Lamina Dura: Bundle Bone Or Radiographic Artifact

LAMINA DURA: BUNDLE BONE OR RADIOGRAPHIC ARTIFACT

GASTON BERENGUER

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2005

Gaston Berenguer

To Carlos Berenguer, Vilma L. Bornot, and Candice Bean

ACKNOWLEDGMENTS

I would like to thank my family and friends for their support in all of my endeavors. I appreciate the opportunity Drs. Herbert J. Towle and Frederic Brown have given me and the guidance Drs. Jonathan Gray and Gregory Horning provided. I am also grateful to Dr. Arthur Vernino for his commitment to the profession of periodontics. I am indebted to my supervisory committee (Dr. Gregory Horning, Dr. Herbert J. Towle,

Dr. Katherine Karpinia, and Dr. Donald Cohen). I would also like to acknowledge Dr.

Linda Young, Dana Lucas, Lisa L. Booher, and Solomon Abraham.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES...... vii

LIST OF FIGURES ...... viii

ABSTRACT...... ix

CHAPTER

1 INTRODUCTION ...... 1

2 BACKGROUND ...... 3

3 AIM OF STUDY ...... 8

4 NULL HYPOTHESIS...... 9

5 METHODS AND MATERIALS ...... 10

6 RESULTS...... 15

Radiographic Osteotomy Results (Phase I) ...... 15 Overall Radiographic versus Histological Results for LD and PDL (Phase II) ...... 15 Statistical Correlation Analysis for LD (Radiographic versus Histological) ...... 16 Statistical Correlation Analysis for PDL (Radiographic versus Histological) ...... 17

7 DISCUSSION...... 22

APPENDIX

A PROTOCOL FOR PHASE I ...... 24

Protocol for Obtaining and Drilling Osteotomies into Cadaver Sections...... 24 Drilling the Pine Wood, Plaster of Paris and Polyvinyl Siloxane Impressions...... 24 Evaluation of Samples ...... 24

B PROTOCOL FOR PHASE II ...... 26

Protocol for Mid-Root Digital Radiography Data Gathering...... 26 Protocol for Sectioning and Preparation of Cadaver Samples...... 26 Protocol for Hematoxylin and Easin (H & E) Staining ...... 27

C LAMINA DURA ANALYSIS...... 28

D PERIODONTAL LIGAMENT ANALYSIS...... 30

LIST OF REFERENCES...... 32

BIOGRAPHICAL SKETCH ...... 35

LIST OF TABLES

Table page

1 Percent negative (-) responses to false LD images due to method used ...... 18

2 Percent negative (-) responses to false LD images due to individual examiners.....18

3 Mean LD recorded by principal investigator ...... 18

4 Mean PDL recorded by principal investigator ...... 18

vii

LIST OF FIGURES

Figure page

1 Osteotomy being created...... 12

2 Radiographic jig...... 12

3 Pine wood block...... 12

4 Plaster of Paris impression ...... 13

5 Polyvinyl siloxane impression ...... 13

6 Digital radiograph with osteotomy...... 13

7 Section embedded in paraffin before staining...... 14

8 Hematoxylin and Eosin stained samples in notebook...... 14

9 Pine wood block radiograph...... 19

10 Plaster of Paris impression radiograph...... 19

11 Polyvinyl siloxane impression radiograph ...... 19

12 Osteotomy ...... 20

13 Straight radiograph...... 20

14 Close up of interproximal #19 and 20...... 20

15 Corresponding histologic section tooth #20...... 21

16 Corresponding histologic section tooth #19 (mesial root) ...... 21

viii

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

LAMINA DURA: BUNDLE BONE OR RADIOGRAPHIC ARTIFACT

Gaston Berenguer

May 2005

Chair: Gregory Horning Major Department: Periodontics

The clinical significance and implication of lamina dura (LD) has long been controversial. Some clinicians think the absence or diminution of LD is diagnostic of such pathoses as hyperparathyroidism, osteoporosis, and periapical or periodontal inflammation. Others suggest the radiographic density increase of the LD indicates occlusal traumatism. In a landmark 1963 article, Manson concluded that the LD about teeth could be a radiographic artifact. The aim of our study was to correlate the presence or absence of radiographic LD with histologic findings.

In Phase I, we tried to create artifactual LD. We evaluated holes (osteotomies) drilled into a pine wood block and cadaver specimens, and also evaluated holes created with Plaster of Paris and polyvinyl siloxane impression materials. In all, 63 cadaver osteotomies were drilled. In Phase II, we examined 18 preserved human-mandibular alveolar-arch dentate sections. Digital radiographs were taken from the straight

(perpendicular to the teeth), -5°, and +5° angles of the cadaver specimens. Measurements were taken of the LD and periodontal ligament (PDL) images. These measurements were

compared to histomorphometric measurements made of mid-root alveolar bone proper and PDL from Hematoxylin and Eosin (H &E) stained sections. In all, 50 surfaces were evaluated.

False images of LD were observed 7 out of 72 (9.7%) possible times. False images were observed in the wood block 2 out of 3 (66.7%) possible times. No false images were noted for the impression materials. False images were observed in the cadaver specimens 5 out of 63 (7.9%) possible times. Radiographic LD images were observed

135 out of 150 (90%) possible times. Corresponding alveolar bone proper was observed

150 out of 150 (100%) possible times. Radiographic image of PDL were observed 130 out of 150 (86.7%) possible times. Corresponding histological PDL were observed 150 out of 150 (100%) possible times. Mean radiographic LD was 386.8 + 198.2 µm. The range was 0 to1398.0 µm. Corresponding mean mid-root alveolar bone proper width was

277.0 + 196.7 µm. The range was 82.0 to 1279.0 µm. Mean radiographic PDL was

109.5 + 45.7 µm. The range was 0 to 297.0 µm. Corresponding mean mid-root histological PDL width was 141.4 + 50.0 µm. The range was 41.0 to 350.0 µm. There was no statistically significant correlation (P<0.05) between LD and corresponding alveolar bone proper. There was a weak correlation (P<0.05) between radiographic and corresponding histological PDL images.

The radiographic lamina dura does not appear to be an artifact, and could not be readily created in specimens. Lamina dura measurements did not significantly correlate

(P<0.05) with corresponding alveolar bone proper. There was a weak correlation

(P<0.05) for radiographic and histological PDL images.

CHAPTER 1 INTRODUCTION

Throughout the literature, strong evidence supports and opposes the concept that

alveolar sockets and their radiographic images have clinical significance and

implications. To many, the absence of lamina dura (LD) is diagnostic of pathology. This

is especially true once it loses its continuity near the apex, where the lamina dura seems

interrupted.1 The classic literature describes the radiopaque socket LD as an image of a

lining-reactive bundle bone responding to external forces applied to its surface by

Sharpey’s fibers.2 The LD was also said to have a direct relationship with occlusal trauma.3 Richey and Orban,4 in 1953 thought the LD indicated changes in periodontal

health. As recently as 1994, Rams et al.5 said the crestal LD could be used in predicting

periodontal health or disease activity. Others think LD (especially the socket LD) is unreliable. In 1963, Manson6 concluded that the LD was a radiographic artifact; a

tangential bony radiopacity of no clinical significance and inconsistent with disease,

trauma or health. In 1981, Greenstein et al.7 thought the LD was unrelated to the

presence or absence of clinical inflammation. Socket and/or crestal lamina dura are also

attributed to physiologic trabecular bone response to trauma or periodontal health, and

are used as a potential diagnostic tool for such systemic diseases.8 Systemic conditions

such as primary hyperparathyroidism, secondary hyperparathyroidism, and primary

osteoporosis have been associated with absence or decrease density of LD.9-12 Renal

osteodystrophy (a disease characterized by uneven bone growth and demineralization)

and Burkitt’s lymphoma also show decreased LD.13-14 History of end-stage renal disease,

1 2

or being on hemodialysis (associated with diabetes mellitus or not) are both associated

with absent or decreased LD.15-16 Gestational hyperparathyroidism also results in mild resorption of the LD and may be a feature of normal pregnancy,17 according to a study

comparing non calcium (Ca+), vitamin D supplemented women versus supplemented

women during their third trimester of pregnancy. White and Pharoah18 said that the LD is

a radiographic image that may vary in sharpness (or lack of) with x-ray beam

angulations, possibly due to disruptions along the LD. They also said that thickness and

density are a product of occlusal stress from tooth force distribution. They did not

discuss widening or thickening of the image as a radiographic angle phenomenon, a true

thickening of the LD, trabeculation adjacent to the LD, or a combination of the three.

Other authors suggest that occlusal trauma, periodontitis, and occlusal trauma related to

fixed prosthesis affect radiographic LD.19-20 Most of these authors conclude that the LD is

thicker when subjected to any of these pathologic changes. At least one author associated

these changes with decreased definition of the LD.21 A comprehensive study is needed

that analysis previous studies and that gives radiographic and histological evidence for or

against thickening of the LD. A closer look at anatomy and radiographic characteristics

of the structures is needed.

CHAPTER 2 BACKGROUND

The alveolar process is a bony structure, part of the maxilla and mandible that

forms, surrounds, and supports teeth. Within this process are the alveoli for the teeth, also known as the tooth socket. Although the alveolar process is considered a separate entity, there is no line of demarcation between the process and the body of the maxilla or the mandible. The alveolar process is made up of three distinct anatomical structures.22

The inner (palatal and lingual) and outer (labial and buccal) compact cortical plates, the alveoli lining bone referred to as the alveolar bone proper or cribiform plate and the cancellous or spongy bone found between the cortical plates and the alveolar bone proper. The alveolar bone proper has been referred to as a bundle bone, cortical bone, and a cribiform plate. The internal cancellous or spongy bone found between the cortical plates is a specifically patterned trabecullar bone that acts as a scaffold. The alveolar process always attempts to conform to root morphology through the alveolar bone proper, the cancellous bone region, and its cortical plates. Since the alveolar process broadens posteriorly, so too does the cancellous or spongy bone region with its cancellous bony spicules. The proximity of teeth to each other, their angulations, and their vertical positioning all influence the anatomy of the alveolar process anatomy.4

On a cellular level, the alveolar process, whether looking at compact cortical plates,

alveolar bone proper, or cancelluous bone, the types of structures identified are

histologically identical.22 Mature bone is deposited in lamellae (thin layers) of ground substance 4-12 µm thick.23 These are easily identifiable. Osteocytes house themselves

3 4

in and between the lamellae, along the length of its planes. The pattern of lamellar

arrangement differs among the different areas or types of bone. The lamellae can be

deposited along the periphery of the bone (circumferential), around a Haversian system

(concentric), or in between the concentric patterns (interstitial).22

The Haversian systems are composed of an osteon (the cylindrical unit of bone) which consists of a centered Haversian canal (housing capillaries) surrounded by concentric lamellae. The Haversian canals are in communication with interconnecting

Volkmann canals, which mainly house blood vessels that anastemose with those in the

Haversian canals. In compact bone, trabecular and alveolar bone proper, the lamellae are deposited in layers incrementally. The outer section of the cortical plates tends to have fewer Haversian systems and a well-organized pattern of parallel lamellae (along the

entire circumference of the bone). The inner section of the cortical plates has more

Haversian systems with well-defined concentric lamellae. The deeper cancelluous bone

pattern is scattered at best and is vascularized by some Haversian systems and the

marrow itself. Its trabeculae vary in thickness and length, with a single trabeculae

consisting of a few lamellae in concentric or parallel layers to each other.5 The

scaffolding, or array of struts, is always adapting by rebuilding to accommodate the alveolar bone proper to the cortical plates. This is how the structure retains its ability to distribute continuously changing forces emitted onto teeth by the ever-changing environment. As for the alveolar bone proper, Volkmann’s canals perforate its surface through the bony housing. Everywhere else, Sharpey’s fibers are identified. One can also readily identify bone resorption, incremental bone deposition lines, or a combination of the two.

The radiographic images, and interpretation of the structures discussed, is perhaps

where most controversy exists. The cortical plates seem to have a light radiopaque

appearance, only truly defined when removed. When cortical plates are partly or fully

compromised, the density of the radiograph images are affected, but the trabecular pattern

remains.23 Bender and Seltzer24 found the intensity of radiographic images, created by

intra-osseous defects, to be dependant on the level of cortical plate invasion (penetration) by these lesions. Lesions confined to cancellous bone; therefore, are not easily detected.

Until either cortical plate is compromised, slight, yet hard to identify radiopacities can be created, but are not of diagnostic quality. The cancellous (spongy) bone, composed of trabeculae and bone marrow, is characterized as having a radiolucent appearance with numerous scattered radiopaque lines. These lines represent a 3-dimentional bony pattern which is the result of superimposed trabeculae bony spicules. Radiopacities corresponding to crestal or tooth socket LD, inferior border of mandible, maxillary sinus, nasal cavity, and inferior alveolar canals, are readily seen on radiographs.

Of all the structures described, the radiographic tooth socket LD (corresponding to the histological alveolar bone proper) has had the most controversy. What is not controversial is that most clinicians believe it identifies the physical alveolar bone proper or cribiform plate housing teeth. What is controversial is its reliability, its validity, and what its thickness, disappearance, or change in size represent. Weinmann5 believed its

affiliation with Sharpey’s fibers, by definition, qualified it as bundle bone (bony which

has penetrating collagen fibers). Although, at one point, it was thought to represent an

area of bone with higher mineral content, Goldman et al.23 said it was the quantity, not

the mineral variation that determined the appearance of LD. Manson6 supported

Goldman et al.,23 who said the radiopaque line corresponded to a plate of bone through

which the x-ray beam passed tangentially, and thus through a greater area of bone than the actual width of the cortical plates. Manson6 also suggested the bone of the socket

wall had the same mineral contents, by mass, as the adjacent bone. He believed there

was no evidence of a special bone with a higher mineral content.6 He also proposed the

idea that the LD image could be a radiographic artifact. Through a series of wood block

and dry skull studies, he documented this possibility. In 1978, Kilpinen and Hakala25

considered the image to be the result of “Mach-band” affect. The “Mach-band” affect is

defined as a radiopaque image that forms at the interface between to areas with different

densities.25 The LD image is at the interface of two such zones, the periodontal ligament

(PDL) and the adjacent cancellous bone. This theory was not evidence based though. A study did confirm the image of a LD would disappearance when the alveolar bone proper was removed.26 A similar study suggested that both the alveolar bone proper and some

adjacent trabecular bone had to be removed to detect a difference in radiographic LD

image.27 One study did manage to obtain a histological alveolar bone proper size range,

0.22-0.54 mm.28

According to the fourth edition of the Jan Lindhe’s text, Clinical Periodontology

and Implant Dentistry, the bony walls of tooth sockets are cortical bony plates, compact

bone, and bundle bone.29 Their reference to bundle bone is, again, by definition. The

references to cortical or compact bone are the very claims Manson6 opposed in 1963. For

most clinicians today, there is little doubt that the size, sharpness and shape of the LD

corresponds, in some way, to tooth form (size and shape), position, and radiographic

angulations of the corresponding alveolar bone proper. The histological evidence for

7 these claims, and the extent to which these variables may influence such images, are at best illusive.

CHAPTER 3 AIM OF STUDY

The aim of our study was to correlate the presence or absence of radiographic lamina dura (LD) with histologic findings. Specific objectives are listed below.

1. To determine if artifactual LD can readily be created in wood, impression materials and cadaver bone

2. To determine the relationship of radiographic LD density and size to histological alveolar bone proper size

3. To determine if radiographic angulations, alveolar bone proper size, adjacent trabecular bone or a combination make a difference on the LD radiographic image

CHAPTER 4 NULL HYPOTHESIS

There is no correlation between radiographic lamina dura (LD) and its corresponding histological alveolar bone properties. LD is a radiographic artifact.

CHAPTER 5 METHODS AND MATERIALS

Twenty human mandibular alveolar-arch specimens, stored in formaldehyde, were

obtained from the Florida State Anatomical Board. The cause of death is unknown.

From these, eighteen sections were obtained for the present study.

In Phase I, we tried to create artifactual lamina dura (LD). We evaluated holes

(osteotomies) drilled into a pine wood block and cadaver specimens (figure 1) and also

evaluated holes created with Plaster of Paris and polyvinyl siloxane impression materials.

In all, 63 cadaver osteotomies were drilled. Inclusion criteria for cadaver specimen included mandibular alveolar-arch section, intact cortical plate, and evidence of

remaining bone at a >50% level. The later was determined by visual and radiographic

qualification of the sample. Digital radiographs were taken on a jig (figure 2),

perpendicular and at a constant distance of 2-3” from the specimens. These were then

evaluated independently on light boxes by three calibrated board certified periodontists.

Holes drilled into the wood block (figure3) and those created with plaster of Paris (figure

4) and light bodied polyvinyl siloxane impression materials (figure 5) were also evaluated. The protocol for obtaining and drilling cadaver sections, drilling the pine wood, and taking the Plaster of Paris and polyvinyl siloxane impressions is described in

Appendix A. In the end, in addition to the pine wood holes and the Plaster of Paris and polyvinyl siloxane impressions, twenty one cadaver osteotomies were created and evaluated.

10 11

In Phase II, we examined 18 preserved human-mandibular alveolar-arch dentate sections. Digital radiographs were taken from the straight (perpendicular to the teeth),

-5°, and +5° angles of the cadaver specimens (figure 6). Measurements were taken of LD images. These radiographic measurements were compared and correlated to histomorphometric measurements taken of mid-root LD from the Hematoxylin and Eosin

(H &E) stained sections (figure 7, 8). In all, 50 surfaces were evaluated. Inclusion criteria were same as in Phase I, in addition to the samples being dentate. The protocol for mid-root digital radiography data gathering and processing of the cadaver samples is described on Appendix B. Due to ease of data collection, measurements were also documented for the periodontal ligament.

Figure 1. Osteotomy being created

Figure 2. Radiographic jig

Figure 3. Pine wood block

Figure 4. Plaster of Paris impression

Figure 5. Polyvinyl siloxane impression

Figure 6. Digital radiograph with osteotomy

Figure 7. Section embedded in paraffin before staining

Figure 8. Hematoxylin and Eosin stained samples in notebook

CHAPTER 6 RESULTS

Radiographic Osteotomy Results (Phase I)

The results from Phase I are shown on tables 1 and 2. False images of LD were observed 7 out of 72 (9.7%) possible times. False images were observed in the wood block 2 out of 3 (66.7%) possible times (figure 9). No false images were noted for the

Plaster of Paris (figure 10) and the polyvynil siloxane(figure 11) impression materials.

The two later where true impression techniques unlike the pine wood block where

drilling was performed. False images were observed in the cadaver specimens 5 out of

63 (7.9%) possible times (figure 12). Analyzing these same cadaver specimens, when eliminating sample J (sample where a tooth was purposely drilled), false images were observed in 3 out of 60 (5.0 %) possible times.

Overall Radiographic versus Histological Results for LD and PDL (Phase II)

Radiographic LD images were observed 135 out of 150 (90%) possible times.

Corresponding alveolar bone proper was observed 150 out of 150 (100%) possible times.

Radiographic image of PDL were observed 130 out of 150 (86.7%) possible times.

Corresponding histological PDL were observed 150 out of 150 (100%) possible times.

Examples of radiographic and histological specimen are demonstrate in figures 13 and

14. Mean radiographic LD was 386.8 + 198.2 µm. The range was 0 to1398.0 µm.

Corresponding mean mid-root alveolar bone proper width was 277.0 + 196.7 µm. The

range was 82.0 to 1279.0 µm. Mean radiographic PDL was 109.5 + 45.7 µm. The range

was 0 to 297.0 µm. Corresponding mean mid-root histological PDL width was 141.4 +

15 16

Statistical Correlation Analysis for LD (Radiographic versus Histological)

For statistical purposes, HO:Rho=0, indicating no correlation. For each surface, three (straight, -5o, +5o) radiographic measurements and a histological measurement were recorded. Plots were constructed to provide insight. The correlations of the three radiographic measurements, the mean of the three measurements, and the histological measurements were computed and tested for significance. Regression methods were used to explore relationships. The basic conclusion is that there is little evidence of a relationship in the radiographic and histological measurements.

The plots provided little support for a relationship between radiographic and histological measurements. For the LD, results from the correlation analysis are shown in Appendix C. Note that none of the correlations between a radiographic measurement and the histologic measurements were significant (P=<0.05)

Using an analysis of variance, it was found that when the radiographic measurement was made from the straight position that for both the “D” (Diffused) versus

“S”(solid) variable and the “I”(Isolated) versus ” N”(non-isolated) variable were significant (P = 0.0114, 0.0166, respectively). Each individual subgroup “D”, “S”, “I”,

“N” and combinations “DI”, “DN”, “SI” and “SN” were analyzed for relationships with corresponding histology. Again, none of the correlations between the radiographic measurement and the histologic measurements were significant (P = <0.05).

Statistical Correlation Analysis for PDL (Radiographic versus Histological)

For statistical purposes, HO:Rho=0, indicating no correlation. For the PDL measurements, the plots provided little support for a relationship between radiographic and histological measurements. Results from the correlation analysis are shown in

Appendix D. There is a significant correlation between the mean PDL radiographic measurement and the histological measurement (P = 0.0498). Although the correlation of

0.279 is significant, it is not strong. It remains very unpredictable.

Table 1. Percent negative (-) responses to false LD images due to method used Samples Pine Wood Plaster of Paris Polyvynil Cadaver Cadaver Block Impression Siloxane Samples Samples w/out sample J Total (-) LD 1/3 3/3 3/3 58/63 57/60 % (-) LD 33.3 100.0 100.0 92.1 95.0

Table 2. Percent negative (-) responses to false LD images due to individual examiners Examiners Total (-) LD % (-) LD Cadaver Samples: Cadaver Samples w/out % (-) LD sample J: %(-) LD I 24/24 100.0 100.0 100.0 II 22/24 91.7 95.2 100.0 III 19/24 79.2 81.0 86.4 Total 65/72 90.3 92.1 95.0

Table 3. Mean LD recorded by principal investigator Alveolar Bone Proper (LD) Mean (µm) SD (µm) Straight LD 417.8 253.4 -5° LD 387.2 267.1 +5° LD 355.4 246.3 Overall LD 386.8 198.2 X-sec LD 277.0 196.7

Table 4. Mean PDL recorded by principal investigator Periodontal Ligament (PDL) Mean (µm) SD (µm) Straight PDL 120.7 50.7 -5° PDL 103.9 66.3 +5° PDL 103.9 59.1 Overall PDL 109.5 45.7 X-sec PDL 141.4 49.9

Figure 9. Pine wood block radiograph

Figure 10. Plaster of Paris impression radiograph

Figure 11. Polyvinyl siloxane impression radiograph

Figure 12. Osteotomy

Figure 13. Straight radiograph

Figure 14. Close up of interproximal #19 and 20

Figure 15. Corresponding histologic section tooth #20

Figure 16. Corresponding histologic section tooth #19(mesial root)

CHAPTER 7 DISCUSSION

The radiographic lamina dura (LD) does not appear to be an artifact, and could not be readily created in specimens. Lamina dura measurements did not significantly correlate (P<0.05) with corresponding alveolar bone proper. There was a weak correlation (P<0.05) for radiographic and histological PDL images.

When looking at the two impression materials, no false radiographic LD image was observed. As for the wood sample, 2 of the 3 examiners noted a LD image. Reasons for this result could be residual wood particles along the edge of the hole, or compressed or burnished wood along the same perimeter. The impression materials could not have had these shortcomings. These findings were also consistent with the cadaver bone osteotomy results which demonstrated it was extremely difficult to visualize a false image of a LD. After sample J (sample where a tooth was purposely drilled) false images were observed in 3 out of 60 (5.0 %) possible times.

Radiographic LD images were observed 135 out of 150 (90%) possible times.

Corresponding alveolar bone proper was observed 150 out of 150 (100%) possible times.

Radiographic image of PDL were observed 130 out of 150 (86.7%) possible times.

Corresponding histological PDL were observed 150 out of 150 (100%) possible times.

Mean radiographic LD was 386.8 + 198.2 µm. The range was 0 to1398.0 µm.

Corresponding mean mid-root alveolar bone proper width was 277.0 + 196.7 µm. The

range was 82.0 to 1279.0 µm. Mean radiographic PDL was 109.5 + 45.7 µm. The range

was 0 to 297.0 µm. Corresponding mean mid-root histological PDL width was 141.4 +

22 23

50.0 µm. The range was 41.0 to 350.0 µm. There was no statistically significant correlation (P<0.05) between LD and corresponding alveolar bone proper. There was a weak correlation (P<0.05) between radiographic and corresponding histological PDL images. In 1937, Coolidge31 concluded the mean PDL width was 150-210µm.31 He determined the PDL became smaller with age. In his study, he noted the mean PDL size for individuals ages 11 to 16 was 0.21 mm and for the age group 50 to 67, it was 0.15 mm. The later number is comparable to 141.4 µm obtained in this study. Although not known, it is very likely the age group in our study was similar.

The lack of LD size correlation between radiographs and histological findings indicates that, not only does radiographic angulation play an important role in size distortion and variation, but that the complex trabecular pattern adjacent to the alveolar bone proper may be contributing to the true thickening of the alveolar bone proper or the illusion of a thicker LD. The illusion is possible due to the proximity of individual trabeculae to the alveolar bone proper. In the end, both scenarios probably result reinforced alveolar bone proper. When present on a radiograph, LD does correspond to a true bony entity. When the image is not present on a radiograph, it can not be assumed the corresponding alveolar bone proper is absent. The LD is therefore, a better positive predictor of its existence than a negative predictor of its absence. In our study, possible experimental errors were examiner bias, accuracy of measurements obtained digitally and histometrically, sectioning approximate location and tissue shrinkage.

APPENDIX A PROTOCOL FOR PHASE I

Protocol for Obtaining and Drilling Osteotomies into Cadaver Sections

• Approval from State Anatomical Board

• Section out 1 to 2” long mandibular sections meeting inclusion criteria

• Take preliminary digital radiographs to continue selection process

• Label specimens with tag and cord

• Using 3i Innovations™, Palm Beach FL, disposable implant drills, multiple osteotomies were created

• Irrigation with saline to minimize residual particles and denaturing of bone matrix

• Digital, straight radiographs were taken at 2 to 3“ distance from samples

Drilling the Pine Wood, Plaster of Paris and Polyvinyl Siloxane Impressions

• Using the same drills, multiple holes were created in the pine wood block

• Impression of different size wooden pegs were made using Plaster of Paris and polyvinyl siloxane impression material

• Digital, straight radiographs taken at 2 to 3“ distance from samples

Evaluation of Samples

Evaluation of radiographs taken from the pine wood sample, both impression materials and 21 cadaver osteotomies were conducted by three board certified periodontists, after calibration. Calibration was performed until evaluators had identical answers. The pine wood block contained six holes and both impression materials

24 25

contained four holes. In these three items, any one surface demonstrating a radiographic image of a lamina dura (LD) resulted as a false positive (+) answer for the complete item.

For any of the specimens, evaluators were instructed to select no (-) if no or an uncertain

LD was appreciated. Only true image of LD was recorded as false positives (+).

APPENDIX B PROTOCOL FOR PHASE II

Protocol for Mid-Root Digital Radiography Data Gathering

One examiner, the principle investigator, took mid-root measurements of tooth surfaces with a consistent image for approximately 4mm. If image was not consistence for specified length, the surface was ignored. It was important to have consistency in the area due to the inaccuracy of histological sectioning. The sections would only be an approximation of the mid-root area of a given surface.

Protocol for Sectioning and Preparation of Cadaver Samples

• Once radiographs were satisfactory of an area and measurements of lamina dura (LD) documented, the corresponding cadaver pieces were thinned, mounted and stained with Hematoxylin and Easin staining technique.

• First, area to be stained was determined

• Using diamond cutting macrotomes, sections were thinned to approximately 3 to 4 mm

• Samples were then labeled and decalcified in decalcifying solution for for approximately one week. Decalcifying solution contained:

• Water • Hydrochloric acid • Ethylenediaminetetraacetic acid, tetrasodium • Sodium tartrate • Potassium sodium tartrate

• Once decalcified, samples were then rinsed with water and stored in 10% neutral buffered formalin:

• Formaldehyde • Methyl alcohol • Buffers • Water

26 27

• Sections were imbedded in paraffin

• Samples were faced (planed)

• Cooled (at room temperature)

• Samples were then cut with microtome to 5 µm, released in water and picked up on a slide

• Paraffin was then melted off on heat element for 10min

• Sample stained with H & E stain cover slip fixed

Protocol for Hematoxylin and Easin (H & E) Staining

• Sequence protocol as per the Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology by Lee G. Luna.30

• Hematoxylin is a natural dye that in combination with salts is a powerful myelin and nuclear stain

• Eosin, a counterstain for Hematoxylin, stains collagen

APPENDIX C LAMINA DURA ANALYSIS

Simple Statistics

Variable N Mean Std Dev Sum Minimum Maximum Label

LDSTR 50 417.74000 253.40971 20887 0 1365 LDSTR LDMINUS5 50 387.24000 267.08835 19362 0 1398 LDMINUS5 LDPLUS5 50 355.42000 246.26020 17771 0 1080 LDPLUS5 MEANLD 50 386.80000 198.21281 19340 0 1130 LDXSEC 50 276.98000 196.73835 13849 82.00000 1279 LDXSEC

Pearson Correlation Coefficients, N = 50

Prob > |r| under H0: Rho=0 LDSTR LDMINUS5 LDPLUS5 MEANLD LDXSEC

LDSTR 1.00000 0.52700 0.36891 0.81564 0.10203 29 LDSTR <.0001 0.0084 <.0001 0.4808

LDMINUS5 0.52700 1.00000 0.30420 0.79973 0.10004 LDMINUS5 <.0001 0.0317 <.0001 0.4894

LDPLUS5 0.36891 0.30420 1.00000 0.70798 0.23641 LDPLUS5 0.0084 0.0317 <.0001 0.0983

MEANLD 0.81564 0.79973 0.70798 1.00000 0.18632 <.0001 <.0001 <.0001 0.1951

LDXSEC 0.10203 0.10004 0.23641 0.18632 1.00000 LDXSEC 0.4808 0.4894 0.0983 0.1951

APPENDIX D PERIODONTAL LIGAMENT ANALYSIS

Simple Statistics

Variable N Mean Std Dev Sum Minimum Maximum Label

PDLSTR 50 120.70000 50.69929 6035 0 225.00000 PDLSTR PDLMINUS5 50 103.90000 66.27902 5195 0 297.00000 PDLMINUS5 PDLPLUS5 50 103.86000 59.11732 5193 0 233.00000 PDLPLUS5 MEANPDL 50 109.48667 45.69400 5474 0 200.33333 PDLXSEC 50 141.36000 49.93660 7068 41.00000 350.00000 PDLXSEC

Pearson Correlation Coefficients, N = 50

Prob > |r| under H0: Rho=0 PDLSTR PDLMINUS5 PDLPLUS5 MEANPDL PDLXSEC

PDLSTR 1.00000 0.42022 0.25356 0.68237 0.24571 31 PDLSTR 0.0024 0.0756 <.0001 0.0854

PDLMINUS5 0.42022 1.00000 0.50907 0.85845 0.17791 PDLMINUS5 0.0024 0.0002 <.0001 0.2164

PDLPLUS5 0.25356 0.50907 1.00000 0.77116 0.23675 PDLPLUS5 0.0756 0.0002 <.0001 0.0979

MEANPDL 0.68237 0.85845 0.77116 1.00000 0.27899 <.0001 <.0001 <.0001 0.0498

PDLXSEC 0.24571 0.17791 0.23675 0.27899 1.00000 PDLXSEC 0.0854 0.2164 0.0979 0.0498

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BIOGRAPHICAL SKETCH

Gaston Berenguer D.M.D. was born in Santiago de Cuba, Cuba, on January 17,

1971. He immigrated to the United States, with his mother Vilma L. Bornot, in 1980.

His father, Carlos Berenguer was able to join them in 1984. In 1991, Gaston became an officer in the United States Army. He graduated from Florida State University

(Tallahassee), College of Arts and Science, with a Bachelor of Science degree in biology in 1995. In 2002, he graduated from the University of Florida (Gainesville), College of

Dentistry, with a Doctor of Dental Medicine degree. He completed a residency in the dental specialty of periodontics, with a Master of Science degree in periodontics. He looks forward to enjoying a full-time career in private practice in South Florida, and a part-time career in academics.