trigger the production of cytokines or an overly active production mechanism
for these molecules may be responsible for excessive amounts of these
molecules. When secreted in excess and when not fully utilized or
metabolized, they may "wash out" into the gingival sulcus. These excess
molecules will then have no actual metabolic purpose and serve only as
markers for metabolic functions if so investigated. This mechanism dictates
that the excess secretion of these molecules reflects a hyperactive production
mechanism where, in our model, excessive resorption of the root will occur,
74
and enough molecules are secreted so that the excess will also be reflected in
the wash out in the sulcus.
2) The other explanation involves a mechanism where the normal uptake or
feedback loop is disabled. If the intended targets are not able to respond to or
bind these molecules they will .accumulate in excess and wash out into the
sulcus. The overall effect.in thispathway will be the reverse of the first. If
the target cells do not bind these molecules, they may remain inactive. In our
.model, increased levels of wash out will reflect a decreased amount o.f
reso.rptive activity.
As we were not able to find a dependable linear model to predict root resorption
from crevicular TNF-x levels, it is difficult to know which pathway is responsible for the
presence of cytokines in the gingival sulcus. Better understanding of the interaction of
these molecules with their targets and the subsequent actions of the targets with regards to
root resorption is also required to validate either model.
GENERAL DISCUSSION
It is well accepted that with orthodontic treatment some degree of root resorption
occurs. As most sites of root resorption are repaired, orthodontic treatment provides
benefits which far outweigh the risks. In fact, minor loss of root structure, even when left
un-repaired, is not a significant dental health problem in orthodontic patients. Rarely,
this resorption is severe and may result in the loss of periodontal support which may
compromise the longevity of the tooth. The mechanism or sequence of events which
75
cause a more pernicious resorption pattern is yet unknown. No specific, or even set of
clinical criteria have been identified as contributory factors to this more severe
occurrence of root resorption.
Previous investigators have identified the cell population and mediators involved
in tooth movement and root resorption. Basic steps involved in the biology of tooth
movemem have been studied. For example, the levels of specific mediators and the
number of clastic cells increases as a response to the application of mechanical stresses to
the PDL. Also, similar processes and increases in mediators is seen where a pathologic
state exists. Given these circumstances, one would expect levels of mediators to increase
where root resorption is progressing unchecked by a proper repair response.
The loss of root structure occurs generally and to a mild degree during tooth
movement. When root loss occurs excessively or at an aggressive rate, the balance
between resorption and repair is impaired. Macroscopically, this is seen as the loss of
significant amounts of root structure. Closer examination by histologic methods or
biochemical sampling shows increased resorptive activity by clastic cells which are being
mediated by increased levels of cytokines.
Our study incorporated a sample where resorption of root structure was clinically
insignificant and unchallenging to the longevity of the teeth examined. The amount of
root resorption was limited to mild occurrences, limited to less than 2.5 mm. TNF-a
production reflected a balance between resorption and repair (deposition) or what is
typical for the gingival status. The variation in TNF-ot production in the gingival sulcus
76
failed to distinguish differences in the severity of root resorption. This indicates that
minor resorption of root structure are not recognized as a pathologic state.
FUTURE STUDIES
Ideally conducted, this study should incorporate a larger sample. This would
increase the chance to incorporate a larger number of teeth with a more severe root
resorption. Abutment teeth for rapid palatal expanders can be used, as they have been
shown to experience significant amounts of resorption due to the high forces generated by
the expander. Teeth planned for extraction could be used as samples. This would allow
histologic, as well as biochemical examination.
The relationship between gingival conditions and the production of crevicular
cytokines is well studied in the literature and demonstrated in the present study: All
attempts should be made to control for these variables. For example, a strict protocol of
oral hygiene should be implemented in the pool of patients. This can include
administration of antibacterial agents (Triclosan, Chlorhexidine, etc.) to control or reduce
gingival inflammation. Elimination of the contribution or affect of these significant
variables allows a much clearer look at the affects of root resorption.
Animal models have been used in the past to evaluate root resorption. Most
histologic and immunohistologic studies involve the use of an animal model.
Unfortunately, it is just as difficult to incorporate large numbers of teeth with pernicious
root resorption in animal models. Closer examination of the involvement and actions of
cytokines in root resorption should be pursued in these animal models. Also, the cell
77
population involved in the process has been studied, but a quantitative assessment of the
cells still needs to be performed.
CONCLUSION
1) The results of this study indicate that assessment of root loss may be performed
reliably and efficiently by use of an ordinal index.
2) A great amount of individual variation exists in the amounts or presence of Tumor
Necrosis Factor-x in the gingival sulcus.
3) The sampling technique for measuring Tumor Necrosis Factor-a in the gingival
sulcus is valid and derived TNF values are consistent with studies of gingival
inflammation.
4) The lack of gingival inflammation or the lack of root resorption may result in the
absence of Tumor Necrosis Factor-c in the gingival sulcus.
5) The results of this study suggest that within the realm of clinically insignificant root
resorption, the levels of Tumor Necrosis Factor -or in the gingival sulcus cannot be
used to approximate the amount of root resorption occurring.
78
Figure 1 Bone: :metasm. C+ont:: but:ion of c):o.kes, ho:ones, d vous groh
c..o.rs in regtion ,d :udon of ne .taiism,
Ten om, !on SH, Role ofc:}okMes= N ccM disorders ofne
:[Rto i992 #349]
79
8O
F:ige 2..= Methods #I. d #2 :ib:r de,:eig root: loss.
8i
.m, bead
coated,
sus!nded: i:n buffer soiution
Figure 3.. i:nNmno:nmgnetic beads. 5 g,m. in diete.r, e coated, wit,h ti.’l’NFa anti.dies
and used to capture T’-c,., moleeui:es om the gingivaI suicus..
82
F:igure, 4, lz,. ,ISA is ’ed,-t:o qut:i" the aot ofTNF este,d :om, the g,mg,, ai
83
Frequency
Distribution of Sampled Teeth
Cumulative %
18
100.00%
16
90.00%
80.00%
14
70.00%
12
60.00%
10
50.00%
40.00%
30.00%
20.00%
10.00%
.00%
2
3
4
5
6
7
8
Figure 5. Distribution of teeth in our sample.
9
10 11
12 13 14
15
84
Distribution of Root Forms
70
60
50
40
30
20
10
Norm al
B lunted
D ilacerated
Pipette
Figure 6. Distribution of normal and deviating root shapes.
N=85
Frequency
Plaque Index
Cumulative %
4O
100.00%
35
90.00%
80.00%
30
70.00%
60.00%
50.00%
40.00%
15
30.00%
10
20.00%
10.00%
.00%
0
2
Figure 7. Distribution of the Plaque Index.
3
85
Frequency
Gingival Index
Cumulative %
100.00%
40
90.00%
35
80.00%
30
70.00%
25
60.00%
20
50.00%
40.00%
15
30.00%
10
20.00%
10.00%
.00%
0
1
2
3
Figure 8. Distribution of the Gingival Index.
Frequency
Periodontal Index
Cumulative %
100.00%
70
90.00%
60
80.00%
50
70.00%
60.00%
50.00%
40.00%
30.00%
20
20.00%
10
10.00%
.oo%
0
1
2
Figure 9. Distribution of the Periodontal Index.
3
More
86
Frequency
Time Since Last Tooth Brushing
Cumulative %
100.00%
25
90.00%
80.00%
20
70.00%
60.00%
15
50.00%
40.00%
10
30.00%
20.00%
10.00%
.00%
1
2
3
4
5
6
7
8
9
10
11 More
Figure 10. Time since subjects last brushed their teeth until the time of sampling (in
hours).
87
Types of Tooth Movement
45
4O
35
3O
15
10
No Movement
Translation
Tipping
Rotation
Figure 11. Types of tooth movement attempted during the period prior to sampling.
88
Force Application
Ii Applied
Remaining
7O
6O
5O
2O
10
None
Moderate
Light
Heavy
Force
Figure 12. Force application determined at the visit prior to sampling (applied) and at
the time of sampling (remaining).
89
Frequency
Time in Treatment
--B- Cumulative
%
100.00%
35
90.00%
30
80.00%
25
70.00%
60.00%
50.00%
40.00%
30.00%
10
20.00%
10.00%
.00%
0
4
8
12
16
20
24
28
Months
Figure 13. Time in treatment from initial banding visit (months).
32
More
90
Amounts of Root Loss
30
25
20
15
10
mm
Figure 14. Amounts of root loss in our sample as determined from method # 1 (RL).
91
Amounts of Root Loss
6O
5O
4O
3O
2O
10
0
1
2
3
RRI
Figure 15. Amounts of root loss as determined from method #3 (RRI).
More
92
Frequency
TNF Values
Cumulative %
100.00%
40
90.00%
35
80.00%
30
70.00%
25
60.00%
50.00%
20
40.00%
15
30.00%
10
20.00%
10.00%
.00%
0
300
600
900
1200
Figure 16. TNF-ct distribution in our sample (picograms).
1500
More
93
Frequency
TNF Index
-e- Cumulative %
100.00%
40
90.00%
35
80.00%
30
70.00%
25
60.00%
50.00%
20
40.00%
15
30.00%
10
20.00%
10.00%
.00%
0
1
2
Figure 17. TNF-c values tabulated as an index.
3
4
5
94
Frequency
Ln TNF+I
Cumulative %
100.00%
40
90.00%
35
80.00%
30
70.00%
25
60.00%
50.00%
20
40.00%
15
30.00%
10
20.00%
10.00%
.00%
0.00 0.89
1.78 2.66 3.55 4.44 5.33 6.21
7.10 More
Figure 18. TNF-a values transformed using the natural logarithm.
95
TNF Distribution with No Zero Values
Frequency
---e- Cumulative %
20
100.00%
18
90.00%
16
80.00%
14
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
2
.00%
300
600
900
1200
1500
More
Figure 19. Distribution: samples with zero TNF-c values are omitted.
96
Frequency
TNF Index with No Zero values
18
100.00%
16
90.00%
80.00%
14
70.00%
12
60.00%
10
50.00%
40.00%
30.00%
20.00%
10.00%
.00%
2
3
4
5
Figure 20. Distribution of tabulated data using the TNF index and omitting samples
with zero values.
97
Ln TNF (no zero values)
[m Frequency
.._.. Cumulative %
100.00%
14
90.00%
12
80.00%
10
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
.00%
0
2.64
3.53
4.42
5.31
6.20
7.10
More
Figure 21. Distribution of transformed data using natural logarithm and omitting
samples with values of zero.
APPENDIX II: TABLES
Reference
Ketcham ’,293
Becks ’396
Rudolph ’364
Henry and
Sample Size
Patients with
Teeth with
Resorption
(percent)
2012
72
4560
Resorption
(percent)
0.5
32
12
Method of
Evaluation
90.5
PA
PA
PA
HIST
Weinman
,5119
Massler and
Malone ’549
Massler and
Perreault ’54
31
Stenvik. and
Mjor ’7.0 29
Plets et al. ’87
32
Goldson and
Henrikson ’75
33
Harry and Sims
’82 28
Dermaut and
Munck ’86 34
708
100
86.4
PA
301
100
86.4
PA
0
HIST
27.5
PA
4
PA
35
50
27.5
42
10
0
0
PA/HIST
15
0
0
PA
Table 1. Incidence of root resorption in non-orthodontically treated individuals.
98
99
Reference
Sampl
Treatment
e Size
Ketcham ’29 3
Becks ’39 6
Rudolph ’36 4
Hemley ’41 81
Massler and Malone
’54 9
Phillips 55 10
Phillips ’55 10
Deshields ’69 12
Stenvik and Mjor ’70
29
Sjolien and Zachrisson
’73 115
Plets et al. ’87 32
Goldson and
Henrikson 75 33
Hollender et al. ’80 94
Ronnerman and
Larsson 81 120
Harry and Sims ’82 28
Kennedy et al. ’83 30
Kennedy et al. ’83 30
69
Edgewise
62
52
35
Edgewise
Edgewise
59
Edgewise
45
42
Fixed
46
46
Begg
100
77
12
23
Edgewise
10
32
32
Unknown
LL
Fixed
92
99
Intrusion
Activator/
Edgewise
Intrusion
Edgewise
Serial Exo
and fixed
Serial Exo
Dermaut and Munck
’86 34
Sharpe et al. ’87 116
20
Intrusion
18
18
153
Edgewise
Edgewise
485
Method of
Evaluation
(percent)
Unknown
Fixed
32
45
Levander and
Malmgren ’88 76
Linge and Linge ’91
Teeth with
Resorption
500
72
439
195
81
Kennedy et al. ’83 30
Copeland and Green
’86 121
Sharpe et al. ’87 116
Patients w/
Resorption
(percent)
19
73
57
21
100
3.5
93.3
31 incisors
83
92
82
93
PA
PA
PA
PA
PA
PA
CEPH
CEPH
HIST
PA
50
PA
PA
100
PA/HIST
39
100
26.5
20.5
CEPH
PA
PA
PA
PA
CEPH
6
Fixed
86
PA
Fixed
20.1
13.3
56
PA
PA
PA
Mixed
16.5
PA
89
83
36
Table 2. Incidence of root resorption subsequent to orthodontic treatment.
100
Reference
Phillips ’55 ] 0
Sjolien and
Zachrisson ’73
I15
Plets et al. ’87
32
Goldson and
Henrikson ’75
33
Hollender et al.
’80 94
Ronnerman and
Larsson ’81
120
Linge and
Linge ’83 35
Copeland and
Green ’86 121
Dermaut and
Munck ’8634
Goldin ’89 42
McFadden et
al. ’89 80
Linge and
Linge ’9136
Mirabella and
Artun ’95 37
Sample size
Treatment
type
Mean
Method of
analysis
62
59
Edgewise
Edgewise
resorption
1.4
0.5-1.8
45
Fixed
1.78
CEPH
42
Begg
6 % with
>2mm
PA
12
Edgewise
>2mm
PA
23
EW and
1-3
PA
CEPH
PA
activator
719
Mixed
0.7
PA
45
Fixed
2.93
CEPH
20
Intrusion
2.5
PA
17
38
Edgewise
Intrusion
CEPH
485
Mixed
1.35
max 1.84mand
0.61
2.5+ mm
343
Fixed
1.47
PA/CEPH
PA
PA
Table 3. Reported amounts of root resorption subsequent to orthodontic treatment.
101
Cytokine
Bone
Bone
formation
resorption
+
Interleukin- 1
+++
Implicated in
Sources
Myeloma
Squamous cancer
Periodontal disease
Rheumato d arthritis
Osteoporosis
Myeloma
Interleukin-6.
Interleukin-9
:F+
Potential
++
Leukocytes
Osteoblasts
Tumors
As above
Leukocytes
Osteoblasts
+
+++
TNF
+
+++
EGF
no effect
+++
TGF c
++
TGF
PDGF
++
Cancer, autoimmune disease
Periodontal disease
Rheumatoid arthritis,
osteoarthritis
Osteoporosis, multiple sclerosis
Leukocytes
Myeloma
Leukocytes
Tumors
Osteoblasts
Tumors?
+++
Squamous cancer
++
Osteoporosis
Osteoblasts.
Cancer ?
Bone Matrix
Leukocytes
Rheumatoid arthritis
Platelets
++
Tumors
Osteoblasts
CSF
?
+
Osteopetrosis
Osteoblasts
Stromal cell
Leukocytes
IFN 7
IGF-1
Rheumatoid arthritis
+++
no effect
Osteoporosis
Lymphocytes
Osteoblasts
Bone matrix
IGF’2
+++
no effect
Osteoporosis
Osteoblasts
Bone matrix
FGF
+++
no effect
?
Bone matrix
Osteoblasts
Table 4. Cytokines with effects on bone metabolism.
Taken from Ralston SH. Role of cytokines in clinical disorders of bone
metabolism.231
102
General
Bone metabolism
Regulation of phospholipid metabolism
Prostaglandin synthesis (synergistic)
Induction of acute phase proteins
Induction of other cytokines
Prostacyclin synthesis
Increase osteoclast recruitment
Expression of human major
histocompatibility complex antigens
Fever induction
Increase osteoclast proliferation
Role in rheumatoid arthritis,
osteoarthitis, endotoxin shock,
tuberculosis, multiple sclerosis,
glomerulonephritis
Activation of inflammatory leukocytes
Modification of vascular permeability
Increase osteoclast activity
Inhibit osteoblast maturation
Decrease collagen synthesis
Increase osteocalcin production
Promotion of fibroblast growth
Induction of collagenase secretion
Increased in periodontal disease process
Table 5. Actions common to both Interleukin-1 and Tumor Necrosis Factor.
103
GCF
Molecular
Orthodontic
Tooth
Signal
Movement
Acid
3X increase
Normal value
Gingivitis
Periodontitis
232
Phosphatase
Alkaline
20 mU/m1233
60 mU/ml
233
90 mU/ml
233
Phosphatase
IL 1-13
IL 6
234
68 ng/ml
153
36 ng/ml
1.7 pg/site
TM
0.38 pg/ug
192
2.9 pg/site
235
10 pg/site
191
0.020 pg/ug
0.41 pg/ug
PGE 2
152
131 ng/ml
TM
194 ng/ml
5 .ng/m1233
192
62 pg/site
3.20 p g/mL
40 ng/ml
6 ng/m1233
149
120 ng/ml
233
10 ng/ml
TM
EGF
0.18 pg/ug
Osteoclacin
3 5 pg/s
55 ng/m1233
TM
0.88 pg/ug
192
19.2 pg/site
3s
1 O0 pg/site
0.063 pg/ug
TM
20ng/ml
234
404 ng/mL
153
81 ng/mL
31 pg/site
149
38 pg/mL
1.13 pg/ug
Table 6. Molecules found in gingival crevicular fluid.
115 ng/m1233
total amt 10X
TM
1.5X
192
increase
9
109 pg/site
0.82 pg/ug
90 pg/s
110 ng/m1233
TM
191
104
Cellular components
Bacteria, desquamated
Mostly from adjacent
epithelial cells,
periodontal tissues
lymphocytes, monocytes,
macrophages,
polymorphonuclear
neutrophils
Carbohydrates
Glucose hexosamine,
hexuronic acid
3-4 times blood levels
Electrolytes
Potassium, sodium,
calcium
Levels correlate with
inflammation
Proteins
Immunoglobulins (IgA,
IgG, IgM), Complement
components (C3 and
C4), Plasma Proteins
(albumin, fibrinogen,
Lower than blood protein
etc.)
Enzymes
Bacterial and metabolic
products
Acid phosphatase, beta
glucuronidase, lysozyme,
cathepsin D, proteases,
alkaline phosphatase,
lactic dehydrogenase
Endotoxins, lactic acid,
urea, hydroxyproline,
hydrogen sulfide
Table 7. Composition of the gingival sulcus. 236.
levels
No significant
correlation with
severity of gingivitis,
pocket depth, or bone
loss.
105
Inclusion criteria
Exclusion criteria
Comprehensive fixed orthodontic
Chronic inflammation of the gingiva
reatment
Permanent or late mixed dentition
Pre-treatment periapical radiographs of
involved teeth
Mid-treatment periapical radiograph
exhibiting modified root anatomy
High caries index
Chronic use of anti-inflammatory
medication
Smokers
Deep periodontal pockets" 5+mm
Refusal to participate in the study
Table 8. Criteria for patient selection and disqualification.
106
Patient:
Date:
Number:
M
Y
Y
Y
Y
Y
Y: Timesince
Y: Tongue Nailbiter Tongue
Y: Skeletal Dental Both
Age:
Sex:
Familial history:
Medical history:
RME
Habit:
Openbite"
Tooth #
Root loss
R*:mm
EARR: 0-4
7
F
N
N
N
N
N
8
9
10
Direct measurement
Corrected
01234
01234
01234 01234
Irregular, blunt, 1/5-1/3,
1/3+
Root
morphology
Trauma
Pathology
RCT
NA
NA
NA
NA
Norm vs. blunt, narrow,
pipette, dilacerated
Restoration
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
NY
Plaque
0123
0123
0123
0123
Gingival Ind 0123
Periodontal
Index
0123
0123
0123
Yes: time since
yes: type
yes: time since
yes." type and time since
Index
Scale 0-8, need x-ray
None: 1-3 mm,
Pocket: In mm
Last time in hours
Sulcus
Depth
Brushing
N P
Movement
ITRTr
ITRTr
ITRTr ITRTr Intrude,
Force
LMH
LMH
LMH
LMH
L M H
LMH
L M H
L M H
CI
N Y
CI
N Y
CI
N Y
CI
N Y
applied
Force ( at
assay)
Time since
Cont / Int
Occlusal
trauma
Tx time
Time to go
tip, rotate,
translate
Light 100gm, Moderate
100-250, Heavy 250+
Force remaining at the
time of assay
Last visit in weeks
Type offorce applied
In months
In months
TNF
Table 9. Sample summary. The above form will be used to collect data at each visit.
107
Score
Changes noted in root anatomy
No discernible erosion or blunting and complete apexification
Irregular root, contour, notably para-apically
Root blunting with minor resorption
Severe root resorption, loss of one fifth to one third of the original length
Extreme resorption exceeding one third of the original length
Table 10. Root resorption index.
Adapted from Levander and Malmgren: 76
PI score Clinical condition
Negative: no overt inflammation
Mild gingivitis" Overt inflammation in the free gingiva
Gingivitis: Inflammation completely circumscribing the tooth with no
apparent break in the attachment or loss of function
Radiograph: early notch-like resorption of the alveolar crest
Pocket formation: Epithelial attachment has been broken. Tooth is firm
and has not drifted. Horizontal bone loss up to half of the root.
Advanced destruction beyond one half of the root with loss of
masticatory function. Mobility and drift.
Table 11. The Periodontal Index (PI). 237
108
GI score
Clinical conditions
Normal gingiva
Mild inflammation, slight change in color, slight edema, no bleeding
on palpation
Moderate inflammation, redness, edema, and glazing, bleeding on
palpation
Severe inflammation, marked redness and edema, ulcerations;
tendency to spontaneous bleeding
Table 12. Gingival Index (GI). 224
Plaque Index
Clinical condition
No plaque in gingival area
A film of plaque adhering to the free gingival margin and adjacent
area of the tooth
Moderate accumulation of soft deposits within the gingival pocket
and on the gingival margin and or adjacent tooth surface,
which can be seen by naked eye
Abundance of soft matter within the gingival pocket and or on the
gingival margin and adjacent tooth surface.
Table 13. Plaque index. 225
109
Frequency
Percentage
Cumulative %
1
1.2
1.18%
1
1.2
2.35%
2
2.4
4.71%
0
0.0
4.71%
6
7.1
11.76%
15
17.6
29.41%
17
20.0
49.41%
17
20.0
69.41%
10
16
I8.8
88.24%
11
6
7.1
95.29%
12
0
0.0
95.29%
13
2
2.4
97.65%
14
2
2.4
100.00%
15
0
0.0
100.00%
Tooth #
Table 14. Distribution of teeth included in our sample. Also refer to figure 5.
N=85
110
Root Shape
Count
Percentage
Normal
68
80.0
Blunted
13
15.3
Dilacerated
1
1.2
Pipette
2
2.4
Table 15. Distribution of normal and deviating root shapes. Also refer to figure 6.
N=85
Plaque Index
Frequency
Cumulative %
Percentage
11
12.94%
12.9
36
55.29%
42.4
37
98.82%
43.5
1
100.00%
1.2
Table 16. Distribution of the plaque index. Also refer to figure 7.
111
Gingival Index
Frequency
Cumulative %
Percentage
12
14.12%
14.1
38
58.82%
44.7
33
97.65%
38.8
2
100.00%
Table 17. Distribution of the Gingival Index. Also see figure 8.
Periodontal Index
Frequency
Cumulative %
Percentage
0
15
17.65%
17.6
63
91.76%
74.1
7
100.00%
0
100.00%
0
100.00%
More
Table 18. Distribution of the Periodontal Index. Also see figure 9.
112
Hours
Frequency
Cumulative %
1
0
.00%
2
10
11.76%
11.8
3
4
16.47%
4.7
4
4
21.18%
4.7
5
11
34.12%
12.9
6
25
63.53%
29.4
7
6
70.59%
7.1
8
7
78.82%
8.2
9
0
78.82%
0.0
10
8
88.24%
9.4
11
10
100.00%
11.8
More
0
100.00%
0
Percentage
Table 19. Time since subjects last brushed their teeth until the time of sampling (in
hours). Also refer to figure 10.
Type of Tooth Movement
Frequency
Percentage
No Movement
16
18.82
Translation
22
25.88
Tipping
45
52.94
Rotation
2
2.35
Table 20. Types of tooth movement attempted during the period prior to sampling.
Also refer to figure 11.
113
Force
Rmaining
Applied
Frequency
Percentage
Frequency
Percentage
None
16
18.8
22
25.9
Light
19
22.4
63
74.1
Moderate
50
58.8
0
0.0
Heavy
0
0.0
0
0.0
Table 21. Force application determined at the visit prior to sampling (applied) and at
the time of sampling (remaining). Also refer to figure 11.
Frequency
Cumulative %
Percentage
0
.OO%
0.0
14
16.47%
16.5
24
44.71%
28.2
12
34
84.71%
40.0
16
0
84.71%
0.0
2O
0
84.71%
0.0
24
0
84.71%
0.0
28
9.
95.29%
10.6
32
0
95.29%
0.0
More
4
100.00%
4.7
Months
Table 22. Time in treatment from initial banding visit (months).
Also refer to figure 13.
114
Distribution
RL
RL*
Mean
0.6871
0.6894
0.8588
Standard Error
0.0684
0.0687
0.0872
Median
0.6
0.6
1
Mode
0.2
0
0
Standard Deviation
0.6307
0.6338
0.8041
Sample Variance
0.3978
0.4017
0.6465
Kurtosis
2.5060
2.5548
-0.9318
Skewness
O. 1074
O. 1528
0.4043
4.5
4.5497
3
Minimum
-1.7
-1.6888
0
Maximum
2.8
2.8609
3
58.4
58.6011
73
85
85
85
0.1360
0.1367
0.1734
Range
Sum
Count
Confidence Level(95.000%)
Table 23. Descriptive statistics of the three methods used to assess root resorption.
Root loss (RL) in method # 1 is the difference in the length of the entire tooth as
measured from pre to post treatment periapical radiographs. Corrected root loss
(RL*) in method #2 incorporates a ratio using crown lengths to remove
magnification error. The Root Resorption Index (RRI) from method #3 is a
subjective measurement of root loss based on an ihdex ranging from 0 to 4. Zero
score indicate no root loss, whereas a score of four indicates loss of root structure
beyond 1/3 of the original length.
115
Post-tx
Pre-tx
Difference between
Pre-tx Tooth Post-tx Tooth
Length
Length
repeated measures Crown length Crown length
(Error)
Mean
-0.1000
-0.0314
-0.1843
-0.1729
0.0468
0.0467
0.0691
0.0584
Median
-0.2
-0.1
-0.2
-0.2
Mode
-0.5
-0.1
-0.4
-0.3
Standard Deviation
0.3919
0.3907
0.5780
0.4884
Sample Variance
0.1536
0.1526
0.3341
0.2385
Kurtosis
-1.1532
5.6252
-0.3900
-0.5615
Skewness
0.2929
1.9189
-0.5049
-0.1033
1.5
2.2
2.4
2
Minimum
-0.8
-0.7
-1.7
-1.2
Maximum
0.7
1.5
0.7
0.8
Sum
-7
-2.2
-12.9
-12.1
Count
70
70
70
70
0.0918
0.0915
0.1354
0.1144
0.9840
0.9886’
0.9955
0.995
Standard Error
Range
Confidence Level
(95.000%)
orrelation
coefficient
Table 24. Descriptive statistics of the difference between repeated measures.
The first 70 direct measurements of the crown length and the entire tooth were
repeated to assess reproducibility. Reliability between the two intervals, where the
same measurement was made, was assessed by calculating the correlation between
the two sets of measurements.
116
RRI (entire
Difference between
RRI
RL*
repeated measures
sample)
Mean
0.0186
0.0177
0.0286
0.0636
Standard Error
0.0530
0.0530
0.0454
0.0414
Median
0.1
0.0979
0
0
Mode
0.1
0.0992
0
0
Standard Deviation
0.4437
0.4435
0.3 796
0.4343
Sample Variance
0.1969
0.1967
0.1441
0.1886
Kurtosis
1.1429
1.0891
4.3419
2.2920
Skewness
0.4005
0.3875
0.3126
0.3478
2.4
2.3956
2
2
Minimum
-0.9
-0.9073
1
1
Maximum
1.5
1.4884
1
1
Sum
1.3
1.2378
2
7
Count
70
70
70
110
0.1039
0.1039
0.0889
0.0812
0.8122
0.8158
0.8872
0.8931
Range
Confidence
Level(95.000 O%)
correlation
Table 25. Repeatability (error) of the three methods for determination of root loss.
RL
RL*
RRI
RL
1.0000
0.9996
0.7244
RL*
0.9996
1.0000
0.7246
RRI
0.7244
0.7246
1.0000
Table 26. Correlation between the three methods used to assess root loss.
117
TNF
Mean
In TNF+I
TNF Index
232.86
1.27
2.95
45.8136
0.1702
0.3159
29
1
3.4012
0
0
0
Standard Deviation
422.3807
1.5690
2.9121
Sample Variance
178405.5
2.4616
8.4805
Kurtosis
20.1074
-0.1829
1.8088
Skewness
3.7713
1.0337
0.0931
2944
5
7.98786
Minimum
0
0
0
Maximum
2944
5
7.98786
19793
108
250.704
85
85
85
89.793
0.334
0.628
Standard Error
Median
Mode
Range
Sum
Count
Confidence Level(95.000%)
Table 27. Distribution of Tumor Necrosis Factor alpha (TNF-) values.
118
Frequency Percentage
TNF-
More
Cumulative %
0
40
47.1
47.06%
300
20
23.5
70.59%
600
16
18.8
89.41%
900
4
4.7
94.12%
1200
3
3.5
97.65%
1500
1
1.2
98.82%
1
1.2
100.00%
Table 28. Distribution of raw TNF-ot values in our sample (picograms).
Also refer to figure 16.
119
TNF index
TNF
Percentage
Count
Cumulative %
(picograms)
0
40
47.1
47.06%
100-199
1
17
20.0
67.06%
200-399
2
9
10.6
77.65%
400-599
3
7
8.2
85.88%
600-999
4
8
9.4
95.29%
1000+
5
4
4.7
100.00%
Table 29. TNF-tx values tabulated using an ordinal index 0-5.
Also refer to figure 17.
Ln (TNF +1)
Percentage
Count
Cumulative %
0.00
40
47.1
47.06%
0.89
0
0
47.06%
1.78
0
0
47.06%
2.66
1
1.2
48.24%
3.55
2
2.4
50.59%
4.44
3
3.5
54.12%
5.33
11
12.9
67.06%
6.21
13
15.3
82.35%
7.10
13
15.3
97.65%
More
2
2.4
100.00%
Table 30. TNF-o values transformed using the natural logarithm.
Also refer to figure 18.
120
Tooth selection"
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
8.53333
8.475
Variance
5.39091
3.33269
45
40
Observations
Hypothesized Mean Difference
df
82
t Stat
0.12943
P(T<=t) one-tail
0.44867
t Critical one-tail
1.66365
P(T<=t) two-tail
0.89733
t Critical two-tail
1.98932
Table 31. T-test for tooth selection.
Student’s two sample t-test for statistical differences in the teeth selected for
sampling between samples with zero TNF-a levels and samples positive for TNF-
121
Root Shape:
Positive for
TF
Zero TNF
Two sample t-test, unequal
variances
Mean
Variance
Observations
0.2
0.35
0.34545
0143846
45
40
Hypothesized Mean Difference
df
t Star
79
1.0987
P(T<=t) one-tail
0.13761
t Critical one-tail
1.66437
P(T<---t) two-tail
0.27523
t Critical two-tail
1.99045
Table 32. T-test for root shape.
Student’s two sample t-test for statistical differences in root morphology between
samples with zero TNF-ct levels and samples positive for TNF-a.
122
Time since brushing"
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
6.71111
5.975
Variance
6.07374
8.84551
45
40
Observations
Hypothesized Mean Difference
df
76
t Star
1.23353
P(T<--t) one-tail
0.11059
t Critical one-tail
1.66515
P(T<=t) two-tail
0.22118
t Critical two-tail
1.99168
Table 33. T-test for time since brushing.
Student’s two sample t-test for statistical differences in time since the patient last
brushed their teeth between samples with zero TNF-c levels and samples positive
for TNF-z.
123
Type of tooth movement:
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
Variance
Observations
1.2
1.525
1.11818
1.12756
45
40
Hypothesized Mean Difference
df
82
t Stat
-1.4112
P(T<=t) one-tail
0.08098
t Critical one-tail
1.66365
P(T<=t) two-tail
0.16197
t Critical two-tail
1.98932
Table 34. T-test for the type of tooth movement.
Student’s two sample t-test for statistical differences in the type of tooth movement
between samples with zero TNF-c levels and samples positive for TNF-a.
124
Time in treatment:
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
12.4889
9.825,
Variance
95.6192
56.0455
45
40
Observations
Hypothesized Mean Difference
df
81
t Stat
1.41865
P(T<=t) one-tail
0.07992
t Critical one-tail
1.66388
P(T<=t) two-tail
0.15984
t Critical two-tail
1.98969
Table 35. T-test for the time in treatment.
Student’s two sample t-test for statistical differences in the time in treatment
between samples with zero TNF-c levels and samples positive for TNF-c.
125
Plaque Index:
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
Variance
Observations
1.4
1.25
0.42727
0.60256
45
40
Hypothesized Mean Difference
df
77
t Stat
0.95716
P(T<=t) one-tail
0.17074
t Critical one-tail
1.66488
P(T<=t) two-tail
0.34148
t Critical two-tail
1.99126
Table 36. T-test for the Plaque Index.
Student’s two sample t-test for statistical differences in the Plaque Index between
samples with zero TNF-c levels and samples positive for TNF-c.
126
Gingival Index:
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
1.64444
0.85
Variance
0.32525
0.3359
45
40
Observations
Hypothesized Mean Difference
df
t Stat
82
6.3555
P(T<--t) one-tail
5.5E-09
t Critical one-tail
1.66365
P(T<---t) two-tail
1.1E-08 *
t Critical two-tail
1.98932
Table 37. T-test for the Gingival Index.
Student’s two sample t-test for statistical differences in the Gingival Index
between samples with zero TNF-c levels and samples positive for TNF-c.
127
Periodontal Index-
Positive for
TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
1.04444
0.75
Variance
0.27071
0.19231
45
40
Observations
Hypothesized Mean Difference
df
83
t Stat
2.83023
P(T<=t) one-tail
0.00292
t Critical one-tail
1.66342
P(T<=t) two-tail
0.00583 *
t Critical two-tail
1.98896
Table 38. T-test for the Periodontal Index.
Student’s two sample t-test for statistical differences in the Periodontal Index
between samples with zero TNF-ct levels and samples positive for TNF-c.
128
Root Loss (method 1)"
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
:Mean
0.9222
0.4225
Variance
0.3886
0.2828
45
40
Observations
Hypothesized Mean Difference
df
83
t Stat
3.98751
P(T<=t) one-tail
0.00007
t Critical one-tail
1.66342
P(T<=t) two-tail
0.00014 *
t Critical two-tail
1.98896
Table 39. T-test for Root Loss (RL).
Student’s two sample t-test for statistical differences in the amount of root loss, as
determined from method #1, between samples with zero TNF-t levels and samples
positive for TNF-ct.
129
Root Loss* (method #2):
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
0.9238
0.4257
Variance
0.3929
0.2873
45
40
Observations
Hypothesized Mean Difference
df
83
t Stat
3.948093
P(T<---t) one-tail
0.000082
t Critical one-tail
1.663420
P(T<=t) two-tail
0.000164 *
t Critical two-tail
1.988960
Table 40. T-test for Corrected Root Loss (RL*).
Student’s two sample t-test for statistical differences in the amount of root loss, as
determined from method #2, between samples with zero TNF-a levels and samples
positive for TNF-c.
130
Root Resorption Index:
Positive for TNF
Zero TNF
Two sample t-test, unequal
variances
Mean
1.1778
0.5
Variance
0.6040
0.4615
45
40
Observations
Hypothesized Mean Difference
df
83
t Stat
4.28994
P(T<=t) one-tail
0.00002
t Critical one-tail
1.66342
P(T<=t) two-tail
0.00005 *
t Critical two-tail
1.98896
Table 41. T-test for the Root Resorption Index
Student’s two sample t-test for statistical differences in the root resorption index,
as determined from method #3, between samples with zero TNF-c levels and
samples positive for TNF-a.
131
Samples with zero TNF omitted
Mean
TNF
Ln TNF
TNF index
437.05
2.35
5.53
77.5512
0.2128
0.1748
Median
320
2
5.7683
Mode
320
1
5.7683
Standard Deviation
508.5371
1.3953
1.1461
Sample Variance
258610.0
1.9468
1.3136
Kurtosis
13.6915
1.0011
0.0976
Skewness
3.1788
0.6031
-0.4550
2931
4
5.4226
Minimum
13
1
2.5649
Maximum
2944
5
7.9875
18793
101
237.9680
43
43
43
151.997
0.417
0.353
Standard Error
Range
Sum
Count
Confidence Level(95.000%)
Table 42. Distribution with samples containing zero TNF values omitted.
TNF
More
Percentage
Frequency
Cumulative %
300
20
46.5
46.51%
600
14
32.6
79.07%
900
4
9.3
88.37%
1200
3
7.0
95.35%
1500
1
23
97.67%
1
2.3
100.00%
Table 43. Distribution of TNF values with samples of zero omitted.
For comparison, refer to table 28.
132
TNF Index
Percentage
Frequency
Cumulative
17
39.5
39.53%
9
20.9
60.47%
6
14.0
74.42%
7
16.3
90.70%
4
9.3
100.00%
Table 44. Distribution of TNF values sorted according to the TNF index with zero
values omitted. For comparison, refer to table 29.
Ln"TF
More
Frequency
Percentage
Cumulative %
2.64
1
2.3
2.33%
3.53
2
4.7
6.98%
4.42
3
7.0
13.95%
5.31
11
25.6
39.53%
6.20
11
25.6
65.12%
7.10
13
30.2
95.35%
2
4.7
100.00%
Table 45. Distribution of TNF data with no samples of zero when transformed using
natural logarithm. For comparison, refer to table 30.
133
PLI
Tooth
GI
PeI
Brs
Rt
0.00
1.00
PLI
-0.02
-0.07
1.00
GI
0.00
-0.12
0.51
1.00
PeI
-0.02
0.01
0.55
0.65
1.00
Brs
0.06
-0.07
0.08
0.13
0.04
1.00
MVMT
0,00
-0.20
0.05
0.00
0.13
-0.18
Fa
0.10
-0.13
0:31
0.10
0.34
-0.17
FR
0,01
-0.05
0.31
0.15
0.37
-0.40
Time
0.01
-0.01
0.22
0.18
0.43
-0,40
C/I
0,07
-0.03
0.32
0.12
0.36
-0.38
TxT
0.01
-0.04
0.-116
0.14
-0.03
0.62
TxL
-0.01
-0.07
-0.09
-0.22
-0.24
-0.39
RRI
0.01
0.05
-0.04
0.24
0.29
0.10
RL
0.06
-0.05
0.01
0.29
0.26
0.12
RE,*
0.07
-0.05
0.01
0.29
0.26
0.13
TNF
0.07
-0.10
0.14
0.49
0.46
-0.09
lnTNF+I
0.05
-0.13
0.13
0.65
0.38
0.06
TNF-Ind
0.11
-0.10
0.13
0.63
0.46
-0.05
TNF
0.08
-0.12
0,14
0.67
0.42
0.01
ranked
Table 46. Correlation coefficients (tables 46-48).
For legend, see table 48.
134
MVMT
Fa
FR
time
C/I
TxT
TxL
MVMT
1.00
Fa
0.69
1.00
FR
0.60
0.80
1.00
Time
0.61
0.70
0.96
1.00
C/I
0.55
0.77
0.97
0:93
1.00
TxT
-0.12
-0,08
-0.35
-0,38
-0.34
1.00
TxL
0.08
0.08
0,15
0.02
0.16
-0.68
1.00
RRI
-0.08
-0.04
0.03
0.10
0.02
0.20
-0.36
RL
-0.08
0.03
0.12
0. ! 1
0.12
0.10
-0.08
RL*
-0.08
0.03
0.12
0.11
0.12
0.10
-0.09
TNF
0.22
0.17
0.17
0.20
0.16
0.02
-0.12
-0.06
-0.03
0.00
0.03
-0.02
0.10
-0.15
TNF-Index
0.09
0,12
0.15
0.19
0.14
0.03
-0.16
TNF ranked
0.00
0,03
0.08
0.11
0.05
0.08
-0.17
InTNF+I
Table 47. Correlation coefficients (tables 46-48).
135
RRI’
RL
RL*
TNF
InTNF+I
TNF
TNF
Index
ranked
RRI
1.00
RL
0.72
1.00
RL*
0.72
1.00
1.00
TNF
0,37
0.22
0,21
1.00
InTNF+I
0.44
0.38
0.37
0.69
1.00
TNF’Index
0.39
0.310
0.29
0.84
0.89
1.00
TNFranked
0,36
0,35
0.43
0.75
0.98
0.96
1
Table 48. Correlation coefficients (tables 46-48).
Legend: Tooth-which teeth were included in the sample, Root- root morphology,
PLI- Plaque Index, GI- Gingival Index, PeI- Periodontal Index, Brs- hours since
the patient last brushed their teeth, MVMT- type of tooth movement, Fa- force
applied to the tooth during the visit prior to the sampling visit, FR- force remaining
at the time of sampling, Time- weeks since the last visit (the number of weeks that
this force was applied), C/I- continuous or interrupted force, TxT- number of
months in treatment, TxL- approximated time in treatment left, RRI- Root
Resorption Index, RL- root loss, RL*- corrected root loss, TNF- raw TNF levels
(picograms/site), lnTNF+ 1- raw TNF values plus one picogram tranformed using
natural logarithm function, TNF Ind- TNF values categorized using the previously
mentioned index, TNF ranked- ranked TNF values using the entire array of TNF
data.
136
PLI
Rt
Tth
GI
PeI
Brs
Tth
1.00
Rt
-0.08
1.00
PLI
-0.05
-0.02
1.00
GI
0.02
-0.13
0.58
1.00
PeI
0.00
0.04
0.49
0.60
1.00
Brs
0.12
-0.09
-0.18
-0.15
-0.24
1.00
-0:03
-0.22
0.26
0.51
0.44
-0.22
Fa
0i06
-0.16
0.31
0.37
0.48
-0.35
FR
0,00
-0.11
0.46
0.46
0.54
-0.47
Time
0,04
-0.10
0.41
0.49
0.63
-0.47
C/I
0.10
-0.09
0.48
0,42
0.53
-0.42
Tx T
0.03
-0.11
-0.15
-0.05
-0.31
0,65
Tx.L
-0,13
0.10
0.04
-0.12
-0.11
-0.47
RL
0.08
-0.18
0.01
0.03
-0.01
0.21
RL*
0.08
-0.18
0.00
0.02
-0.01
0.22
RRI
0.11
-0.18
-0.13
0.16
0.16
0.11
TNF
0:09
-0.06
0.14
0.60
0.49
-0.29
Tnf-I
0.18
0.00
0.09
0.58
0.49
-0.39
InTNF+I
0.17
-0.09
0.15
0.68
0.46
-0.38
InTNF
0.17
-0.09
0.15
0.68
0.46
-0.38
TNF rank
0,19
-0.05
0.13
0.65
0.48
-0.41
Mvmt
Table 49. Correlation coefficients, omitting samples with TNF values of Zero (tables
49-51). For legend see table 51.
137
Mvmt
Fa
Fn
Time
C/I
Tx T
Tx"L
Fa
0.66
1.00
FR
0.65
0.85
1.00
Time
0.65
0.77
0.96
1.00
C/I
0,55
0.79
0.95
0.91
1.00
Tx T
-0.11
-0.22
-0.33
-0.35
-0.32
1.00
Tx L
0.05
0,11
0.10
-0,01
0.10
-0.72
1.00
RL
0.12
0.15
0.20
0.17
0.23
0.11
0.03
RL*
0.10
0.15
0.20
0.17
0.23
0.12
0.02
RRI
0.22
0.18
0.21
0.27
0.22
0.18
-0.18
TNF
0.49
0,37
0.32
0.37
0.32
-0.07
-0.08
Tnf-I
0.47
0.46
0.42
:0.49
0.43
-0.12
-0.14
InTNF+I
0.44
0,37
0,34
0.41
0.32
-0.16
-0.07
InTNF
0.44
0,37
0.34
0:41
0.32
-0.16
-0,07
TNF rank.
0,46
0.45
0.42
0,49
0.41
-0.12
-0.14
Table 50. Correlation coefficients, omitting samples with TNF values of Zero (tables
49-51).
138
RL
RL*
RRI
TNF
Tnf-I
InTNF+I InTNF TNF rank
RL
1.00
RL*
1.00
1.00
RRI
0.74
0.74
1.00
TNF
0,03
0.02
0.32
1.00
Tnf-I
0.01
0,01
0.28
0,80
1.00
InTNF+I
0,01
0.00
0.28
0.78
0.88
1.00
InTNF
0,01
0,00
0,28:
0.78
0.88
1.00
1.00
TNF rank
0.02
0,01
0.29
0.77
0.95
0.97
0.97
1.00
Table 51. Correlation coefficients, omitting samples with TNF values of Zero (tables
49-51).
Legend" Tooth-which teeth were included in the sample, Root- root morphology,
PLI- Plaque Index, GI- Gingival Index, PeI- Periodontal Index, Brs- hours since
the patient last brushed their teeth, MVMT- type of tooth movement, Fa- force
applied to the tooth during the visit prior to the sampling visit, FR- force remaining
at the time of sampling, Time- weeks since the last visit (the number of weeks that
this force was applied), C/I- continuous or interrupted force, TxT- number of
months in treatment, TxL- approximated time in treatment left, RRI- Root
Resorption Index, RL- root loss, RL*- corrected root loss, TNF- raw TNF levels
(picograms/site), lnTNF+I- raw TNF values plus one picogram tranformed using
natural logarithm function, TNF Ind- TNF values categorized using the previously
mentioned index, TNF ranked- ranked TNF values using the entire array of TNF
data.
139
Patient
Coefficient
Patient
Coefficient
TS
0.9115
DSM
0.3004
GM
0.8974
MM
0.1237
RP
0.8432
JA
0.0099
LR
0.7358
YP
0.0000
DS
0.7177
JO
0.0000
SM
0.4855
BG
-0.0411
BS
0.4460
LU
-0.0793
DM
0.4155
KM
-0.3042
SG
0.3678
KZ
-0.3674
HJ
-0.5521
Table 52. Correlation coefficients between Root Loss and raw TNF scores were
calculated for each patient (n=19).
140
Mean
Standard Error
Median
Mode
Standard Deviation
Sample Variance
Kurtosis
Skewness
Range
Minimum
Maximum
Sum
Count
Confidence
Tooth
8.51
0.227
9.00
8.00
2.091
4.372
1.439
-0.149
12
2
14
723
85
0.451
RT
PLI
0.27
0.068
0.00
0.00
0.625
0.390
8.053
2.737
3
0
3
23
85
0.135
GI
1.33
0.077
1.00
2.00
0.714
0.509
-0.642
-0.379
3
0
3
113
85
0.154
1.27
0.076
1.00
1.00
0.697
0.485
-0.865
-0.425
2
0
2
108
85
0.150
PeI
Brs
0.91
6.36
0.055 0.296
1.00
6.00
1.00
6.00
0.503 2.725
0.253 7.425
0.914 -0.705
-0.185 0.209
2
9
0
2
2
11
541
77
85
85
0.108 0.588
Level(95.0%)
Table 53. Distribution of clinical variables (tables 53-55).
MVMT
Mean
Standard Error
Median
Mode
Standard Deviation
Sample Variance
Kurtosis
Skewness
Range
Minimum
Maximum
Sum
Count
Confidence
Fa
FR
1.41
0.74
1.35
0.116 0.084 0.048
2.00
1.00
1.00
2.00
1.00
1.00
1.066 0.776 0.441
1.136 0.602 0.194
-0.964 -0.778 -0.761
0.576 -0.872 -1.121
1
3
2
0
0
0
1
2
3
120
63
115
85
85
85
0.230 0.167 0.095
time
3.51
0.236
4.00
4.00
2.175
4.729
-0.914
-0.833
6
0
6
298
85
0.469
Level(95.0%)
Table 54. Distribution of clinical variables (tables 53-55).
TxL
C/I
TxT
0.73
0.048
1.00
1.00
0.447
0.200
-0.917
-1.051
1
0
1
62
85
0.096
11.24 13.98
0.957 0.947
9.00 19.00
12.00 19.00
8.826 8.733
77.896 76.261
1.655 -1.261
1.570 -0.573
25
34
2
0
36
25
955
1188
85
85
1.904 1.884
141
RL* RRI TNF InTNF+I TNF-Index TNF ranked
1.27
44.059
0.69 0.69
0.86 232.86
2.95
45.8
0.068 0.069 0.087
0.316
0.170
2.398
RL
Mean
Standard
Error
Median
Mode
Standard
0.60 0.60
1.10 0.00
0.631 0.634
1.00
0.00
0.804
29.00
0.00
422.4
3.40
0.00
2.912
1.00
0.00
1.569
43
23
22.109
0.398 0.402
0.646 178405
8.480
2.462
488.806
-1.809
0.093
8
0
8
251
85
1
-0.183
1.034
5
0
5
108
85
0
-1.418
0.390
62
23
85
3745
85
4.700
Deviation
Sample
Variance
Kurtosis
Skewness
Range
Minimum
Maximum
Sum
Count
Confidence
2.506 2.555 -0.932
0.107 0.153 0.404
5
3
5
0
-2
-2
3
3
3
59
73
58
85
85
85
0.136 0.137 0.173
20.11
3.77
2944
0
2944
19793
85
91
Level(95.0%)
Table 55. Distribution of root loss measures and TNF values (tables 53-55).
142
Regression Statistics
Multiple R
0.6083
R Square
0.3701
Adjusted R Square
0.3467
Standard Error
341.3873
85
Observations
ANOVA
SS
df
Regression
F
MS
Significance F
3 5545892.325 1848630.775 15.8619
Residual
81
9440167.981
Total
84
14986060.31
Coefficients
Standard
3.34426E-08
116545.284
t Stat
P-value Lower 95%
Error
Upper
95%
Intercept
-355.442
95.030 -3.7403 0.00034
-544.522
-166.362
GI
MVMT
258.657
55.066 4.6972 0.00001
149.093
368.221
96.452
35.068 2.7504 0.00734
26.677
166.226
RR
150.392
47.870 3.1417 0.00235
55.146
245.638
Table 56. Regression model" Y= TNF, X= GI + MVMT + RRI
Backwards stepwise multivariable regression with raw TNF-ct values as the dependent
variable and the Gingival Index, type of tooth movement, and the Root Resorption
Index as the independent variables.
143
Regression Statistics
Multiple R
0.5579
R Square
0.3112
Adjusted R Square
0.2944
Standard Error
354.79
85
Observations
ANOVA
SS
df
Regression
2
F
MS
4664255
2332128 18.52723
Residual
82 10321805 125875.7
Total
84 14986060
Coefficients Standard
Significance F
2.3E-07
t Stat P-value Lower 95% Upper 95%
Error
Intercept
GI
RR
-219.11
84.26 -2.60 0.01104
-386.73
-51.48
261.36
57.22
4.57 0.00002
147.53
375.18
139.59
49.58
2.82 0.00610
40.96
238.23
Table 57. Regression model: Y= TNF, X GI + RRI
Backwards stepwise multivariable regression with raw TNF-c values as the dependent
variable and the Gingival Index and the Root Resorption Index as the independent
variables.
144
Regres’io’n Statistics
Multiple R
0.5429
R Square
0.2947
Adjusted R Square
0.2775
Standard Error
449.20
43
Observations
ANOVA
df
Regression
1
SS
3457458
41
Total
42 11730344
Coefficient Standard
Intercept
GI
F
Significance F
3457458 17.13499
0.000169
8272885 2O1777.7
Residual
s
MS
t Stat
P-value
Error
-218.267 143.7973 -1.51788 0.136719
Lower
Upper
95%
95%
-508.672 72.13784
409.1723 98.84719 4.139443 0.000169 209.5462 6O8.7984
Table 58. Regression model" Y= TNF, X= GI
Backwards stepwise multivariable regression with raw TNF-c values as the dependent
variable and the Gingival Index as the independent variable.
145
Regression Statistics
Multiple R
0.3688
R Square
0.1360
Adjusted R Square
0.1256
Standard Error
394.97
85
Observations
ANOVA
SS
Regression
1
MS
2038027
2038027 13.06424
Residual
83 12948034 156000.4
Total
84 14986060
Intercept
RR
F
Coefficient
Standard
s
Error
t Stat
Significance F
0.000515
P-value Lower 95% Upper 95%
66.4848
62.8815 1.0573
0.2934
-58.5840
191.5536
193.7231
53.5969 3.6144
0.0005
87.1211
300.3252
Table 59. Regression model" Y= TNF, X RRI
Backwards stepwise multivariable regression with raw TNF-(z values as the dependent
variable and the Root Resorption Index as the independent variable.
146
Regression Statistics
Multiple R
0.7472
R Square
0.5582
Adjusted R Square
0.5419
Standard Error
14.9643
85
Observations
ANOVA
SS
df
Regression
F
MS
Significance F
3 22921.45 7640.483 34.1201
2.33E-14
18138.26 223.9291
Residual
81
Total
84 41059.71
Coefficient
Standard
s
Error
t Stat
P-value Lower 95% Upper 95%
Intercept
PLI
18.1524
4.0852
4.4435
0.0000
10.0242
26.2807
-6.8085
2.7192 -2.5039
0.0143
-12.2189
-1.3981
GI
22.8988
2.8657
7.9908
0.0000
17 1970
28.6005
RR
6.8266
2.1335
3.1997
0.0020
2.5816
11.0716
Table 60. Regression model" Y= ranked TNF, X PLI + GI + RRI
Backwards stepwise multivariable regression with ranked TNF-ot values as the
dependent variable and the Plaque Index, Gingival Index, and Root Resorption
Index as the independent variables.
147
Regression Statistics
Multiple R
0.7239
R Square
0.,241
Adjusted R Square
0.5124
Standard Error
15.4376
Observations
85
ANOVA
SS
Regression
F
MS
Significance F
21517.58 10758.79 45.14455
Residual
82 19542.13 238.3186
Total
84 41059.71
Coefficient
Standard
t Stat
6.02E-14
P-value Lower 95% Upper 95%
Error
Intercept
13.1083
3.6663 3.5753
0.0006
5.8148
20.4018
GI
19.0298
2.4897 7.6434
0.0000
14.0770
23.9826
RR
7.8845
2.1574 3.6547
0.0005
3.5928
12.1763
Table 61. Regression model" Y= ranked TNF, X GI + RRI
Backwards stepwise multivariable regression with ranked TNF-c values as the
dependent variable and the Gingival Index and the Root Resorption Index as the
independent variables.
148
Regression Statistics
Multiple R
0.6682
R Square
0.4465
Adjusted R Square
0.4399
Standard Error
16.547
85
Observations
ANOVA
Regression
1
F
MS
SS
df
Significance F
18334.46 18334.46 66.96341
Residual
83 22725.25 273.7982
Total
84 41059.71
Coefficient
Standard
s
Error
t Stat
2.81E-12
P-value Lower 95% Upper 95%
Intercept
17.1166
3.7498 4.5646
0.0000
9.6583
24.5748
GI
21.2046
2.5913 8.1831
0.0000
16.0507
26.3585
Table 62. Regression model: Y= ranked TNF, X GI
Backwards stepwise multivariable regression with ranked TNF-c values as the
dependent variable and the Gingival Index as the independent variable.
149
Regression’Statistics
Multiple R
0.4511
R Square
0.2035
Adjusted R Square
0.1841
Standard Error
19.997
43
Observations
ANOVA
Regression
Significance F
1 4189.509 4189.509 10.47657
0.002395
16395.62 399.8931
Residual
41
Total
42 20585.13
Intercept
RR
F
MS
SS
Coefficient
Standard
s
Error
t Stat
P-value Lower 95% Upper 95’
35.6748
4.8561
7.3464
0.0000
25.8677
45.4819
12.2321
3.7791
3.2368
0.0024
4.6000
19.8643
Table 63. Regression model: Y= ranked TNF, X RRI
Backwards stepwise multivariable regression with ranked TNF-ot values as the
dependent variable and the Root Resorption Index as the independent variable.
150
Regression Statistics
Multiple R
0.7363
R Square
0.5421
Adjusted R Square
0.5251
Standard Error
2.0068
85
Observations
ANOVA
Regression
F
MS
SS
3 386.1534 128.7178 31.96169
Residual
81 326.2O75 4.027253
Total
84 712.3609
Intercept
PLI
GI
RR
Significance F
Coefficient
Standard
s
Error
t Stat
9.86E-14
P-value Lower 95% Upper 95%
-0.4185
0.5478 -0.7638
0.4472
-1.5085
0.6716
-0.8709
0.3647 -2.3881
0.0193
-1.5964
-0.1453
2.9122
0.3843
7.5779
0.0000
2.1476
3.6769
0.9611
0.2861
3.3592
0:0012
0.3918
1.5304
Table 64. Regression model: Y= In (TNF+I), X= PLI + GI + RRI
Backwards stepwise multivariable regression using transformed TNF-c values (natural
logarithm) as the dependent variable and the Plaque Index, the Gingival Index, and
the Root Resorption Index as the independent variables.
151
R’egression’ Statistics
Multiple R
0.7140
R Square
0.5098
Adjusted R Square
O.4979
Standard Error
2.0635
85
Observations
ANOVA
df
Regression
F
MS
SS
Significance
2 363.1854 181.5927 42.64503
Residual
82 349.1755 4.258238
Total
84 712.3609
Coefficient
Standard
t Stat
F
2.01E-13
P-value Lower 95% Upper 95%
Error
Intercept
GI
RR
-1.0636
0.4901 -2.1703
0.0329
-2.0386
-0.0887
2.4174
0.3328
7.2637
0.0000
1.7553
3.0794
1 .O964
0.2884
3.8020
0.0003
0.5228
1.6701
Table 65. Regression model: Y= In (TNF+I), X= GI + RRI
Backwards stepwise multivariable regression using transformed TNF-c values (natural
logarithm) as the dependent variable and the Gingival Index and the Root
Resorption Index as the independent variables.
152
Regresio’n Statistics
Multiple R
0.6507
R Square
0.4234
Adjusted R Square
0.4165
Standard Error
2.2245
85
Observations
ANOVA
Regression
1 301.6301 301.6301 60.95306
Residual
83 410.7308 4.948564
Total
84 712.3609
Coefficient Standa’d
s
Intercept
GI
Significance F
F
SS
-0.5062
2.7198
t Siat’
P-value
1.57E-11
Lower 95% Upper 95"
Error
0.5041 -1.0042
0.3182
-1.5089
0.4964
7.8072
0.0000
2.0269
3.4127
0.3484
Table 66. Regression model" Y= In (TNF+I), X= GI
Backwards stepwise multivariable regression using transformed TNF-a values (natural
logarithm) as the dependent variable and the Gingival Index as the independent
variable.
153
Regression’ Statistics
Multiple R
O.44O96
R Square
0.19445
Adjusted R Square
0.18474
Standard Error
2.62941
Observations
ANOVA
df
Regression
1
’SS
F
MS
138.5161
138.5161 20.03475
Residual
83 573.8448 6.913792
Total
84 712.3609
Coefficient Standa’rd
s
Intercept
RR
Significance F
t Stat
2.4E-05
P-value Lower 95%
UPper 95%
Error
1.57785
0.41862 3.76919 0.00031
0.74524
2.41O47
1.59708
0.35681 4.47602 0.00002
0.88740
2.30676
Table 67. Regression model" Y= In (TNF+I), X= RRI
Backwards stepwise multivariable regression using transformed TNF-ot values (natural
logarithm) as the dependent variable and the Root Resorption Index as the
independent variable.
154
Regression Statistics
Multiple R
0.6984
R Square
0.4877
Adjusted R Square
0.4687
Standard Error
1.1436
85
Observations
ANOVA
df
Regression
3 100.8464 33.61548 25.70426
8.81E-12
105.93 1.307778
Residual
81
Total
84 206.7765
Intercept
PLI
GI
RR
Significance F
F
MS
SS
Coefficient
Standard
s
Error
t Stat
P-value Lower 95% Upper 95
-0.4386
0.3122 -1.4048
0.1639
-1.0597
0.1826
-0.4622
0.2078 -2.2244
0.0289
-0.8757
-0.0488
1.5432
0.2190
7.0469
0.0000
1.1075
1.9790
0.4225
0.1630
2.5913
0.0113
0.0981
0.7469
Table 68. Regression model: Y= TNF Index, X PLI + GI + RRI
Backwards stepwise multivariable regression using the TNF Index as the dependent
variable and the Plaque Index, the Gingival Index, and the Root Resorption Index
as the independent variables.
155
Regression Statistics
Multiple R
0.6025
R Square
0.3630
Adjusted R Square
0.3475
Standard Error
410.80
43
Observations
ANOVA
Regression
1
F
MS
SS
3942702
Significance
3942702 23.3636
F
1.91E-05
6918918 168754.1
Residual
41
Total
42 10861620
Coefficients Standard
t Stat
P-value Lower 9’5% Upper 95%
Error
Intercept
G!
-359.048
475.445
176.212 -2.038
98.363
4.834
0.0481
-714.916
-3.180
0.0000
276.798
674.093
Table 69. Regression model: Y=TNF-0’s, X=GI
Backwards stepwise multivariable regression using TNF-c values, where samples with
values of zero were omitted, as the dependent variable and the Gingival Index as
the independent variable.
156
Regression Statistics
Multiple R
0.3190
R Square
t).1018
Adjusted R Square
0.0799
Standard Error
487.81
43
Observations
ANOVA
Regression
1
1105391
Significance F
1105391 4.645345
0.03706
9756229 237956.8
Residual
41
Total
42 1O861620
Coefficient Standard
s
Intercept
RR
F
MS
SS
df
t Stat
P-value
Error
Lower
Upper
95%
95%
186.77
137.91
1.354
0.1830
(91.74)
465.28
215.24
99.86
2.155
0.0371
13.56
416.92
Table 70. Regression model" Y=TNF-0’s, X=RRI
Backwards stepwise multivariable regression using TNF-c values, where samples with
values of zero were omitted, as the dependent vail’able and the Root Resorption
Index as the independent variables.
157
Regression Statistics
Multiple R
0.7599
R Square
0.5775
Adjusted R Square
0.5450
Standard Error
8.4488
43
Observations
ANOVA
Regression
F
MS
SS
3 3804.723 1268.241
Residual
39 2783.905 71.38218
Total
42 6588.628
Coefficient
stan’dard
s
Error
Significance F
17.76691
t Stat
1.99E-07
P-value Lower 95% Upper 95%
Intercept
10.7394
5.5237
1.9442
0.0591
-0.4333
21.9121
GI
10.9822
2.0794
5.2814
0.0000
6.7762
15.1882
Brs
RR
-1.7637
0.5391 -3.2714
0.002:2
-2.8541
-0.6732
2.1926
0’.0344
0.3005
7.4553
3.8779
1.7686
Table 71. Regression model" Y= Ranked TNF-0’S, X=GI + BRS + RR_I
Backwards stepwise multivariable regression using ranked TNF-c values, where
samples with values of zero were omitted, as the dependent variable and the
Gingival Index, hours since the patient last brushed their teeth, and the Root
Resorption Index as the independent variables.
158
Regression Statistics
Multiple R
0.6540
R Square
0.4277
Adjusted R Square
0.4137
Standard Error
9.5900
43
Observations
ANOVA
df
Regression
F
MS
SS
1 2817.925 2817.925 30.64016
Residual
41 3770.703 91.96836
Total
42 6588.628
Intercept
GI
Significan"ce F
Coefficient
Standard
s
Error
t Stat
1.98E-06
P-value Lower 95% Upper 95%
0.6240
4.1137 0.1517
0.8802
-7.6837
8.9317
12.7107
2.2963 5.5354
0.0000
8.0733
17.3481
Table 72. Regression model" Y= Ranked TNF-0’s, X=GI
Backwards stepwise multivariable regression using ranked TNF-c values, where
samples with values of zero were omitted, as the dependent variable and the
Gingival Index as the independent variable.
159
Regression Statistics
Multiple IR
O.4059
R Square
0.1647
Adjusted R Square
0.1444
Standard Error
11.5855
43
Observations
ANOVA
Regression
1
1085.434 1085.434 8.086722
Residual
41 5503.194 134.2242
Total
42 6588.628
Intercept
Brs
Significance F
F
MS
SS
df
Coefficient
Standard
s
Error
t Stat
0.006924
P-value Lower 95% Upper 95%
35.6087
5.1320
6.9386
0.0000
25.2445
45.9730
-2.0601
0.7244 -2.8437
0.0069
-3.5231
-0.5971
Table 73. Regression model: Y= Ranked TNF-0’s, X=BRS
Backwards stepwise multivariable regression using ranked TNF-a values, where
samples with values of zero were omitted, as the dependent variable and the hours
since the patient last brushed their teeth as the independent variable.
160
Regression Statistics
Multiple R
0.2866
R Square
0.0822
Adjusted R Square
0.0598
Standard Error
12.145
43
Observations
ANOVA
df
Regression
SS
F
MS
1 541.3384 541.3384 3.670219
Residual
41 6047.289 147.4949
Total
42 6588.628
Intercept
RR
Significance F
Coefficient
Standard
s
Error
t Stat
0.062384
P-value Lower 95% Upper 95%
16.3684
3.4334 4.7674
0.0000
9.4346
23.3023
4.7632
2.4863 1.9158
0.0624
-0.2580
9.7843
Table 74. Regression model" Y= Ranked TNF-0’s, X=RRI
Backwards stepwise multivariable regression using ranked TNF-(x values, where
samples with values of zero were omitted, as the dependent variable and the Root
Resorption Index as the independent variable.
APPENDIX III. CONSENT FORMS
INFORMED CONSENT FOR ADULTS
Title: Levels of cytokines present in the gingival sulcus of orthodontically treated teeth
Investigators" EF Rossomondo DDS, PhD, Louis Norton DMD, Mike McKay DDS, and
Robert Marzban DDS
I understand that I have been asked to participate in a research study conducted by Dr.
Rossomando and colleagues of the University of Connecticut Health Center. The
purpose of this study is to develop a device for the diagnosis of root resorption. Although
I will receive no direct benefit from this research, this work may lead to the development
of better diagnostic tools for orthodontics.
My participation in this research will require the collection of fluid from between my
gums and teeth. The fluid will be collected from around the gums by putting a small
amount of very small protein-coated particles in the space between the gums and teeth.
The particles have an iron center so that they can be taken out with a small magnet. I
understand that these procedures will take about five minutes and I might be asked to
allow the researcher to take repeated monthly samples at my convenience. At that time
the doctor will examine me for any signs of trouble from the first procedure. If the doctor
finds any problem, the second procedure will not be done. These procedures will not
require any anesthesia and have no known risks. In some cases, only about one-half of
the beads are recovered. The other half do not remain and are washed out of the mouth.
These studies have been in progress for more than five years and no harmful effects have
161
162
been seen. Nonetheless, unexpected trouble from side effects, namely an irritaion of the
gums, is a possibility. I understand that I will receive a complete examination of the
mouth for signs of oral disease and if any problems are found I will be informed and
directed to appropriate individuals who advise me on treatment.
Any reports or presentations about this research will not idemify me by name. Best
efforts will be made to maintain confidentiality, although the Federal Food and Drug
Administration has the fight to examine research records involved in drug or diagnostic
device development and testing. I understand that this study has not been approved by
the Federal Food and Drug Administration.
I understand that I may withdraw from this study at any time without prejudice to care
at the University of Connecticut Health Center.
The University of Connecticut Health Center (UCHC) does not provide insurance
coverage to compensate me if I am injured during this research. However, I may still be
eligible for compensation. If I am injured, I can file a claim against the State of
Connecticut seeking compensation. For a description of this process, or available
compensation options, I may contact Rose Marie Howes, Executive Secretary,
Institutional Review Board (RIB) at the UCHC, at 679- 142.
The UCHC does not offer free care. However, treatment for a research related injury
may obtained at UCHC for the usual fee.
If I have any questions about this study, of my fights as a research subject, I may call
Dr. Edward Rossomando (679-2622) at the University of Connecticut Health Center.
163
I, the undersigned, have understood the above explanation, give consent to my
voluntary participation in this research project, and have received a copy of this consent
Date
Signature of the investigator
Signature of the subject
Date
Witness
Date
164
INFORMED CONSENT FOR DEPENDENTS
Title" Levels of cytokines present in the gingival sulcus of orthodontically treated teeth
Investigators: EF Rossomando DDS PhD, DDS, Louis Norton DMD, Mike McKay DDS,
and RobertMarzban DDS
I understand that I have been asked to allow my child to participate in a research
study conducted by Dr. Rossomando and his colleagues of potential problems with
orthodontic treatment. Although my child will receive no direct benefit from this
research, this work may lead to the development of better diagnostic tools for
orthodontics.
Participation in this research will require the collection of fluid from between gums
and teeth. The fluid will be collected from around the gums by putting a small amount of
very small protein-coated particles in the space between the gums and teeth. The
particles have an iron center so that they can be taken out with a small magnet. I
understand that these procedures will take about 5 minutes. These procedures will not
require any anesthesia and have no known risks since this technique has been used in
over 200 patients. In some cases only about one half of these beads are recovered. The
other half do not remain and are washed out. These studies have been in progress for
more than four years and we assume no harmful effects have ever been seen.
Any reports or presentations about this research will not identify my dependent by
name. Best efforts will be made to maintain confidentiality, although the Federal Food
and Drug Administration has the fight to examine research records involved in drug or
diagnostic device development and testing. I understand that this study has not been
approved by the Federal Food and Drug Administration.
165
I understand that my child may withdraw from this study at any time without
prejudice to care at the University of Connecticut Health Center.
The University of Connecticut Health Center (UCHC) does not provide insurance
coverage for compensation in the highly unlikely event of injury during this research.
However, my child may still be eligible for compensation. If my child is injured, I can
file a claim against the State of Connecticut seeking compensation. For a description of
this process, or available compensation options, I may contact Rose Marie Howes,
Executive Secretary, Institutional Review Board (IRB) at the UCHC, at 679-2142.
The UCHC does not offer free care. However, treatment for a research related injury
may obtained at UCHC for the usual fee.
If I need additional information, I may contact Rose Marie Howes, Executive
Secretary of the IRB at 679-2142. The Executive Secretary can review the matter with
me, identify the resources that may be available, and provide me with further inforation
as to how to proceed.
If I have any questions about this study, or my child’s fights as a research subject, I
may call Dr. Rossomando at 679-2664 at the University of Connecticut Health Center.
I, the undersigned, have understood the above expl[nation, give consent to my
child’s voluntary participation in this research project, and have received a copy of this
consent form.
Investigator
Date
Guardian
Date
Witness
Date
166
If the subject is a minor:
My researcher, Dr. Marzban or Dr. Norton, and parents have talked to me about being
a part of a study that will collect fluid from around.my teeth. They are interested to know
how much of a particle (called TNF) is in the fluid around my teeth. How much TNF
they find may be a reason why teeth shorten while being moved. I understand the reason
for the study and why I am being asked to take part in it. I have been told that as a
subject in the study I will have the following performed: cotton rolls will be placed
between my tooth and cheek and also between my tooth and tongue; my tooth will be
dried with air; little particles will be placed near my tooth and gum with a long thin straw;
after about a minute or two, the particles will be collected with a magnet that is smaller
than a pencil. I understand that these procedures will not hurt. I know that I can ask
questions about this study at any time. I also know that I can decide not to be in this
study, or after entering the study I can decide that I want to be taken out of it.
Whatever I decide to do, I know my researcher will not be angry with me and that my
dentist will continue to treat me as his patient.
Date
Subject
Investigator
Date
Witness
Date
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